bluuebunny/crossref_metadata_2025 huggingface dataset:
Protection against hemorrhagic shock in mice genetically deficient in poly(ADP-ribose)polymerase
Authors: Liaudet Lucas , Soriano Francisco Garcia , Szabó Éva , Virág László , Mabley Jon G. , Salzman Andrew L. , Szabó Csaba
Published: July 2002

Hemorrhagic shock (HS) and resuscitation leads to widespread production of oxidant species. Activation of the enzyme poly(ADP-ribose) polymerase (PARP) has been shown to contribute to cell necrosis and organ failure in various disease conditions associated with oxidative stress. We tested the hypothesis whether PARP activation plays a role in the multiple organ dysfunction complicating HS and resuscitation in a murine model of HS and resuscitation by using mice genetically deficient in PARP (PARP−/−) and their wild-type littermates (PARP+/+). Animals were bled to a mean blood pressure of 45 mmHg (1 mmHg = 133 Pa) and resuscitated after 45 min with isotonic saline (2× volume of shed blood). There was a massive activation of PARP, detected by poly(ADP-ribose) immunohistochemistry, which localized to the areas of the most severe intestinal injury, i.e., the necrotic epithelial cells at the tip of the intestinal villi, and colocalized with tyrosine nitration, an index of peroxynitrite generation. Intestinal PARP activation resulted in gut hyperpermeability, which developed in PARP+/+but not PARP−/−mice. PARP−/−mice were also protected from the rapid decrease in blood pressure after resuscitation and showed an increased survival time, as well as reduced lung neutrophil sequestration. The beneficial effects of PARP suppression were not related to a modulation of the NO pathway nor to a modulation of signaling through IL-6, which similarly increased in both PARP+/+and PARP−/−mice exposed to HS. We propose that PARP activation and associated cell injury (necrosis) plays a crucial role in the intestinal injury, cardiovascular failure, and multiple organ damage associated with resuscitated HS.
Timing of hepatocyte entry into DNA synthesis after partial hepatectomy is cell autonomous
Authors: Weglarz Teresa C. , Sandgren Eric P.
Published: July 2002

After surgical removal of two-thirds of the liver, remaining hepatocytes replicate and restore hepatic mass within 2 weeks. This process must be initiated by signals extrinsic to the hepatocyte, but it remains unclear whether subsequent events leading to DNA synthesis (S phase) are regulated by circulating or locally produced growth factors (a noncell autonomous response), or by a program intrinsic to the hepatocyte itself (a cell autonomous response). To identify the type of mechanism regulating passage to S, we exploited the difference between rat and mouse hepatocytes in the timing of DNA synthesis after partial hepatectomy, which peaks 12–16 h earlier posthepatectomy in rat compared with mouse. Four groups of animals received two-thirds partial hepatectomies: rats, mice, mice with chimeric livers composed of both transplanted rat hepatocytes and endogenous mouse hepatocytes, and mice with chimeric livers composed of both transplanted and endogenous mouse hepatocytes. Following two-thirds partial hepatectomy, both donor and endogenous hepatocytes in mouse/mouse chimeric livers displayed kinetics of DNA synthesis characteristic of the mouse, indicating that transplantationper sedid not affect the response to subsequent partial hepatectomy. In contrast, rat hepatocytes in chimeric mouse livers displayed rat kinetics despite their presence in a mouse host. Thus, factors intrinsic to the hepatocyte must regulate the timing of entry into DNA synthesis. This result defines the process as cell autonomous and suggests that locally or distantly produced cytokines or growth factors may have a permissive but not an instructive role in progression to S.
Spoken word production: A theory of lexical access
Authors: Levelt Willem J. M.
Published: July 2002

A core operation in speech production is the preparation of words from a semantic base. The theory of lexical access reviewed in this article covers a sequence of processing stages beginning with the speaker's focusing on a target concept and ending with the initiation of articulation. The initial stages of preparation are concerned with lexical selection, which is zooming in on the appropriate lexical item in the mental lexicon. The following stages concern form encoding, i.e., retrieving a word's morphemic phonological codes, syllabifying the word, and accessing the corresponding articulatory gestures. The theory is based on chronometric measurements of spoken word production, obtained, for instance, in picture-naming tasks. The theory is largely computationally implemented. It provides a handle on the analysis of multiword utterance production as well as a guide to the analysis and design of neuroimaging studies of spoken utterance production.
The animal species–body size distribution of Marion Island
Authors: Gaston Kevin J. , Chown Steven L. , Mercer Richard D.
Published: July 2002

