bluuebunny/crossref_metadata_2025 huggingface dataset:
Replication of enhancer-deficient amphotropic murine leukemia virus in human cells
Authors: Reuss Frank U. , Berdel Bianca , Ploss Martin , Heber Ricarda
Published: July 2002

Amphotropic murine leukemia virus (MLV) replicates in cells from various mammalian species, including humans, and is a potential contaminant in MLV vector preparations for human gene transfer studies. The generation of replication-competent virus is considered less likely with vectors that delete the viral transcription elements. This conclusion is based on data obtained in rodents, where MLV replication depends on the expression of viral genes under the control of 75-bp enhancer elements in the long terminal repeat. We demonstrate here that in some human cells replication of amphotropic MLV is possible in the absence of these enhancer elements. Replication of the enhancer-deficient virus MLV-(MOA)ΔE is observed in selected human sarcoma and B lymphoma lines and proceeds at a lower rate than that of the intact virus. No insertion of a foreign promoter or enhancer into the long terminal repeat was detected. Our data suggest the presence of a secondary enhancer element within the MLV provirus that can in selected human cells mediate virus transcription and replication in the absence of the 75-bp U3 enhancers.
Batrachotoxin alkaloids from passerine birds: A second toxic bird genus (
Ifrita kowaldi
) from New Guinea
Authors: Dumbacher J. P. , Spande T. F. , Daly J. W.
Published: July 2002

Batrachotoxins, including many congeners not previously described,
were detected, and relative amounts were measured by using HPLC-mass
spectrometry, in five species of New Guinean birds of the genus
Pitohui
as well as a species of a second toxic bird
genus,
Ifrita kowaldi
. The alkaloids, identified in
feathers and skin, were batrachotoxinin-A
cis
-crotonate
(1), an allylically rearranged 16-acetate (2), which can form
from 1 by sigmatropic rearrangement under basic conditions,
batrachotoxinin-A and an isomer (3 and 3a, respectively), batrachotoxin
(4), batrachotoxinin-A 3′-hydroxypentanoate (5), homobatrachotoxin (6),
and mono- and dihydroxylated derivatives of homobatrachotoxin. The
highest levels of batrachotoxins were generally present in the contour
feathers of belly, breast, or legs in
Pitohui dichrous
,
Pitohui kirhocephalus,
and
Ifrita kowaldi
.
Lesser amounts are found in head, back, tail, and wing feathers.
Batrachotoxin (4) and homobatrachotoxin (6) were found only in feathers
and not in skin. The levels of batrachotoxins varied widely for
different populations of
Pitohui
and
Ifrita
, a
result compatible with the hypothesis that these birds are sequestering
toxins from a dietary source.
On the impossibility of predicting the behavior of rational agents
Authors: Foster Dean P. , Young H. Peyton
Published: July 2002

A foundational assumption in economics is that people are rational: they choose optimal plans of action given their predictions about future states of the world. In games of strategy this means that each player's strategy should be optimal given his or her prediction of the opponents' strategies. We demonstrate that there is an inherent tension between rationality and prediction when players are uncertain about their opponents' payoff functions. Specifically, there are games in which it is impossible for perfectly rational players to learn to predict the future behavior of their opponents (even approximately) no matter what learning rule they use. The reason is that in trying to predict the next-period behavior of an opponent, a rational player must take an action this period that the opponent can observe. This observation may cause the opponent to alter his next-period behavior, thus invalidating the first player's prediction. The resulting feedback loop has the property that, a positive fraction of the time, the predicted probability of some action next period differs substantially from the actual probability with which the action is going to occur. We conclude that there are strategic situations in which it is impossible
in principle
for perfectly rational agents to learn to predict the future behavior of other perfectly rational agents based solely on their observed actions.
A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding
Authors: Lim Heon Man , Lee Hee Jung , Roy Siddhartha , Adhya Sankar
Published: July 2002

Isomerization of a closed to open complex of a promoter upon
RNA polymerase binding involves base unpairing at the −10 region.
After potassium permanganate sensitivity of unpaired thymine residues,
we studied base unpairing at the −10 region during isomerization upon
RNA polymerase binding at the
P1
and
P3
promoters of the
gal
operon. Substitution of adenine by
2-amino purine (2-AP) at the invariable A⋅T base pair at the −11
position of
P1
and
P3
prevented unpairing
not only at that position but also at the other downstream positions,
suggesting a “master” role of the adenine base at −11 of the
template strand in overall base unpairing. 2-AP at −11 did not inhibit
the formation of RNA polymerase⋅promoter complex and subsequent
isomerization of the polymerase. Substitution of adenine by 2-AP at
several other positions did not affect thymine unpairing. Changing the
position of the amino group from C6 in adenine to C2 in 2-AP is
mutational only at the master switch position, −11.
HLA-G expression during preimplantation human embryo development.
Authors: Jurisicova A , Casper R F , MacLusky N J , Mills G B , Librach C L
Published: July 2002

