bluuebunny/crossref_metadata_2025 huggingface dataset:
Vaccination with a Nogo-A-derived peptide after incomplete spinal-cord injury promotes recovery via a T-cell-mediated neuroprotective response: Comparison with other myelin antigens
Authors: Hauben Ehud , Ibarra Antonio , Mizrahi Tal , Barouch Rina , Agranov Eugenia , Schwartz Michal
Published: July 2002

The myelin-associated protein Nogo-A has received more research attention than any other inhibitor of axonal regeneration in the injured central nervous system (CNS). Circumvention of its inhibitory effect, by using antibodies specific to Nogo-A, has been shown to promote axonal regrowth. Studies in our laboratory have demonstrated that active or passive immunization of CNS-injured rats or mice with myelin-associated peptides induces a T-cell-mediated protective autoimmune response, which promotes recovery by reducing posttraumatic degeneration. Here, we show that neuronal degeneration after incomplete spinal-cord contusion in rats was substantially reduced, and hence recovery was significantly promoted, by posttraumatic immunization with p472, a peptide derived from Nogo-A. The observed effect seemed to be mediated by T cells and could be reproduced by passive transfer of a T cell line directed against the Nogo-A peptide. Thus, it seems that after incomplete spinal-cord injury, immunization with a variety of myelin-associated peptides, including those derived from Nogo-A, can be used to evoke a T cell-mediated response that promotes recovery. The choice of peptide(s) for clinical treatment of spinal-cord injuries should be based on safety considerations; in particular, the likelihood that the chosen peptide will not cause an autoimmune disease or interfere with essential functions of this peptide or other proteins. From a therapeutic point of view, the fact that the active cellular agents are T cells rather than antibodies is an advantage, as T cell production commences within the time window required for a protective effect after spinal-cord injury, whereas antibody production takes longer.
Mutagenesis of a functional chimeric gene in yeast identifies mutations in the simian virus 40 large T antigen J domain
Authors: Fewell Sheara W. , Pipas James M. , Brodsky Jeffrey L.
Published: July 2002

Simian virus 40 large T antigen contains an amino terminal J domain that catalyzes T antigen-mediated viral DNA replication and cellular transformation. To dissect the role of the J domain in these processes, we exploited the genetic tools available only in the yeast
Saccharomyces cerevisiae
to isolate 14 loss-of-function point mutations in the T antigen J domain. This screen also identified mutations that, when engineered into simian virus 40, resulted in T antigen mutants that were defective for the ability to support viral growth, to transform mammalian cells in culture, to dissociate the p130–E2F4 transcription factor complex, and to stimulate ATP hydrolysis by hsc70, a hallmark of J domain-containing molecular chaperones. These data correlate the chaperone activity of the T antigen J domain with its roles in viral infection and cellular transformation and support a model by which the viral J domain recruits the cytoplasmic hsc70 molecular chaperone in the host to rearrange multiprotein complexes implicated in replication and transformation. More generally, this study presents the use of a yeast screen to identify loss-of-function mutations in a mammalian virus and can serve as a widely applicable method to uncover domain functions of mammalian proteins for which there are yeast homologues with selectable mutant phenotypes.
Genomic evidence for a complete sexual cycle in
Candida albicans
Authors: Tzung Keh-Weei , Williams Roy M. , Scherer Stewart , Federspiel Nancy , Jones Ted , Hansen Nancy , Bivolarevic Vesna , Huizar Lucas , Komp Caridad , Surzycki Ray , Tamse Raquel , Davis Ronald W. , Agabian Nina
Published: July 2002