Body size is one of the most significant features of animals. Not only is it correlated with many life history and ecological traits, but it also may influence the abundance of species within, and their membership of, assemblages. Understanding of the latter processes is frequently based on a comparison of model outcomes with the frequency of species of different body mass within natural assemblages. Consequently, the form of these frequency distributions has been much debated. Empirical data usually concern taxonomically delineated groups, such as classes or orders, whereas the processes ultimately apply to whole assemblages. Here, we report the most complete animal species–body size distribution to date for those free-living species breeding on sub-Antarctic Marion Island and using the terrestrial environment. Extending over 15 orders of magnitude of variation in body mass, this distribution is bimodal, with separate peaks for invertebrates and vertebrates. Under logarithmic transformation, the distribution for vertebrates is not significantly skewed, whereas that for invertebrates is right-skewed. Contrary to expectation based on a fractal or pseudofractal environmental structure, the decline in the richness of species at the smallest body sizes is a real effect and not a consequence of unrecorded species or of species introductions to the island. The scarcity of small species might well be a consequence of their large geographic ranges.
Topology to geometry in protein folding: β-Lactoglobulin
Authors: Fernández Ariel , Colubri Andrés , Berry R. Stephen
Published: July 2002

Evolution of protein structure from random coil to native is first
represented topologically by its time-dependent sequences of
discretized Ramachandran basins occupied by successive backbone
residues. Introducing energetic and entropic criteria at each instant
of observation transforms the description from a structurally ambiguous
topological representation to an unambiguous geometric picture of the
folding process. The method is applied with success to folding of
β-lactoglobulin, traditionally perplexing because of its reputed
nonhierarchical folding pattern. This molecule passes through a stage,
ca.
0.1 μs duration, of transient, “flickering”
α-helical structure, until a bit of tertiary structure forms that
stabilizes the system long enough to allow it to pass to its native
β-sheet.
Explaining mortality rate plateaus
Authors: Weitz Joshua S. , Fraser Hunter B.
Published: July 2002

We propose a stochastic model of aging to explain deviations from exponential growth in mortality rates commonly observed in empirical studies. Mortality rate plateaus are explained as a generic consequence of considering death in terms of first passage times for processes undergoing a random walk with drift. Simulations of populations with age-dependent distributions of viabilities agree with a wide array of experimental results. The influence of cohort size is well accounted for by the stochastic nature of the model.
Different cleavage sites are aligned differently in the active site of M1 RNA, the catalytic subunit of Escherichia coli RNase P.
Authors: Kufel J , Kirsebom L A
Published: July 2002

We have studied RNase P RNA (M1 RNA) cleavage of model tRNA precursors that are cleaved at two independent positions. Here we present data demonstrating that cleavage at both sites depends on the 2'-OH immediately 5' of the respective cleavage site. However, we show that the 2-amino group of a guanosine at the cleavage site plays a significant role in cleavage at one of these sites but not at the other. These data suggest that these two cleavage sites are handled differently by the ribozyme. This theory is supported by our finding that the cross-linking pattern between Ml RNA and tRNA precursors carrying 4-thioU showed distinct differences, depending on the location of the 4-thioU relative to the respective cleavage site. These findings lead us to suggest that different cleavage sites are aligned differently in the active site, possibly as a result of different binding modes of a substrate to M1 RNA. We discuss a model in which the interaction between the 3'-terminal "RCCA" motif (first three residues interact) of a tRNA precursor and M1 RNA plays a significant role in this process.
Apolipoprotein B RNA editing enzyme-deficient mice are viable despite alterations in lipoprotein metabolism.
Authors: Morrison J R , Pászty C , Stevens M E , Hughes S D , Forte T , Scott J , Rubin E M
Published: July 2002

RNA editing in the nucleus of higher eukaryotes results in subtle changes to the RNA sequence, with the ability to effect dramatic changes in biological function. The first example to be described and among the best characterized, is the cytidine-to-uridine editing of apolipoprotein B (apo-B) RNA. The editing of apo-B RNA is mediated by a novel cytidine deaminase, apobec-1, which has acquired the ability to bind RNA. The stop translation codon generated by the editing of apo-B RNA truncates the full-length apo-B100 to form apo-B48. The recent observations of tumor formation in Apobec-1 transgenic animals, together with the fact that Apobec-1 is expressed in numerous tissues lacking apo-B, raises the issue of whether this enzyme is essential for a variety of posttranscriptional editing events. To directly test this, mice were created with a null mutation in Apobec-1 using homologous recombination in embryonic stem cells. Mice, homozygous for this mutation, were viable and made apo-B100 but not apo-B48. The null animals were fertile, and a variety of histological, behavioral, and morphological analyses revealed no phenotype other than abnormalities in lipoprotein metabolism, which included an increased low density lipoprotein fraction and a reduction in high density lipoprotein cholesterol. These studies demonstrate that neither apobec-1 nor apo-B48 is essential for viability and suggest that the major role of apobec-1 may be confined to the modulation of lipid transport.
Haemonchus contortus GA1 antigens: related, phospholipase C-sensitive, apical gut membrane proteins encoded as a polyprotein and released from the nematode during infection.
Authors: Jasmer D P , Perryman L E , McGuire T C
Published: July 2002