HLA-G is a nonclassical class I major histocompatibility complex molecule with a restricted pattern of expression that includes the placental extravillus cytotrophoblast cells in direct contact with maternal tissues. Circumstantial evidence suggests that HLA-G may play a role in protection of the semiallogeneic human fetus. We examined whether HLA-G is expressed during the critical period of preimplantation human development and whether expression of this molecule could be correlated with the cleavage rate of embryos. Using reverse transcription PCR on surplus human embryos and unfertilized oocytes from patients undergoing in vitro fertilization we detected HLA-G heavy chain mRNA in 40% of 148 of blastocysts tested. The presence of HLA-G mRNA was also detected in unfertilized oocytes and in early embryos, but not in control cumulus oophorus cells. beta 2-Microglobulin mRNA was also found in those embryos expressing HLA-G. In concordance with our mRNA data, a similar proportion of embryos stained positive for HLA-G utilizing a specific monoclonal antibody. Interestingly, expression of HLA-G mRNA was associated with an increased cleavage rate, as compared to embryos lacking HLA-G transcript. Thus, HLA-G could be a functional homologue of the mouse Qa-2 antigen, which has been implicated in differences in the rate of preimplantation embryo development. To our knowledge, the presence of HLA-G mRNA and protein in human preimplantation embryos and oocytes has not been reported previously. The correlation of HLA-G mRNA expression with cleavage rate suggests that this molecule may play an important role in human pre-embryo development.
Evidence for a preformed transducer complex organized by the B cell antigen receptor.
Authors: Wienands J , Larbolette O , Reth M
Published: July 2002

The B cell antigen receptor (BCR) consists of the membrane-bound immunoglobulin (mIg) molecule and the Ig-alpha/Ig-beta heterodimer, which functions as signaling subunit of the receptor. Stimulation of the BCR activates protein tyrosine kinases (PTKs) that phosphorylate a number of substrate proteins, including the Ig-alpha/Ig-beta heterodimer of the BCR itself. How the PTKs become activated after BCR engagement is not known at present. Here, we show that BCR-negative J558L cells treated with the protein tyrosine phosphatase inhibitor pervanadate/H2O2 display only a weak substrate phosphorylation. However, in BCR-positive transfectants of J558L, treatment with pervanadate/H2O2 induces a strong phosphorylation of several substrate proteins. Treatment with pervanadate/H2O2 does not result in receptor crosslinking, yet the pattern of protein phosphorylation is similar to that observed after BCR stimulation by antigen. The response requires cellular integrity because tyrosine phosphorylation of most substrates is not visible in cell lysates. Cells that express a BCR containing an Ig-alpha subunit with a mutated immunoreceptor tyrosine-based activation motif display a delayed response. The data suggest that, once expressed on the surface, the BCR organizes protein tyrosine phosphatases, PTKs, and their substrates into a transducer complex that can be activated by pervanadate/H202 in the absence of BCR crosslinking. Assembly of this preformed complex seems to be a prerequisite for BCR-mediated signal transduction.
Olfactory marker protein (OMP) gene deletion causes altered physiological activity of olfactory sensory neurons.
Authors: Buiakova O I , Baker H , Scott J W , Farbman A , Kream R , Grillo M , Franzen L , Richman M , Davis L M , Abbondanzo S , Stewart C L , Margolis F L
Published: July 2002

Olfactory marker protein (OMP) is an abundant, phylogentically conserved, cytoplasmic protein of unknown function expressed almost exclusively in mature olfactory sensory neurons. To address its function, we generated OMP-deficient mice by gene targeting in embryonic stem cells. We report that these OMP-null mice are compromised in their ability to respond to odor stimull, providing insight to OMP function. The maximal electroolfactogram response of the olfactory neuroepithelium to several odorants was 20-40% smaller in the mutants compared with controls. In addition, the onset and recovery kinetics following isoamyl acetate stimulation are prolonged in the null mice. Furthermore, the ability of the mutants to respond to the second odor pulse of a pair is impaired, over a range of concentrations, compared with controls. These results imply that neural activity directed toward the olfactory bulb is also reduced. The bulbar phenotype observed in the OMP-null mouse is consistent with this hypothesis. Bulbar activity of tyrosine hydroxylase, the rate limiting enzyme of catecholamine biosynthesis, and content of the neuropeptide cholecystokinin are reduced by 65% and 50%, respectively. This similarity to postsynaptic changes in gene expression induced by peripheral olfactory deafferentation or naris blockade confirms that functional neural activity is reduced in both the olfactory neuroepithelium and the olfactory nerve projection to the bulb in the OMP-null mouse. These observations provide strong support for the conclusion that OMP is a novel modulatory component of the odor detection/signal transduction cascade.
Increased glycogen accumulation in transgenic mice overexpressing glycogen synthase in skeletal muscle.
Authors: Manchester J , Skurat A V , Roach P , Hauschka S D , Lawrence J C
Published: July 2002

To investigate the role of glycogen synthase in controlling glycogen accumulation, we generated three lines of transgenic mice in which the enzyme was overexpressed in skeletal muscle by using promoter-enhancer elements derived from the mouse muscle creatine kinase gene. In all three lines, expression was highest in muscles composed primarily of fast-twitch fibers, such as the gastrocnemius and anterior tibialis. In these muscles, glycogen synthase activity was increased by as much as 10-fold, with concomitant increases (up to 5-fold) in the glycogen content. The uridine diphosphoglucose concentrations were markedly decreased, consistent with the increase in glycogen synthase activity. Levels of glycogen phosphorylase in these muscles increased (up to 3-fold), whereas the amount of the insulin-sensitive glucose transporter 4 either remained unchanged or decreased. The observation that increasing glycogen synthase enhances glycogen accumulation supports the conclusion that the activation of glycogen synthase, as well as glucose transport, contributes to the accumulation of glycogen in response to insulin in skeletal muscle.
Association of a novel human FE65-like protein with the cytoplasmic domain of the beta-amyloid precursor protein.
Authors: Guénette S Y , Chen J , Jondro P D , Tanzi R E
Published: July 2002