Candida albicans
is a diploid fungus that has become
a medically important opportunistic pathogen in immunocompromised
individuals. We have sequenced the
C. albicans
genome to
10.4-fold coverage and performed a comparative genomic analysis between
C. albicans
and
Saccharomyces cerevisiae
with the objective of assessing whether
Candida
possesses a genetic repertoire that could support a complete sexual
cycle. Analyzing over 500 genes important for sexual differentiation in
S. cerevisiae
, we find many homologues of genes that are
implicated in the initiation of meiosis, chromosome recombination, and
the formation of synaptonemal complexes. However, others are striking
in their absence.
C. albicans
seems to have homologues
of all of the elements of a functional pheromone response pathway
involved in mating in
S. cerevisiae
but lacks many
homologues of
S. cerevisiae
genes for meiosis. Other
meiotic gene homologues in organisms ranging from filamentous fungi to
Drosophila melanogaster
and
Caenorhabditis elegans
were also found
in the
C. albicans
genome, suggesting potential
alternative mechanisms of genetic exchange.
Inhibition of growth and metastatic progression of pancreatic carcinoma in hamster after somatostatin receptor subtype 2 (sst2) gene expression and administration of cytotoxic somatostatin analog AN-238
Authors: Benali Naoual , Cordelier Pierre , Calise Denis , Pagès Philippe , Rochaix Philippe , Nagy Attila , Estève Jean-Pierre , Pour Parviz M. , Schally Andrew V. , Vaysse Nicole , Susini Christiane , Buscail Louis
Published: July 2002

The sst2 somatostatin receptor mediates the antiproliferative effects of somatostatin analogs. The present study demonstrates that stable expression of sst2 in the hamster pancreatic cancer cells PC-1 and PC-1.0 activates an autocrine negative loop leading to an
in vitro
inhibition of cell proliferation.
In vivo
studies conducted in Syrian golden hamsters after orthotopic implantation of PC-1.0 cells showed that both tumor growth and metastatic progression of allografts containing 100% of sst2-expressing cells were significantly inhibited for up to 20 days after implantation, as compared with control allografts that did not express sst2. A local antitumor bystander effect was observed after induction of mixed tumors containing a 1:3 ratio of sst2-expressing cells to control cells. Tumor volume and incidence of metastases of mixed tumors were significantly reduced at day 13 post implantation. This effect decreased with time as at day 20, growth of mixed tumors was similar to that of control tumors. After administration of the cytotoxic somatostatin conjugate AN-238 on day 13, antitumor bystander effect observed in mixed tumors was significantly extended to day 20. We also observed that
in vitro
invasiveness of sst2-expressing PC-1.0 cells was significantly reduced. Tyrosine dephosphorylation of E-cadherin may participate in restoring the E-cadherin function, reducing in turn pancreatic cancer cell motility and invasiveness. This dephosphorylation depends on the tyrosine phosphatase src homology 2-containing tyrosine phosphatase 1 (SHP-1) positively coupled to sst2 receptor. The inhibitory effect of sst2 gene expression on pancreatic cancer growth and invasion combined with chemotherapy with targeted cytotoxic somatostatin analog administration provides a rationale for a therapeutic approach to gene therapy based on
in vivo
sst2 gene transfer.
Genomic analysis of orthologous mouse and human olfactory receptor loci
Authors: Lane Robert P. , Cutforth Tyler , Young Janet , Athanasiou Maria , Friedman Cynthia , Rowen Lee , Evans Glen , Axel Richard , Hood Leroy , Trask Barbara J.
Published: July 2002

Olfactory receptor (OR) genes represent ≈1% of genomic coding sequence in mammals, and these genes are clustered on multiple chromosomes in both the mouse and human genomes. We have taken a comparative genomics approach to identify features that may be involved in the dynamic evolution of this gene family and in the transcriptional control that results in a single OR gene expressed per olfactory neuron. We sequenced ≈350 kb of the murine P2 OR cluster and used synteny, gene linkage, and phylogenetic analysis to identify and sequence ≈111 kb of an orthologous cluster in the human genome. In total, 18 mouse and 8 human OR genes were identified, including 7 orthologs that appear to be functional in both species. Noncoding homology is evident between orthologs and generally is confined within the transcriptional unit. We find no evidence for common regulatory features shared among paralogs, and promoter regions generally do not contain strong promoter motifs. We discuss these observations, as well as OR clustering, in the context of evolutionary expansion and transcriptional regulation of OR repertoires.
The central cytoplasmic loop of the major facilitator superfamily of transport proteins governs efficient membrane insertion
Authors: Weinglass Adam B. , Kaback H. Ronald
Published: July 2002