It was previously shown that the Haemonchus contortus apical gut surface proteins p46, p52, and p100 induced protective immunity to challenge infections in goats. Here, it is shown that the three proteins are all encoded by a single gene (GA1) and initially expressed in adult parasites as a polyprotein (p100GA1). p46GA1 and p52GA1 are related proteins with 47% sequence identity, including a cysteine-containing region, which appears to confer secondary structure to these proteins, and a region with sequence similarity to bacterial Tolb proteins. GA1 protein expression is regulated during the life cycle at the level of transcript abundance. Only p52GA1 has characteristics of a glycosylinositolphospholipid membrane-anchored protein. However, both p46GA1 and p52GA1 were released from the gut membrane by phosphatidylinositol specific-phospholipase C, suggesting that p46GA1 membrane association depends on interactions with a glycosylinositolphospholipid gut membrane protein. Finally, GA1 proteins occur in abomasal mucus of infected lambs, demonstrating possible presentation to the host immune system during H. contortus infection. The results identify multiple characteristics of the GA1 proteins that should be considered for design of recombinant antigens for vaccine trials and that implicate a series of cellular processes leading to modification and expression of GA1 proteins at the nematode apical gut surface.
Protein p4 represses phage phi 29 A2c promoter by interacting with the alpha subunit of Bacillus subtilis RNA polymerase.
Authors: Monsalve M , Mencía M , Salas M , Rojo F
Published: July 2002

Regulatory protein p4 from Bacillus subtilis phage phi 29 represses the strong viral A2c promoter (PA2c) by preventing promoter clearance; it allows RNA polymerase to bind to the promoter and form an initiated complex, but the elongation step is not reached. Protein p4 binds at PA2c immediately upstream from RNA polymerase; repression involves a contact between both proteins that holds the RNA polymerase at the promoter. This contact is held mainly through p4 residue Arg120, which is also required for activation of the phi 29 late A3 promoter. We have investigated which region of RNA polymerase contacts protein p4 at PA2c. Promoter repression was impaired when a reconstituted RNA polymerase lacking the 15 C-terminal residues of the alpha subunit C-terminal domain was used; this polymerase was otherwise competent for transcription. Binding cooperativity assays indicated that protein p4 cannot interact with this mutant RNA polymerase at PA2c. Protein p4 could form a complex at PA2c with purified wild-type alpha subunit, but not with a deletion mutant lacking the 15 C-terminal residues. Our results indicate that protein p4 represses PA2c by interacting with the C-terminal domain of the alpha subunit of RNA polymerase. Therefore, this domain of the alpha subunit can receive regulatory signals not only from transcriptional activators, but from repressors also.
A thermodynamic coupling mechanism for GroEL-mediated unfolding.
Authors: Walter S , Lorimer G H , Schmid F X
Published: July 2002

Chaperonins prevent the aggregation of partially folded or misfolded forms of a protein and, thus, keep it competent for productive folding. It was suggested that GroEL, the chaperonin of Escherichia coli, exerts this function 1 unfolding such intermediates, presumably in a catalytic fashion. We investigated the kinetic mechanism of GroEL-induced protein unfolding by using a reduced and carbamidomethylated variant of RNase T1, RCAM-T1, as a substrate. RCAM-T1 cannot fold to completion, because the two disulfide bonds are missing, and it is, thus, a good model for long-lived folding intermediates. RCAM-T1 unfolds when GroEL is added, but GroEL does not change the microscopic rate constant of unfolding, ruling out that it catalyzes unfolding. GroEL unfolds RCAM-T1 because it binds with high affinity to the unfolded form of the protein and thereby shifts the overall equilibrium toward the unfolded state. GroEL can unfold a partially folded or misfolded intermediate by this thermodynamic coupling mechanism when the Gibbs free energy of the binding to GroEL is larger than the conformational stability of the intermediate and when the rate of its unfolding is high.
The role of CD4-Lck in T-cell receptor antagonism: evidence for negative signaling.
Authors: Racioppi L , Matarese G , D'Oro U , De Pascale M , Masci A M , Fontana S , Zappacosta S
Published: July 2002

Small changes in the complex between a peptide and a molecule of the major histocompatibility complex generate ligands able to partially activate (partial agonist) or even inhibit (antagonist) T-cell functions. T-cell receptor engagement of antagonist complex results in a partial zeta chain phosphorylation without activation of the associated ZAP-70 kinase. Herein we show that, despite a strong inhibition of both inositol phospholipid hydrolysis and extracellular increasing antagonist concentrations increased the activity of the CD4-Lck kinase. Addition of anti-CD4 antibody to culture medium prevented inhibitory effects induced by antagonist ligand. We propose that CD4-Lck activation triggered by antagonist complexes may act in a dominant negative mode, thus overriding stimulatory signals coming from agonist ligand. These findings identify a new T-cell signaling profile that may explain the ability of some T-cell receptor variant ligands to inhibit specific biological activities or trigger alternative activation programs.
Significance of some previously unrecognized apomorphies in the nasal region of Homo neanderthalensis.
Authors: Schwartz J H , Tattersall I
Published: July 2002