We identified a novel human homologue of the rat FE65 gene, hFE65L, by screening the cytoplasmic domain of beta-amyloid precursor protein (beta PP) with the "interaction trap." The cytoplasmic domains of the beta PP homologues, APLP1 and APLP2 (amyloid precursor-like proteins), were also tested for interaction with hFE65L. APLP2, but not APLP1, was found to interact with hFE65L. We confirmed these interactions in vivo by successfully coimmunoprecipatating endogenous beta PP and APLP2 from mammalian cells overexpressing a hemagglutinin-tagged fusion of the C-terminal region of hFE65L. We report the existence of a human FE65 gene family and evidence supporting specific interactions between members of the beta PP and FE65 protein families. Sequence analysis of the FE65 human gene family reveals the presence of two phosphotyrosine interaction (PI) domains. Our data show that a single PI domain is sufficient for binding of hFE65L to the cytoplasmic domain of beta PP and APLP2. The PI domain of the protein, Shc, is known to interact with the NPXYp motif found in the cytoplasmic domain of a number of different growth factor receptors. Thus, it is likely that the PI domains present in the C-terminal moiety of the hFE65L protein bind the NPXY motif located in the cytoplasmic domain of beta PP and APLP2.
Agrobacterium tumefaciens-mediated transformation of yeast.
Authors: Piers K L , Heath J D , Liang X , Stephens K M , Nester E W
Published: July 2002

Agrobacterium tumefaciens transfers a piece of its Ti plasmid DNA (transferred DNA or T-DNA) into plant cells during crown gall tumorigenesis. A. tumefaciens can transfer its T-DNA to a wide variety of hosts, including both dicotyledonous and monocotyledonous plants. We show that the host range of A. tumefaciens can be extended to include Saccharomyces cerevisiae. Additionally, we demonstrate that while T-DNA transfer into S. cerevisiae is very similar to T-DNA transfer into plants, the requirements are not entirely conserved. The Ti plasmid-encoded vir genes of A. tumefaciens that are required for T-DNA transfer into plants are also required for T-DNA transfer into S. cerevisiae, as is vir gene induction. However, mutations in the chromosomal virulence genes of A. tumefaciens involved in attachment to plant cells have no effect on the efficiency of T-DNA transfer into S. cerevisiae. We also demonstrate that transformation efficiency is improved 500-fold by the addition of yeast telomeric sequences within the T-DNA sequence.
Down-regulation of pituitary receptors for luteinizing hormone-releasing hormone (LH-RH) in rats by LH-RH antagonist Cetrorelix.
Authors: Halmos G , Schally A V , Pinski J , Vadillo-Buenfil M , Groot K
Published: July 2002

Antagonists of luteinizing hormone-releasing hormone (LH-RH), unlike the LH-RH agonists, suppress gonadotropins and sex steroid secretion immediately after administration, without initial stimulatory effects. [Ac-D-Nal(2)1,D-Ph(4Cl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-R H (SB-75; Cetrorelix) is a modern, potent antagonistic analog of LH-RH. In this study, the binding characteristics of receptors for LH-RH in membrane fractions from rat anterior pituitaries were investigated after a single injection of Cetrorelix at a dose of 100 microg per rat. To determine whether the treatment with Cetrorelix can affect the concentration of measurable LH-RH binding sites, we applied an in vitro method to desaturate LH-RH receptors by chaotropic agents such as manganous chloride (MnCl2) and ammonium thiocyanate (NH4SCN). Our results show that the percentages of occupied LH-RH receptors at 1, 3, and 6 h after administration of Cetrorelix were approximately 28%, 14%, and 10%, respectively, of total receptors. At later time intervals, we could not detect occupied LH-RH binding sites. Ligand competition assays, following in vitro desaturation, demonstrated that rat pituitary LH-RH receptors were significantly (P < 0.01) down-regulated for at least 72 h after administration of Cetrorelix. The lowest receptor concentration was found 3-6 h after Cetrorelix treatment and a recovery in receptor number began within approximately 24 h. The down-regulation of LH-RH binding sites induced by Cetrorelix was accompanied by serum LH and testosterone suppression. Higher LH-RH receptor concentrations coincided with elevated serum hormone levels at later time intervals. Our results indicate that administration of LH-RH antagonist Cetrorelix produces a marked down-regulation of pituitary receptors for LH-RH and not merely an occupancy of binding sites.
Activation of CD4+ T lymphocytes form interleukin 2-deficient mice by costimulatory B7 molecules.
Authors: Razi-Wolf Z , Höllander G A , Reiser H
Published: July 2002