Deletion of 5 residues (Δ5) from the central cytoplasmic loop of the lactose permease of
Escherichia coli
has no significant effect on expression or activity, whereas Δ12 leads to increased rates of permease turnover after membrane insertion and decreased transport activity, and Δ20 abolishes insertion and activity. By expressing Δ12 or Δ20 in two halves, both expression and activity are restored to levels approximating wild type. Replacing deleted residues with random hydrophilic amino acids also leads to full recovery. However, introduction of hydrophobic residues decreases expression and activity in a context-dependent manner. Thus, a minimum length of the central cytoplasmic loop is vital for proper insertion, stability, and efficient transport activity, because of constraints at the cytoplasmic ends of helices VI and VII. Furthermore, the results are consistent with the idea that the middle cytoplasmic loop provides a temporal delay between insertion of the first six helices into the membrane before insertion of the second six helices.
Use of cDNA subtraction and RNA interference screens in combination reveals genes required for germ-line development in
Caenorhabditis elegans
Authors: Hanazawa Momoyo , Mochii Makoto , Ueno Naoto , Kohara Yuji , Iino Yuichi
Published: July 2002

Caenorhabditis elegans
is an ideal organism for the study of the molecular basis of fundamental biological processes such as germ-line development, especially because of availability of the whole genome sequence and applicability of the RNA interference (RNAi) technique. To identify genes involved in germ-line development, we produced subtracted cDNA pools either enriched for or deprived of the cDNAs from germ-line tissues. We then performed differential hybridization on the high-density cDNA grid, on which about 7,600 nonoverlapping expressed sequence tag (EST) clones were spotted, to identify a set of genes specifically expressed in the germ line. One hundred and sixty-eight clones were then tested with the RNAi technique. Of these, 15 clones showed sterility with a variety of defects in germ-line development. Seven of them led to the production of unfertilized eggs, because of defects in spermatogenesis (4 clones), or defects in the oocytes (3 clones). The other 8 clones led to failure of oogenesis. These failures were caused by germ-line proliferation defect (Glp phenotype), meiotic arrest, and defects in sperm–oocyte switch (Mog phenotype) among others. These results demonstrate the efficacy of the screening strategy using the EST library combined with the RNAi technique in
C. elegans
.
A sea urchin genome project: Sequence scan, virtual map, and additional resources
Authors: Cameron R. Andrew , Mahairas Gregory , Rast Jonathan P. , Martinez Pedro , Biondi Ted R. , Swartzell Steven , Wallace James C. , Poustka Albert J. , Livingston Brian T. , Wray Gregory A. , Ettensohn Charles A. , Lehrach Hans , Britten Roy J. , Davidson Eric H. , Hood Leroy
Published: July 2002

Results of a first-stage Sea Urchin Genome Project are summarized here. The species chosen was
Strongylocentrotus purpuratus
, a research model of major importance in developmental and molecular biology. A virtual map of the genome was constructed by sequencing the ends of 76,020 bacterial artificial chromosome (BAC) recombinants (average length, 125 kb). The BAC-end sequence tag connectors (STCs) occur an average of 10 kb apart, and, together with restriction digest patterns recorded for the same BAC clones, they provide immediate access to contigs of several hundred kilobases surrounding any gene of interest. The STCs survey >5% of the genome and provide the estimate that this genome contains ≈27,350 protein-coding genes. The frequency distribution and canonical sequences of all middle and highly repetitive sequence families in the genome were obtained from the STCs as well. The 500-kb
Hox
gene complex of this species is being sequenced in its entirety. In addition, arrayed cDNA libraries of >10
5
clones each were constructed from every major stage of embryogenesis, several individual cell types, and adult tissues and are available to the community. The accumulated STC data and an expanding expressed sequence tag database (at present including >12,000 sequences) have been reported to GenBank and are accessible on public web sites.
Identification of a conserved erythroid specific domain of histone acetylation across the α-globin gene cluster
Authors: Anguita Eduardo , Johnson Colin A. , Wood William G. , Turner Bryan M. , Higgs Douglas R.
Published: July 2002