For many years, the Neanderthals have been recognized as a distinctive extinct hominid group that occupied Europe and western Asia between about 200,000 and 30,000 years ago. It is still debated, however, whether these hominids belong in their own species, Homo neanderthalensis, or represent an extinct variant of Homo sapiens. Our ongoing studies indicate that the Neanderthals differ from modern humans in their skeletal anatomy in more ways than have been recognized up to now. The purpose of this contribution is to describe specializations of the Neanderthal internal nasal region that make them unique not only among hominids but possibly among terrestrial mammals in general as well. These features lend additional weight to the suggestion that Neanderthals are specifically distinct from Homo sapiens.
Nested expression domains for odorant receptors in zebrafish olfactory epithelium
Authors: Weth Franco , Nadler Walter , Korsching Sigrun
Published: July 2002

The mapping of high-dimensional olfactory stimuli onto the
two-dimensional surface of the nasal sensory epithelium constitutes the
first step in the neuronal encoding of olfactory input. We have used
zebrafish as a model system to analyze the spatial distribution of
odorant receptor molecules in the olfactory epithelium by quantitative
in situ
hybridization. To this end, we have cloned 10
very divergent zebrafish odorant receptor molecules by PCR. Individual
genes are expressed in sparse olfactory receptor neurons. Analysis of
the position of labeled cells in a simplified coordinate system
revealed three concentric, albeit overlapping, expression domains for
the four odorant receptors analyzed in detail. Such regionalized
expression should result in a corresponding segregation of functional
response properties. This might represent the first step of spatial
encoding of olfactory input or be essential for the development of the
olfactory system.
Studies of retrograde memory: A long-term view
Authors: Warrington Elizabeth K.
Published: July 2002

Studies of retrograde amnesia are reviewed. First, the issues of
temporal gradients of retrograde amnesia are discussed. Second, the
question of the anatomical substrates of this syndrome are considered.
Finally, some evidence for fractionation of different classes of
memoranda within the retrograde time period are presented.
Recurrent G-to-A substitution in a single codon of SREBP cleavage-activating protein causes sterol resistance in three mutant Chinese hamster ovary cell lines
Authors: Nohturfft Axel , Hua Xianxin , Brown Michael S. , Goldstein Joseph L.
Published: July 2002

Oxygenated sterols such as 25-hydroxycholesterol kill Chinese
hamster ovary cells because they inhibit the proteolytic processing of
sterol regulatory element binding proteins (SREBPs), a pair of
membrane-bound transcription factors that activate genes controlling
cholesterol synthesis and uptake from lipoproteins. The unprocessed
SREBPs remain membrane-bound, they cannot activate the cholesterol
biosynthetic pathway, and the cells die of cholesterol deprivation.
Several sterol-resistant hamster cell lines have been isolated
previously by chemical mutagenesis and selection for resistance to
killing by 25-hydroxycholesterol. We recently identified the defect in
one such cell line (25-RA cells) as a point mutation in a newly
discovered membrane protein of 1276 amino acids, designated SREBP
cleavage-activating protein (SCAP). The mutation in the 25-RA cells
resulted from a G-to-A transition in codon 443 of the
SCAP
gene, changing aspartic acid to asparagine.
Wild-type SCAP, when overexpressed by transfection, stimulates the
proteolytic processing of both SREBPs. The D443N substitution is an
activating mutation that increases the activity of SCAP and renders it
resistant to inhibition by 25-hydroxycholesterol. We here report the
identical G-to-A transition in two additional lines of Chinese hamster
ovary cells that were mutagenized and isolated by a similar protocol.
The three mutations occurred independently as indicated by haplotype
analysis of the mutant genes using two intragenic sequence
polymorphisms. All three cell lines were mutagenized with alkylating
agents (nitrosoethylurea or ethylmethane sulfonate) that favor G-to-A
transitions. Nevertheless, the finding of the same nucleotide
substitution at the same location in all three cell lines indicates
that SCAP may be unique in its ability to stimulate SREBP cleavage, and
residue 443 is a crucial determinant of the protein’s ability to be
inhibited by 25-hydroxycholesterol.
The mouse mammary tumor virusenvgene is the source of a CD8+T-cell-stimulating peptide presented by a major histocompatibility complex class I molecule in a murine thymoma
Authors: Malarkannan Subramaniam , Serwold Thomas , Nguyen Vu , Sherman Linda A. , Shastri Nilabh
Published: July 2002