Interleukin 2 (IL-2)-deficient (IL-2-/-) mice develop hemolytic anemia and chronic inflammatory bowel disease. Importantly, the induction of disease in IL-2-deficient mice is critically dependent on CD4+ T cells. We have studied the requirements of T cells from IL-2-deficient mice for costimulation with B7 antigens. Stable B7-1 or B7-2 chinese hamster ovary (CHO) cell transfectants could synergize with anti-CD3 monoclonal antibody (mAb) to induce the proliferation of CD4+ T cells from IL-2-/- mutant mice. Further mechanistic studies established that B7-induced activation resulted in surface expression of the alpha chain of the IL-2 receptor. B7-induced proliferation occurred independently of IL-4 and was largely independent of the common gamma chain of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors. Finally, anti-B7-2 but not anti-B7-1 mAb was able to inhibit the activation of IL-2-/- T cells induced by anti-CD3 mAb in the presence of syngeneic antigen-presenting cells. The results of our experiments indicate that IL-2-/- CD4+ T cells remain responsive to B7 stimulation and raise the possibility that B7 antagonists have a role in the prevention/treatment of inflammatory bowel disease.
Prevention of autoimmune disease due to lymphocyte modulation by the B-subunit ofEscherichia coli heat-labile enterotoxin
Authors: Williams Neil A. , Stasiuk Lisa M. , Nashar Toufic O. , Richards Claire M. , Lang Allison K. , Day Michael J. , Hirst Timothy R.
Published: July 2002

We demonstrate that the receptor binding moiety ofEscherichia coliheat-labile enterotoxin (EtxB) can completely prevent autoimmune disease in a murine model of arthritis. Injection of male DBA/1 mice at the base of the tail with type II collagen in the presence of complete Freund’s adjuvant normally leads to arthritis, as evidenced by inflammatory infiltration and swelling of the joints. A separate injection of EtxB at the same time as collagen challenge prevented leukocyte infiltration, synovial hyperplasia, and degeneration of the articular cartilage and reduced clinical symptoms of disease by 82%. The principle biological property of EtxB is its ability to bind to the ubiquitous cell surface receptor GM1 ganglioside, and to other galactose-containing glycolipids and galactoproteins. The importance of receptor interaction in mediating protection from arthritis was demonstrated by the failure of a non-receptor-binding mutant of EtxB to elicit any protective effect. Analysis of T cell responses to collagen, in cultures of draining lymph node cells, revealed that protection was associated with a marked increase in interleukin 4 production concomitant with a reduction in interferon γ levels. Furthermore, in protected mice there was a significant reduction in anti-collagen antibody levels as well as an increase in the IgG1/IgG2a ratio. These observations show that protection is associated with a shift in the Th1/Th2 balance as well as a general reduction in the extent of the anti-type II collagen immune response. This suggests that EtxB-receptor-mediated modulation of lymphocyte responses provides a means of preventing autoimmune disease.
Probing the role of packing specificity in protein design
Authors: Dahiyat Bassil I. , Mayo Stephen L.
Published: July 2002

By using a protein-design algorithm that quantitatively considers side-chain packing, the effect of specific steric constraints on protein design was assessed in the core of the streptococcal protein G β1 domain. The strength of packing constraints used in the design was varied, resulting in core sequences that reflected differing amounts of packing specificity. The structural flexibility and stability of several of the designed proteins were experimentally determined and showed a trend from well-ordered to highly mobile structures as the degree of packing specificity in the design decreased. This trend both demonstrates that the inclusion of specific packing interactions is necessary for the design of native-like proteins and defines a useful range of packing specificity for the design algorithm. In addition, an analysis of the modeled protein structures suggested that penalizing for exposed hydrophobic surface area can improve design performance.
Minisatellite instability in severe combined immunodeficiency mouse cells
Authors: Imai Hiroshi , Nakagama Hitoshi , Komatsu Kenshi , Shiraishi Taizo , Fukuda Hirokazu , Sugimura Takashi , Nagao Minako
Published: July 2002

We have recently found that okadaic acid, which shows strong inhibitory activity on protein serine/threonine phosphatases and tumor-promoting activity
in vivo
and
in vitro
, induces minisatellite mutation (MSM). Human tumors and chemically induced counterparts in experimental animals are also sometimes associated with MSM. In the present study, we demonstrated minisatellite (MS) instability in severe combined immunodeficiency (SCID) cells in which the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is impaired. Cells from a SCID fibroblast cell line transformed by simian virus 40 large tumor antigen, SC3VA2, and from an embryonal SCID fibroblast cell line, SC1K, were cloned and propagated to 10
7
to 10
8
cells, and then subjected to subcloning. After propagation of each subclone to 10
7
to 10
8
cells, DNA samples were digested with
Hin
fI and analyzed by Southern blotting using the Pc-1 MS sequence as a probe. Under low-stringency conditions, about 40 MS bands were detected, with 45% ± 6% and 37% ± 3% of SC3VA2 and SC1K cells, respectively, having MSM. In contrast, cells from the RD13B2 cell line, which was established from SCVA2 by introducing human chromosome 8q fragments, on which DNA-PKcs is known to reside, to complement the SCID phenotype, showed a very low frequency of MSM (3% ± 3%). The high frequencies of MSM in SC3VA2 and SC1K were significant, with no difference between the two. The present study clearly demonstrates that MS instability exists in SCID fibroblasts, suggesting that DNA-PKcs might be involved in the stable maintenance of MS sequences in the genome.
Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein ɛ-deficient mice
Authors: Yamanaka Ryuya , Barlow Carrolee , Lekstrom-Himes Julie , Castilla Lucio H. , Liu Pu P. , Eckhaus Michael , Decker Thomas , Wynshaw-Boris Anthony , Xanthopoulos Kleanthis G.
Published: July 2002