We have analyzed the pattern of core histone acetylation across 250 kb of the telomeric region of the short arm of human chromosome 16. This gene-dense region, which includes the α-globin genes and their regulatory elements embedded within widely expressed genes, shows marked differences in histone acetylation between erythroid and non-erythroid cells. In non-erythroid cells, there was a uniform 2- to 3-fold enrichment of acetylated histones, compared with heterochromatin, across the entire region. In erythroid cells, an ≈100-kb segment of chromatin encompassing the α genes and their remote major regulatory element was highly enriched in histone H4 acetylated at Lys-5. Other lysines in the N-terminal tail of histone H4 showed intermediate and variable levels of enrichment. Similar broad segments of erythroid-specific histone acetylation were found in the corresponding syntenic regions containing the mouse and chicken α-globin gene clusters. The borders of these regions of acetylation are located in similar positions in all three species, and a sharply defined 3′ boundary coincides with the previously identified breakpoint in conserved synteny between these species. We have therefore demonstrated that an erythroid-specific domain of acetylation has been conserved across several species, encompassing not only the α-globin genes but also a neighboring widely expressed gene. These results contrast with those at other clusters and demonstrate that not all genes are organized into discrete regulatory domains.
CR16 forms a complex with N-WASP in brain and is a novel member of a conserved proline-rich actin-binding protein family
Authors: Ho Hsin-Yi Henry , Rohatgi Rajat , Ma Le , Kirschner Marc W.
Published: July 2002

The Neuronal Wiskott–Aldrich syndrome protein (N-WASP) has emerged
as a central regulator of the actin cytoskeleton with abilities to
integrate multiple upstream signal inputs and transmit them to the
Arp2/3 complex. Here, we demonstrate that native N-WASP is present in
a tight complex with a proline-rich protein, CR16, which shares ≈25%
identity with WASP interacting protein. CR16 is encoded by a gene
previously cloned as a glucocorticoid-regulated mRNA from a rat
hippocampal cDNA library. Although N-WASP is expressed ubiquitously,
full-length CR16 protein is found predominately in the brain. CR16 and
N-WASP colocalize in primary hippocampal neurons and at the tips of
their growth cone filopodia.
In vitro
, CR16 directly
binds both monomeric and filamentous actin but does not affect the
kinetics of actin polymerization mediated by N-WASP and the Arp2/3
complex. Sequence homologues of CR16 are found not only in other
vertebrates but also in the invertebrate
Caenorhabditis elegans
and in yeast.
Thus, CR16 and WASP interacting protein belong to a family of
N-WASP-binding proteins.
TAF
II
55 binding to TAF
II
250 inhibits its acetyltransferase activity
Authors: Gegonne Anne , Weissman Jocelyn D. , Singer Dinah S.
Published: July 2002

The general transcription factor, TFIID, consists of the TATA-binding protein (TBP) associated with a series of TBP-associated factors (TAFs) that together participate in the assembly of the transcription preinitiation complex. One of the TAFs, TAF
II
250, has acetyltransferase (AT) activity that is necessary for transcription of MHC class I genes: inhibition of the AT activity represses transcription. To identify potential cellular factors that might regulate the AT activity of TAF
II
250, a yeast two-hybrid library was screened with a TAF
II
250 segment (amino acids 848-1279) that spanned part of its AT domain and it's the domain that binds to the protein, RAP74. The TFIID component, TAF
II
55, was isolated and found to interact predominantly with the RAP74-binding domain. TAF
II
55 binding to TAF
II
250 inhibits its AT activity. Importantly, the addition of recombinant TAF
II
55 to
in vitro
transcription assays inhibits TAF
II
250-dependent MHC class I transcription. Thus, TAF
II
55 is capable of regulating TAF
II
250 function by modulating its AT activity.
Extraordinary variation in a diversified family of immune-type receptor genes
Authors: Hawke Noel A. , Yoder Jeffrey A. , Haire Robert N. , Mueller M. Gail , Litman Ronda T. , Miracle Ann L. , Stuge Tor , Shen Linling , Miller Norman , Litman Gary W.
Published: July 2002