CD8+cytotoxic T cells recognize their targets by the presence of unique peptide bound to a major histocompatibility complex (MHC) class I molecules on the cell surface. The MHC molecules normally display thousands of distinct peptides, making it difficult to identify individual antigenic peptides, their protein precursors, and their relative importance in the T-cell response. Here we used the EL-4 tumor-specificlacZ-inducible KZ30.6 T cell as a probe for detecting the peptide/MHC ligand that was generated in cells transfected with an EL-4 cDNA library. These expression screens allowed identification of a mouse mammary tumor virus (MMTV) transcript as the source of the antigenic peptide presented by the KbMHC molecule. The antigenic activity was encoded within the MMTVenvgene and was defined by the octapeptide ANYDFICV (AFV8). Synthetic AFV8 stimulated KZ30.6 T cells at picomolar concentrations and coeluted with one of two active peptides in HPLC-fractionated extracts of EL-4 cells. The AFV8/Kbcomplex was also recognized by two other EL-4-specific T cells. The results illustrate a novel strategy for identifying T-cell-stimulating antigens and suggest that the MMTVenvgene and its naturally processed AFV8 peptide product can serve as a model for study of antigen processing and tumor immunotherapy.
Mutagenesis of the potato ADPglucose pyrophosphorylase and characterization of an allosteric mutant defective in 3-phosphoglycerate activation.
Authors: Greene T W , Chantler S E , Kahn M L , Barry G F , Preiss J , Okita T W
Published: July 2002

ADPglucose pyrophosphorylase (glucose-1-phosphate adenylyltransferase; ADP:alpha-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) catalyzes a key regulatory step in alpha-glucan synthesis in bacteria and higher plants. We have previously shown that the expression of the cDNA sequences of the potato tuber large (LS) and small (SS) subunits yielded a functional heterotetrameric enzyme capable of complementing a mutation in the single AGP (glgC) structural gene of Escherichia coli. This heterologous complementation provides a powerful genetic approach to obtain biochemical information on the specific roles of LS and SS in enzyme function. By mutagenizing the LS cDNA with hydroxylamine and then coexpressing with wild-type SS in an E. coli glgC- strain, >350 mutant colonies were identified that were impaired in glycogen production. One mutant exhibited enzymatic and antigen levels comparable to the wild-type recombinant enzyme but required 45-fold greater levels of the activator 3-phosphoglycerate for maximum activity. Sequence analysis identified a single nucleotide change that resulted in the change of Pro-52 to Leu. This heterologous genetic system provides an efficient means to identify residues important for catalysis and allosteric functioning and should lead to novel approaches to increase plant productivity.
Checkpoints in the progression of autoimmune disease: lessons from diabetes models.
Authors: André I , Gonzalez A , Wang B , Katz J , Benoist C , Mathis D
Published: July 2002

In the last few years, data from experiments employing transgenic models of autoimmune disease have strengthened a particular concept of autoimmunity: disease results not so much from cracks in tolerance induction systems, leading to the generation of anti-self repertoire, as from the breakdown of secondary systems that keep these cells in check. T cells with anti-self specificities are readily found in disease-free individuals but ignore target tissues. This is also the case in some transgenic models, in spite of overwhelming numbers of autoreactive cells. In other instances, local infiltration and inflammation result, but they are well tolerated for long periods of time and do not terminally destroy target tissue. We review the possible molecular and cellular mechanisms that underlie these situations, with a particular emphasis on the destruction of pancreatic beta cells in transgenic models of insulin-dependent disease.
Human immunodeficiency virus type 1 infection despite prior immunization with a recombinant envelope vaccine regimen.
Authors: McElrath M J , Corey L , Greenberg P D , Matthews T J , Montefiori D C , Rowen L , Hood L , Mullins J I
Published: July 2002

With efforts underway to develop a preventive human immunodeficiency virus type 1 (HIV-1) vaccine, it remains unclear which immune responses are sufficient to protect against infection and whether prior HIV-1 immunity can alter the subsequent course of HIV-1 infection. We investigated these issues in the context of a volunteer who received six HIV-1LAI envelope immunizations and 10 weeks thereafter acquired HIV-1 infection through a high-risk sexual exposure. In contrast to nonvaccinated acutely infected individuals, anamnestic HIV-1-specific B- and T-cell responses appeared within 3 weeks in this individual, and neutralizing antibody preceded CD8+ cytotoxic responses. Despite an asymptomatic course and an initial low level of detectable infectious virus, a progressive CD4+ cell decline and dysfunction occurred within 2 years. Although vaccination elicited immunity to HIV-1 envelope, which was recalled upon HIV-1 exposure, it was insufficient to prevent infection and subsequent immunodeficiency.
A glutamate residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions
Authors: Schnappauf Georg , Sträter Norbert , Lipscomb William N. , Braus Gerhard H.
Published: July 2002

Chorismate mutase acts at the first branchpoint of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast
Saccharomyces cerevisiae
with
Escherichia coli
chorismate mutase/prephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid residues. The resulting enzymes exhibit the necessity of these residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest activity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.
A peptide export–import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay
Authors: Perego Marta
Published: July 2002

The phosphorelay signal transduction system activates developmental transcription in sporulation of
Bacillus subtilis
by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the
phrA
gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export–import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase–prepeptide gene pairs in
B. subtilis
, suggesting that the use of this mechanism may be widespread in signal transduction.
A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens
Authors: Nicholson Lindsay B. , Murtaza Anwar , Hafler Brian P. , Sette Alessandro , Kuchroo Vijay K.
Published: July 2002

Experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) residues 139–151 (HSLGKWLGHPDKF) can be prevented by treatment with a T cell receptor (TCR) antagonist peptide (L144/R147) generated by substituting at the two principal TCR contact residues in the encephalitogenic peptide. The TCR antagonist peptide blocks activation of encephalitogenic Th1 helper cellsin vitro, but the mechanisms by which the antagonist peptide blocks EAEin vivoare not clear. Immunization with L144/R147 did not inhibit generation of PLP-(139–151)-specific T cellsin vivo. Furthermore, preimmunization with L144/R147 protected mice from EAE induced with the encephalitogenic peptides PLP-(178–191) and myelin oligodendrocyte protein (MOG) residues 92–106 and with mouse myelin basic protein (MBP). These data suggest that the L144/R147 peptide does not act as an antagonistin vivobut mediates bystander suppression, probably by the generation of regulatory T cells. To confirm this we generated T cell lines and clones from animals immunized with PLP-(139–151) plus L144/R147. T cells specific for L144/R147 peptide were crossreactive with the native PLP-(139–151) peptide, produced Th2/Th0 cytokines, and suppressed EAE upon adoptive transfer. These studies demonstrate that TCR antagonist peptides may have multiple biological effectsin vivo. One of the principal mechanisms by which these peptides inhibit autoimmunity is by the induction of regulatory T cells, leading to bystander suppression of EAE. These results have important implications for the treatment of autoimmune diseases where there are autopathogenic responses to multiple antigens in the target organ.
Paracrine stimulation of interstitial collagenase (MMP-1) in the human endometrium by interleukin 1α and its dual block by ovarian steroids
Authors: Singer Christian F. , Marbaix Etienne , Kokorine Isabelle , Lemoine Pascale , Donnez Jacques , Eeckhout Yves , Courtoy Pierre J.
Published: July 2002

In the cycling human endometrium, the expression of interstitial collagenase (MMP-1) and of several related matrix metalloproteinases (MMPs) follows the late-secretory fall in sex steroid plasma concentrations and is thought to be a critical step leading to menstruation. The rapid and extensive lysis of interstitial matrix that precedes menstrual shedding requires a strict control of these proteinases. However, the mechanism by which ovarian steroids regulate endometrial MMPs remains unclear. We report here that, in the absence of ovarian steroids, MMP-1 expression in endometrial fibroblasts is markedly stimulated by medium conditioned by endometrial epithelial cells. This stimulation can be prevented by antibodies directed against interleukin 1α (IL-1α) but not against several other cytokines. Ovarian steroids inhibit the release of IL-1α and repress MMP-1 production by IL-1α-stimulated fibroblasts. In short-term cultures of endometrial explants obtained throughout the menstrual cycle, the release of both IL-1α and MMP-1 is essentially limited to the perimenstrual phase. We conclude that epithelium-derived IL-1α is the key paracrine inducer of MMP-1 in endometrial fibroblasts. However, MMP-1 production in the human endometrium is ultimately blocked by ovarian steroids, which act both upstream and downstream of IL-1α, thereby exerting an effective control via a “double-block” mechanism.
A network of interacting transcriptional regulators involved in
Drosophila
neural fate specification revealed by the yeast two-hybrid system
Authors: Alifragis Pavlos , Poortinga Gretchen , Parkhurst Susan M. , Delidakis Christos
Published: July 2002

Neural fate specification in
Drosophila
is promoted by the products of the proneural genes, such as those of the
achaete–scute complex
, and antagonized by the products of the
Enhancer of split
[E(spl)]
complex
,
hairy,
and
extramacrochaetae
. As all these proteins bear a helix-loop-helix (HLH) dimerization domain, we investigated their potential pairwise interactions using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Da, Ac, Sc, and Extramacrochaetae. We show that the seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. We further show that a subset of E(spl) proteins can heterodimerize with Da, another subset can heterodimerize with proneural proteins, and yet another with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. We investigated the structural requirements for some of these interactions by site-specific and deletion mutagenesis.
TnTIN and TnTAP: Mini-transposons for site-specific proteolysis
in vivo
Authors: Ehrmann Michael , Bolek Petra , Mondigler Martin , Boyd Dana , Lange Roland
Published: July 2002

Tobacco etch virus (TEV) protease recognizes a 7-aa consensus sequence, Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser, where Xaa can be almost any amino acyl residue. Cleavage occurs between the conserved Gln and Ser residues. Because of its distinct specificity, TEV protease can be expressed in the cytoplasm without interfering with viability. Polypeptides that are not natural substrates of TEV protease are proteolyzed if they carry the appropriate cleavage site. Thus, this protease can be used to study target proteins in their natural environment
in vivo
, as well as
in vitro
. We describe two Tn
5
-based mini-transposons that insert TEV protease cleavage sites at random into target proteins. TnTIN introduces TEV cleavage sites into cytoplasmic proteins. TnTAP facilitates the same operation for proteins localized to the bacterial cell envelope. By using two different target proteins, SecA and TolC, we show that such modified proteins can be cleaved
in vivo
and
in vitro
by TEV protease. Possible applications of the site-specific proteolysis approach are topological studies of soluble as well as of inner and outer membrane proteins, protein inactivation, insertion mutagenesis experiments, and protein tagging.
Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control
Authors: Sosnowski Ronald G. , Tu Eugene , Butler William F. , O’Connell James P. , Heller Michael J.
Published: July 2002