Polymorphonuclear leukocytes are essential for host defense to infectious diseases. CCAAT/enhancer binding protein ɛ (C/EBPɛ) is preferentially expressed in granulocytes and lymphoid cells. Mice with a null mutation in C/EBPɛ develop normally and are fertile but fail to generate functional neutrophils and eosinophils. Opportunistic infections and tissue destruction lead to death by 3–5 months of age. Furthermore, end-stage mice develop myelodysplasia, characterized by proliferation of atypical granulocytes that efface the bone marrow and result in severe tissue destruction. Thus, C/EBPɛ is essential for terminal differentiation and functional maturation of committed granulocyte progenitor cells.
Mode matches and their locations in the hydrophobic free energy sequences of peptide ligands and their receptor eigenfunctions
Authors: Mandell Arnold J. , Selz Karen A. , Shlesinger Michael F.
Published: July 2002

Patterns in sequences of amino acid hydrophobic free energies predict secondary structures in proteins. In protein folding, matches in hydrophobic free energy statistical wavelengths appear to contribute to selective aggregation of secondary structures in “hydrophobic zippers.” In a similar setting, the use of Fourier analysis to characterize the dominant statistical wavelengths of peptide ligands’ and receptor proteins’ hydrophobic modes to predict such matches has been limited by the aliasing and end effects of short peptide lengths, as well as the broad-band, mode multiplicity of many of their frequency (power) spectra. In addition, the sequence locations of the matching modes are lost in this transformation. We make new use of three techniques to address these difficulties: (
i
) eigenfunction construction from the linear decomposition of the lagged covariance matrices of the ligands and receptors as hydrophobic free energy sequences; (
ii
) maximum entropy, complex poles power spectra, which select the dominant modes of the hydrophobic free energy sequences or their eigenfunctions; and (
iii
) discrete, best bases, trigonometric wavelet transformations, which confirm the dominant spectral frequencies of the eigenfunctions and locate them as (absolute valued) moduli in the peptide or receptor sequence. The leading eigenfunction of the covariance matrix of a transmembrane receptor sequence locates the same transmembrane segments seen in
n
-block-averaged hydropathy plots while leaving the remaining hydrophobic modes unsmoothed and available for further analyses as secondary eigenfunctions. In these receptor eigenfunctions, we find a set of statistical wavelength matches between peptide ligands and their G-protein and tyrosine kinase coupled receptors, ranging across examples from 13.10 amino acids in acid fibroblast growth factor to 2.18 residues in corticotropin releasing factor
.
We find that the wavelet-located receptor modes in the extracellular loops are compatible with studies of receptor chimeric exchanges and point mutations. A nonbinding corticotropin-releasing factor receptor mutant is shown to have lost the signatory mode common to the normal receptor and its ligand. Hydrophobic free energy eigenfunctions and their transformations offer new quantitative physical homologies in database searches for peptide-receptor matches.
Nested DNA inversion as a paradigm of programmed gene rearrangement
Authors: Dworkin Joel , Blaser Martin J.
Published: July 2002

Programmed gene rearrangements are employed by a variety of microorganisms, including viruses, prokaryotes, and simple eukaryotes, to control gene expression. In most instances in which organisms mediate host evasion by large families of homologous gene cassettes, the mechanism of variation is not thought to involve DNA inversion. Here we report that
Campylobacter fetus
, a pathogenic Gram-negative bacterium, reassorts a single promoter, controlling surface-layer protein expression, and one or more complete ORFs strictly by DNA inversion. Rearrangements were independent of the distance between sites of inversion. These rearrangements permit variation in protein expression from the large surface-layer protein gene family and suggest an expanding paradigm of programmed DNA rearrangements among microorganisms.
Transloading of tumor antigen-derived peptides into antigen-presenting cells
Authors: Buschle Michael , Schmidt Walter , Zauner Wolfgang , Mechtler Karl , Trska Barbara , Kirlappos Helen , Birnstiel Max L.
Published: July 2002

The discovery of a steadily growing number of tumor antigens (TAs) has made generic, cell-free, peptide-based cancer vaccines a possible alternative to cytokine-transfected autologous cellular cancer vaccines. The major drawback of peptide vaccines, however, is the poor immunogenicity of peptides. It is commonly thought that for the induction of an effective anticancer immune response, antigen-presenting cells (APCs) have to display TA-derived peptides to T lymphocytes. Polycationic amino acids have been employed in the past to enhance transport of proteins into cells. In a systematic study, the ability of different cationic polymers to transfer fluorescence-tagged peptides to APCs was investigated. We were able to show that several compounds enhance uptake of fluorescence-labeled peptides by APCs to different degrees. The most efficient compound identified, polyarginine (pArg), enhanced peptide delivery by more than 2 logs as compared with cells treated with peptide alone, whereas polylysine (pLys) treatment resulted in approximately 10-fold increased levels of fluorescence. Augmentation of peptide uptake was concentration-dependent, and the molecular weight of pArg or pLys also influenced peptide delivery. Furthermore, highly negatively charged peptides appear to be delivered with higher efficiency, although neutral peptides were also taken up at enhanced rates. Whereas peptide uptake mediated by pLys appears to be due to an at least transient permeabilization of cell membranes, peptide delivery in the presence of pArg may rely on endocytic processes. TA-derived peptides applied as cancer vaccines in conjunction with polycations afforded antitumor protection in animal models.
Globular cluster ages
Authors: Jimenez Raul
Published: July 2002