Immune inhibitory receptor genes that encode a variable (V) region, a unique V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in two lineages of bony fish. In the present study, eleven related genes encoding distinct structural forms have been identified inIctalurus punctatus(channel catfish), a well characterized immunological model system that represents a third independent bony fish lineage. Each of the different genes encodes an N-terminal V region but differs in the number of extracellular Ig domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues in transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails. Variation in the numbers of genomic copies of the different gene types, their patterns of expression, and relative levels of expression in mixed leukocyte cultures (MLC) is reported. V region-containing immune-type genes constitute a far more complex family than recognized originally and include individual members that might function in inhibitory or, potentially activatory manners.
Signal transduction in the archaeon Halobacterium salinarium is processed through three subfamilies of 13 soluble and membrane-bound transducer proteins.
Authors: Zhang W , Brooun A , McCandless J , Banda P , Alam M
Published: July 2002

Eubacterial transducers are transmembrane, methyl-accepting proteins central to chemotaxis systems and share common structural features. We identified a large family of transducer proteins in the Archaeon Halobacterium salinarium using a site-specific multiple antigenic peptide antibody raised against 23 amino acids, representing the highest homology region of eubacterial transducers. This immunological observation was confirmed by isolating 13 methyl-accepting taxis genes using a 27-mer oligonucleotide probe, corresponding to conserved regions between the eubacterial and first halobacterial phototaxis transducer gene htrI. On the basis of the comparison of the predicted structural domains of these transducers, we propose that at least three distinct subfamilies of transducers exist in the Archaeon H. salinarium: (i) a eubacterial chemotaxis transducer type with two hydrophobic membrane-spanning segments connecting sizable domains in the periplasm and cytoplasm; (ii) a cytoplasmic domain and two or more hydrophobic transmembrane segments without periplasmic domains; and (iii) a cytoplasmic domain without hydrophobic transmembrane segments. We fractionated the halobacterial cell lysate into soluble and membrane fractions and localized different halobacterial methyl-accepting taxis proteins in both fractions.
Intracerebral injection of human immunodeficiency virus type 1 coat protein gp120 differentially affects the expression of nerve growth factor and nitric oxide synthase in the hippocampus of rat.
Authors: Bagetta G , Corasaniti M T , Aloe L , Berliocchi L , Costa N , Finazzi-Agrò A , Nisticò G
Published: July 2002

We have studied the neuropathological characteristics of the brain of rats receiving daily intracerebroventricular administration of freshly dissolved human immunodeficiency virus type 1 recombinant protein gp120 (100 ng per rat per day) given for up to 14 days. Histological examination of serial brain sections revealed no apparent gross damage to the cortex or hippocampus, nor did cell counting yield significant neuronal cell loss. However, the viral protein caused after 7 and 14 days of treatment DNA fragmentation in 10% of brain cortical neurons. Interestingly, reduced neuronal nitric oxide synthase (NOS) expression along with significant increases in nerve growth factor (NGF) were observed in the hippocampus, where gp120 did not cause neuronal damage. No changes in NGF and NOS expression were seen in the cortex, where cell death is likely to be of the apoptotic type. The present data demonstrate that gp120-induced cortical cell death is associated with the lack of increase of NGF in the cerebral cortex and suggest that the latter may be important for the expression of neuropathology in the rat brain. By contrast, enhanced levels of NGF may prevent or delay neuronal death in the hippocampus, where reduced NOS expression may be a reflection of a subcellular insult inflicted by the viral protein.
Pepsin-inhibitory activity of the uterine serpins
Authors: Mathialagan Nagappan , Hansen Thomas R.
Published: July 2002