We have demonstrated that controlled electric fields can be used to regulate transport, concentration, hybridization, and denaturation of single- and double-stranded oligonucleotides. Discrimination among oligonucleotide hybrids with widely varying binding strengths may be attained by simple adjustment of the electric field strength. When this approach is used, electric field denaturation control allows single base pair mismatch discrimination to be carried out rapidly (<15 sec) and with high resolution. Electric field denaturation takes place at temperatures well below the melting point of the hybrids, and it may constitute a novel mechanism of DNA denaturation.
High-resolution functional mapping of a cloned gene by genetic footprinting
Authors: Singh Ila R. , Crowley Rachel Adams , Brown Patrick O.
Published: July 2002

We describe an efficient method for introducing and analyzing a comprehensive set of mutations in a cloned gene to map its functional organization. The technique, genetic footprinting, uses a retroviral integrase to generate a comprehensive library of mutants, each of which bears a single insertion of a defined oligonucleotide at a random position in the gene of interest. This mutant library is selected for gene function
en masse
. DNA samples are isolated from the library both before and after selection, and the mutations represented in each sample are then analyzed. The analysis is designed so that a mutation at a particular location gives rise to an electrophoretic band of discrete mobility. For the whole library, this results in a ladder of bands, each band representing a specific mutation. Mutants in which the inserted sequence disrupts a feature that is required for the selected function,
ipso facto,
fail the selection. The corresponding bands are therefore absent from the ladder of bands obtained from the library after selection, giving rise to a footprint representing features of the gene that are essential for the selected function. Because the sequence of the inserted oligonucleotide is known, and its position can be inferred precisely from the electrophoretic mobility of the corresponding band, the precise location and sequence of mutations that disrupt gene function can be determined without isolating or sequencing individual mutants. This method should be generally applicable for saturation mutagenesis and high-resolution functional mapping of cloned DNA sequences.
A single and early migration for the peopling of the Americas supported by mitochondrial DNA sequence data
Authors: Bonatto Sandro L. , Salzano Francisco M.
Published: July 2002

To evaluate the number and time of the migration(s) that colonized the New World we analyzed all available sequences of the first hypervariable segment of the human mitochondrial DNA control region, including 544 Native Americans. Sequence and population trees showed that the Amerind, Na-Dene, and Eskimo are significantly closer among themselves than anyone is to Asian populations, with the exception of the Siberian Chukchi, that in some analyses are closer to Na-Dene and Eskimo. Nucleotide diversity analyses based on haplogroup A sequences suggest that Native Americans and Chukchi originated from a single migration to Beringia, probably from east Central Asia, that occurred ≈30,000 or ≈43,000 years ago, depending on which substitution rate is used, with 95% confidence intervals between ≈22,000 and ≈55,000 years ago. These results support a model for the peopling of the Americas in which Beringia played a central role, where the population that originated the Native Americans settled and expanded. Some time after the colonization of Beringia they crossed the Alberta ice-free corridor and peopled the rest of the American continent. The collapse of this ice-free corridor during a few thousand years ≈14,000–20,000 years ago isolated the people south of the ice-sheets, who gave rise to the Amerind, from those still in Beringia; the latter originated the Na-Dene, Eskimo, and probably the Siberian Chukchi.
A coupled proton-transfer and twisting-motion fluorescence probe for lipid bilayers
Authors: Mateo C. Reyes , Douhal Abderrazzak
Published: July 2002

A new and sensitive molecular probe, 2-(2′-hydroxyphenyl)imidazo[1,2-a]pyridine (HPIP), for monitoring structural changes in lipid bilayers is presented. Migration of HPIP from water into vesicles involves rupture of hydrogen (H) bonds with water and formation of an internal H bond once the probe is inside the vesicle. These structural changes of the dye allow the occurrence of a photoinduced intramolecular proton-transfer reaction and a subsequent twisting/rotational process upon electronic excitation of the probe. The resulting large Stokes-shifted fluorescence band depends on the twisting motion of the zwitterionic phototautomer and is characterized in vesicles of dimyristoyl-phosphatidylcholine and in dipalmitoyl-phosphatidylcholine at the temperature range of interest and in the presence of cholesterol. Because the fluorescence of aqueous HPIP does not interfere in the emission of the probe within the vesicles, HPIP proton-transfer/twisting motion fluorescence directly allows us to monitor and quantify structural changes within bilayers. The static and dynamic fluorescence parameters are sensitive enough to such changes to suggest this photostable dye as a potential molecular probe of the physical properties of lipid bilayers.
A unified theory of carcinogenesis based on order–disorder transitions in DNA structure as studied in the human ovary and breast
Authors: Malins Donald C. , Polissar Nayak L. , Schaefer Stefan , Su Yingzhong , Vinson Mark
Published: July 2002