We review two new methods to determine the age of globular clusters
(GCs). These two methods are more accurate than the classical isochrone
fitting technique. The first method is based on the morphology of the
horizontal branch and is independent of the distance modulus of the
globular cluster. The second method uses a careful binning of the
stellar luminosity function and determines simultaneously the distance
and age of the GC. We find that the oldest galactic GCs have an age of
13.5 ± 2 gigayears (Gyr). The absolute minimum age for the oldest
GCs is 10.5 Gyr (with 99% confidence) and the maximum 16.0 Gyr (with
99% confidence). Therefore, an Einstein–De Sitter Universe (Ω = 1)
is not totally ruled out if the Hubble constant is about 65 ± 10
K
m
s
−1
Mpc
−1
.
Intraspecific heterochrony and life history evolution: Decoupling somatic and sexual development in a facultatively paedomorphic salamander
Authors: Ryan Travis J. , Semlitsch Raymond D.
Published: July 2002

Morphological features such as size and shape are the most common focus in studies of heterochronic change. Frequently, these easily observed and measured features are treated as a major target of selection, potentially ignoring traits more closely related to fitness. We question the primacy of morphological data in studies of heterochrony, and instead suggest that principal sources of fitness, such as life history characteristics, are not only the chief targets of selection, but changes in them may necessitate changes in other (subordinate) elements of the organism. We use an experimental approach to investigate the timing of metamorphosis and maturation in a facultatively paedomorphic salamander,
Ambystoma talpoideum
. We determine that individuals possessing the well-known paedomorphic phenotype are peramorphic with regard to maturation, through the process of predisplacement (an earlier onset of maturation). Combining the well studied ecology of dimorphic
A. talpoideum
populations with theories of heterochronic mechanisms and life history evolution, we conclude that age at maturation is the principal target of selection and that morphological changes are secondary effects. Increased attention to the intimate connection between life history evolution and heterochrony is the most promising route to a better understanding of both.
Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice
Authors: Schnermann Jurgen , Chou Chung-Lin , Ma Tonghui , Traynor Timothy , Knepper Mark A. , Verkman A. S.
Published: July 2002

To investigate the role of aquaporin-1 (AQP1) water channels in proximal tubule function,
in vitro
proximal tubule microperfusion and
in vivo
micropuncture measurements were done on AQP1 knockout mice. The knockout mice were generated by targeted gene disruption and found previously to be unable to concentrate their urine in response to water deprivation. Unanesthetized knockout mice consumed 2.8-fold more fluid than wild-type mice and had lower urine osmolality (505 ± 40 vs. 1081 ± 68 milliosmolar). Transepithelial osmotic water permeability (P
f
) in isolated microperfused S2 segments of proximal tubule from AQP1 knockout [−/−] mice was 0.033 ± 0.005 cm/s (SE,
n
= 6 mice, 37°C), much lower than that of 0.15 ± 0.03 cm/s (
n
= 8) in tubules from wild-type [+/+] mice (
P
< 0.01). In the presence of isosmolar luminal perfusate and bath solutions, spontaneous fluid absorption rates (nl/min/mm tubule length) were 0.31 ± 0.12 (−/−,
n
= 5) and 0.64 ± 0.15 (+/+,
n
= 8). As determined by free-flow micropuncture, the ratios of tubular fluid-to-plasma concentrations of an impermeant marker TF/P in end proximal tubule fluid were 1.36 ± 0.05 (−/−,
n
= 8 mice [53 tubules]) and 1.95 ± 0.09 (+/+,
n
= 7 mice [40 tubules]) (
P
< 0.001), corresponding to 26 ± 3% [−/−] and 48 ± 2% [+/+] absorption of the filtered fluid load. In collections of distal tubule fluid, TF/P were 2.8 ± 0.3 [−/−] and 4.4 ± 0.5 [+/+], corresponding to 62 ± 4% [−/−] and 76 ± 3% [+/+] absorption (
P
< 0.02). These data indicate that AQP1 deletion in mice results in decreased transepithelial proximal tubule water permeability and defective fluid absorption. Thus, the high water permeability in proximal tubule of wild-type mice is primarily transcellular, mediated by AQP1 water channels, and required for efficient near-isosmolar fluid absorption.
Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA
Authors: Deretic Dusanka , Schmerl Sonia , Hargrave Paul A. , Arendt Anatol , McDowell J. Hugh
Published: July 2002

Several mutations that cause severe forms of the human disease autosomal dominant retinitis pigmentosa cluster in the C-terminal region of rhodopsin. Recent studies have implicated the C-terminal domain of rhodopsin in its trafficking on specialized post-Golgi membranes to the rod outer segment of the photoreceptor cell. Here we used synthetic peptides as competitive inhibitors of rhodopsin trafficking in the frog retinal cell-free system to delineate the potential regulatory sequence within the C terminus of rhodopsin and model the effects of severe retinitis pigmentosa alleles on rhodopsin sorting. The rhodopsin C-terminal sequence QVS(A)PA is highly conserved among different species. Peptides that correspond to the C terminus of bovine (amino acids 324–348) and frog (amino acids 330–354) rhodopsin inhibited post-Golgi trafficking by 50% and 60%, respectively, and arrested newly synthesized rhodopsin in the trans-Golgi network. Peptides corresponding to the cytoplasmic loops of rhodopsin and other control peptides had no effect. When three naturally occurring mutations: Q344ter (lacking the last five amino acids QVAPA), V345M, and P347S were introduced into the frog C-terminal peptide, the inhibitory activity of the peptides was no longer detectable. These observations suggest that the amino acids QVS(A)PA comprise a signal that is recognized by specific factors in the trans-Golgi network. A lack of recognition of this sequence, because of mutations in the last five amino acids causing autosomal dominant retinitis pigmentosa, most likely results in abnormal post-Golgi membrane formation and in an aberrant subcellular localization of rhodopsin.
Free recombination within
Helicobacter pylori
Authors: Suerbaum Sebastian , Smith John Maynard , Bapumia Khairun , Morelli Giovanna , Smith Noel H. , Kunstmann Erdmute , Dyrek Isabelle , Achtman Mark
Published: July 2002

Sequences of three gene fragments (
flaA
,
flaB
, and
vacA
) from
Helicobacter pylori
strains isolated from patients in Germany, Canada, and South Africa were analyzed for diversity and for linkage equilibrium by using the Homoplasy Test and compatibility matrices. Horizontal genetic exchange in
H. pylori
is so frequent that different loci and polymorphisms within each locus are all at linkage equilibrium. These results indicate that
H. pylori
is panmictic. Comparisons with sequences from
Escherichia coli
,
Neisseria meningitidis
, and
Drosophila melanogaster
showed that recombination in
H. pylori
was much more frequent than in other species. In contrast, when multiple family members infected with
H. pylori
were investigated, some strains were indistinguishable at all three loci. Thus,
H. pylori
is clonal over short time periods after natural transmission.
Quantitative insight into proliferation and differentiation of oligodendrocyte type 2 astrocyte progenitor cells
in vitro
Authors: Yakovlev Andrej Y. , Boucher Kenneth , Mayer-Proschel Margot , Noble Mark
Published: July 2002

As part of our attempts at understanding fundamental principles that underlie the generation of nondividing terminally differentiated progeny from dividing precursor cells, we have developed approaches to a quantitative analysis of proliferation and differentiation of oligodendrocyte type 2 astrocyte (O-2A) progenitor cells at the clonal level. Owing to extensive previous studies of clonal differentiation in this lineage, O-2A progenitor cells represent an excellent system for such an analysis. Previous studies have resulted in two competing hypotheses; one of them suggests that progenitor cell differentiation is symmetric, the other hypothesis introduces an asymmetric process of differentiation. We propose a general model that incorporates both such extreme hypotheses as special cases. Our analysis of experimental data has shown, however, that neither of these extreme cases completely explains the observed kinetics of O-2A progenitor cell proliferation and oligodendrocyte generation
in vitro
. Instead, our results indicate that O-2A progenitor cells become competent for differentiation after they complete a certain number of critical mitotic cycles that represent a period of symmetric development. This number varies from clone to clone and may be thought of as a random variable; its probability distribution was estimated from experimental data. Those O-2A cells that have undergone the critical divisions then may differentiate into an oligodendrocyte in each of the subsequent mitotic cycles with a certain probability, thereby exhibiting the asymmetric type of differentiation.
Functional topography of nascent RNA in elongation intermediates of RNA polymerase
Authors: Komissarova Natalia , Kashlev Mikhail
Published: July 2002

To determine the dynamics of transcript extrusion from
Escherichia coli
RNA polymerase (RNAP), we used degradation of the RNA by RNases T1 and A in a series of consecutive elongation complexes (ECs). In intact ECs, even extremely high doses of the RNases were unable to cut the RNA closer than 14–16 nt from the 3′ end. Our results prove that all of the cuts detected within the 14-nt zone are derived from the EC that is denatured during inactivation of the RNases. The protected zone monotonously translocates along the RNA after addition of new nucleotides to the transcript. The upstream region of the RNA heading toward the 5′ end is cleaved and dissociated from the EC, with no effect on the stability and activity of the EC. Most of the current data suggest that an 8- to 10-nt RNA⋅DNA hybrid is formed in the EC. Here, we show that an 8- to 10-nt RNA obtained by truncating the RNase-generated products further with either GreB or pyrophosphate is sufficient for the high stability and activity of the EC. This result suggests that the transcript–RNAP interaction that is required for holding the EC together can be limited to the RNA region involved in the 8- to 10-nt RNA⋅DNA hybrid.
Metabotropic glutamate receptor-initiated translocation of protein kinase p90rsk to polyribosomes: A possible factor regulating synaptic protein synthesis
Authors: Angenstein Frank , Greenough William T. , Weiler Ivan Jeanne
Published: July 2002

Maintenance of lasting synaptic efficacy changes requires protein synthesis. We report here a mechanism that might influence translation control at the level of the single synapse. Stimulation of metabotropic glutamate receptors in hippocampal slices induces a rapid protein kinase C-dependent translocation of multifunction kinase p90rsk to polyribosomes; concomitantly, there is enhanced phosphorylation of at least six polyribosome binding proteins. Among the polyribosome bound proteins are the p90rsk-activating kinase ERK-2 and a known p90rsk substrate, glycogen synthase kinase 3β, which regulates translation efficiency via eukaryotic initiation factor 2B. Thus metabotropic glutamate receptor stimulation could induce synaptic activity-dependent translation via translocation of p90rsk to ribosomes.
ARC, an inhibitor of apoptosis expressed in skeletal muscle and heart that interacts selectively with caspases
Authors: Koseki Takeyoshi , Inohara Naohiro , Chen Shu , Núñez Gabriel
Published: July 2002