Fourier transform-infrared/statistics models demonstrate that the malignant transformation of morphologically normal human ovarian and breast tissues involves the creation of a high degree of structural modification (disorder) in DNA, before restoration of order in distant metastases. Order–disorder transitions were revealed by methods including principal components analysis of infrared spectra in which DNA samples were represented by points in two-dimensional space. Differences between the geometric sizes of clusters of points and between their locations revealed the magnitude of the order–disorder transitions. Infrared spectra provided evidence for the types of structural changes involved. Normal ovarian DNAs formed a tight cluster comparable to that of normal human blood leukocytes. The DNAs of ovarian primary carcinomas, including those that had given rise to metastases, had a high degree of disorder, whereas the DNAs of distant metastases from ovarian carcinomas were relatively ordered. However, the spectra of the metastases were more diverse than those of normal ovarian DNAs in regions assigned to base vibrations, implying increased genetic changes. DNAs of normal female breasts were substantially disordered (e.g., compared with the human blood leukocytes) as were those of the primary carcinomas, whether or not they had metastasized. The DNAs of distant breast cancer metastases were relatively ordered. These findings evoke a unified theory of carcinogenesis in which the creation of disorder in the DNA structure is an obligatory process followed by the selection of ordered, mutated DNA forms that ultimately give rise to metastases.
Identification of viral mutants by mass spectrometry
Authors: Lewis J. Kathleen , Bendahmane Mohammed , Smith Thomas J. , Beachy Roger N. , Siuzdak Gary
Published: July 2002

A method to identify mutations of virus proteins by using protein mass mapping is described. Comparative mass mapping was applied to a structural protein of the human rhinovirus Cys1199 → Tyr mutant and to genetically engineered mutants of tobacco mosaic virus. The information generated from this approach can rapidly identify the peptide or protein containing the mutation and, in cases when nucleic acid sequencing is required, significantly narrows the region of the genome that must be sequenced. High-resolution matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and tandem mass spectrometry were used to identify amino acid substitutions. This method provides valuable information for those analyzing viral variants and, in some cases, offers a rapid and accurate alternative to nucleotide sequencing.
T cells recognizing leukemic CD34
+
progenitor cells mediate the antileukemic effect of donor lymphocyte infusions for relapsed chronic myeloid leukemia after allogeneic stem cell transplantation
Authors: Smit Willem M. , Rijnbeek Marion , van Bergen Cornelis A. M. , Fibbe Willem E. , Willemze Roel , Falkenburg J. H. Frederik
Published: July 2002

Adoptive immunotherapy with donor lymphocyte infusions (DLI) is an effective treatment for relapsed chronic myeloid leukemia (CML) after allogeneic stem cell transplantation. To identify the effector and target cell populations responsible for the elimination of the leukemic cells
in vivo
we developed an assay to measure the frequency of T lymphocyte precursor cells capable of suppressing leukemic progenitor cells. Target cells in this assay were CML cells that were cultured in the presence of stem cell factor, interleukin 3, granulocyte–macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. [
3
H]thymidine incorporation at day 7 represented the proliferation of the progeny of the CD34
+
CML progenitor cells, and not of the more mature CD34
−
CML cells. Effector cells were mononuclear cells, which were used in a limiting dilution analysis to measure the frequencies of CML progenitor cell-inhibitory lymphocyte precursors (PCILp) in peripheral blood of seven patients before and after DLI for relapsed CML. In the six patients who entered complete remission, a 5- to 100-fold increase of PCILp was found during the clinical response. In the patient with resistant relapse the frequency of PCILp was <10 per ml before and after DLI. Leukemia-reactive helper T lymphocyte precursor frequencies remained unchanged after DLI. A significant increase in cytotoxic T lymphocyte precursor frequency against more mature leukemic cells was found in only two responding patients. These results indicate that T cells specifically directed against CD34
+
CML progenitor cells mediate the antileukemic effect of DLI.
Sex pheromone of the oleander scale,
Aspidiotus nerii
: Structural characterization and absolute configuration of an unusual functionalized cyclobutane
Authors: Einhorn Jacques , Guerrero Angel , Ducrot Paul-Henri , Boyer François-Didier , Gieselmann Mary , Roelofs Wendell
Published: July 2002

The sex pheromone emitted by the female oleander scale,
Aspidiotus nerii
(Homoptera, Diaspididae), has been isolated and characterized as (1
R,
2
S
)-
cis
-2-isopropenyl-1-(4′-methyl-4′-penten-1′-yl)cyclobutaneethanol acetate by using advanced MS and NMR spectroscopic methods, as well as a variety of microderivatization sequences. The structure has been confirmed by stereo- and enantioselective synthesis of the four possible stereoisomers. The absolute configuration has been determined by comparison of the activity of the
cis
(1
S,
2
R
) and (1
R,
2
S
) enantiomers with that exhibited by the natural material in greenhouse bioassays and field tests. The structure of this sesquiterpenoid pheromone is new in the coccids and in the pheromone field in general.