We have identified and characterized ARC, apoptosis repressor with caspase recruitment domain (CARD). Sequence analysis revealed that ARC contains an N-terminal CARD fused to a C-terminal region rich in proline/glutamic acid residues. The CARD domain of ARC exhibited significant homology to the prodomains of apical caspases and the CARDs present in the cell death regulators Apaf-1 and RAIDD. Immunoprecipitation analysis revealed that ARC interacts with caspase-2, -8, and
Caenorhabditis elegans
CED-3, but not with caspase-1, -3, or -9. ARC inhibited apoptosis induced by caspase-8 and CED-3 but not that mediated by caspase-9. Further analysis showed that the enzymatic activity of caspase-8 was inhibited by ARC in 293T cells. Consistent with the inhibition of caspase-8, ARC attenuated apoptosis induced by FADD and TRADD and that triggered by stimulation of death receptors coupled to caspase-8, including CD95/Fas, tumor necrosis factor-R1, and TRAMP/DR3. Remarkably, the expression of human ARC was primarily restricted to skeletal muscle and cardiac tissue. Thus, ARC represents an inhibitor of apoptosis expressed in muscle that appears to selectively target caspases. Delivery of ARC by gene transfer or enhancement of its endogenous activity may provide a strategy for the treatment of diseases that are characterized by inappropriately increased cell death in muscle tissue.
Sustainedex vivoexpansion of hematopoietic stem cells mediated by thrombopoietin
Authors: Yagi Mayumi , Ritchie Kindred A. , Sitnicka Ewa , Storey Carl , Roth Gerald J. , Bartelmez Stephen
Published: July 2002

The hematopoietic stem cell (HSC) is defined as a cell that can either self-replicate or generate daughter cells that are destined to commit to mature cells of different specific lineages. Self-replication of the most primitive HSC produces daughter cells that possess a long (possibly unlimited) clonal lifespan, whereas differentiation of HSC produces daughter cells that demonstrate a progressive reduction of their clonal lifespan, a loss of multilineage potential, and lineage commitment. Previous studies indicated that the proliferation of HSCex vivofavors differentiation at the expense of self-replication, eventually resulting in a complete loss of HSC. In contrast, transplantation studies have shown that a single HSC can repopulate the marrow of a lethally irradiated mouse, demonstrating that self-renewal of HSC occursin vivo. Thrombopoietin (TPO) has been shown to function both as a proliferative and differentiative factor for megakaryocytes and as a survival and weakly proliferative factor for HSC. Our studies focused on the effects of exogenous TPO on HSC in mouse long-term bone marrow cultures (LTBMC). Previous results indicate that HSC decline in LTBMC in the absence of TPO. In contrast, the continuous presence of TPO resulted in the generation of both long- and short-term repopulating HSC as detected by anin vivocompetitive repopulation assay. HSC were generated over a 4-month period at concentrations similar to normal bone marrow. Our results demonstrate that TPO can mediate the self-replication of HSC in LTBMC, and provide proof that HSC can self-replicateex vivo.
Reciprocal secretion of proteins by the bacterial type III machines of plant and animal pathogens suggests universal recognition of mRNA targeting signals
Authors: Anderson Deborah M. , Fouts Derrick E. , Collmer Alan , Schneewind Olaf
Published: July 2002

Bacterial pathogens of both animals and plants use type III secretion machines to inject virulence proteins into host cells. Although many components of the secretion machinery are conserved among different bacterial species, the substrates for their type III pathways are not. The
Yersinia
type III machinery recognizes some secretion substrates via a signal that is encoded within the first 15 codons of
yop
mRNA. These signals can be altered by frameshift mutations without affecting secretion of the encoded polypeptides, suggesting a mechanism whereby translation of
yop
mRNA is coupled to the translocation of newly synthesized polypeptide. We report that the type III machinery of
Erwinia chrysanthemi
cloned in
Escherichia coli
recognizes the secretion signals of
yopE
and
yopQ. Pseudomonas syringae
AvrB and AvrPto, two proteins exported by the recombinant
Erwinia
machine, can also be secreted by the
Yersinia
type III pathway. Mapping AvrPto sequences sufficient for the secretion of reporter fusions in
Yersinia
revealed the presence of an mRNA secretion signal. We propose that 11 conserved components of type III secretion machines may recognize signals that couple mRNA translation to polypeptide secretion.
An
Escherichia coli
strain with all chromosomal rRNA operons inactivated: Complete exchange of rRNA genes between bacteria
Authors: Asai Tsuneaki , Zaporojets Dmitry , Squires Craig , Squires Catherine L.
Published: July 2002

Current global phylogenies are built predominantly on rRNA
sequences. However, an experimental system for studying the evolution
of rRNA is not readily available, mainly because the rRNA genes are
highly repeated in most experimental organisms. We have constructed an
Escherichia coli
strain in which all seven chromosomal rRNA
operons are inactivated by deletions spanning the 16S and 23S coding
regions. A single
E. coli
rRNA operon carried by a
multicopy plasmid supplies 16S and 23S rRNA to the cell. By using this
strain we have succeeded in creating microorganisms that contain only a
foreign rRNA operon derived from either
Salmonella
typhimurium
or
Proteus vulgaris
, microorganisms that
have diverged from
E. coli
about 120–350 million years
ago. We also were able to replace the
E. coli
rRNA operon
with an
E. coli
/yeast hybrid one in which the GTPase
center of
E. coli
23S rRNA had been substituted by the
corresponding domain from
Saccharomyces cerevisiae
. These
results suggest that, contrary to common belief, coevolution of rRNA
with many other components in the translational machinery may not
completely preclude the horizontal transfer of rRNA genes.