Among the major products secreted by the uteri of cattle, sheep,
and pigs during pregnancy are glycoproteins with amino acid sequences
that place them in the serpin (
ser
ine
p
roteinase
in
hibitor) superfamily of
proteins. The inferred amino acid sequences for bovine uterine serpin
(boUS-1) and ovine uterine serpin (ovUS-1) exhibit about 72% sequence
identity to each other but only about 50% and 56% identity,
respectively, to two distinct porcine uterine serpins (poUS-1 and
poUS-2). Despite these differences in primary structure, the uterine
serpins possess well-conserved reactive center loop regions that
contain several motifs present in the propeptide regions of
pepsinogens. One such motif, VVVK, aligns with the first 4 amino acids
of the aspartic proteinase inhibitor pepstatin. Although no inhibitory
activity toward any serine proteinase has been found, at least one of
the uterine serpins, ovUS-1, can bind specifically to immobilized
pepsin A and can weakly inhibit the proteolytic activities of pepsin A
and C (but not cathepsins D and E). OvUS-1 is the first specific
inhibitor of aspartic proteinases to be identified in vertebrates and
provides another example of a serpin with “crossover” activity.
The pregnancy-associated glycoproteins (PAGs), which are secreted by
the trophoblast layer of the placentas of ungulate species and are
inactive members of the aspartic proteinase family, can also bind
ovUS-1 and may be the natural target partners for the uterine serpins.
Convex polytopes and quantization of symplectic manifolds
Authors: Vergne Michèle
Published: July 2002

Quantum mechanics associate to some symplectic manifolds
M
a quantum model
Q
(
M
), which is a
Hilbert space. The space
Q
(
M
) is the quantum
mechanical analogue of the classical phase space
M
. We
discuss here relations between the volume of
M
and the
dimension of the vector space
Q
(
M
). Analogues
for convex polyhedra are considered.
Presentation of a cytosolic antigen by major histocompatibility complex class II molecules requires a long-lived form of the antigen
Authors: Guéguen Maryse , Long Eric O.
Published: July 2002

Class I and class II molecules of the major histocompatibility complex present peptides to T cells. Class I molecules bind peptides that have been generated in the cytosol by proteasomes and delivered into the endoplasmic reticulum by the transporter associated with antigen presentation. In contrast, class II molecules are very efficient in the presentation of antigens that have been internalized and processed in endosomal/lysosomal compartments. In addition, class II molecules can present some cytosolic antigens by a TAP-independent pathway. To test whether this endogenous class II presentation pathway was linked to proteasome-mediated degradation of antigen in the cytosol, the N-end rule was utilized to produce two forms of the influenza virus matrix protein with differentin vivohalf-lives (10 min vs. 5 h) when expressed in human B cells. Whereas class I molecules presented both the short- and the long-lived matrix proteins, class II molecules presented exclusively the long-lived form of antigen. Thus, rapid degradation of matrix protein in the cytosol precluded its presentation by class II molecules. These data suggest that the turnover of long-lived cytosolic proteins, some of which is mediated by delivery into endosomal/lysosomal compartments, provides a mechanism for immune surveillance by CD4+T cells.
Functional dissection of the
Drosophila
Enhancer of split protein, a suppressor of neurogenesis
Authors: Giebel Bernd , Campos-Ortega José A.
Published: July 2002

The
Enhancer of split
[
E(spl)
] gene complex of
Drosophila
comprises seven related genes encoding a special type of basic helix–loop–helix proteins, the function of which is to suppress the neural developmental fate. One of these proteins is E(spl) itself. To gain insight into the structural requirements for E(spl) function, we have expressed a large number of deletion variants in transgenic flies. Three protein domains were identified as essential for suppression of bristle development: the carboxyl-terminal tetrapeptide WRPW, the region comprising the putative helix III and helix IV, and the region between helix IV and the WRPW motif. Lack of the basic helix–loop–helix domain, helix III or IV, only partially inhibits the suppressor activity of the protein. Truncated variants that lack all the regions carboxyl-terminal to helix IV elicit the development of additional neural progenitors, and thus act as dominant-negative variants. All these results suggest that E(spl) suppresses neural development by direct interaction with other proteins, such as groucho and the proneural proteins.
Effect of tissue factor deficiency on mouse and tumor development
Authors: Toomey John R. , Kratzer Kenneth E. , Lasky Nina M. , Broze George J.
Published: July 2002

Previous reports suggest that tissue factor (TF) may play an essential role in embryonic vascular development and tumor angiogenesis. To further examine this relationship, the morphology of fully developed TF-deficient embryos and the growth of TF-deficient teratomas and teratocarcinomas were analyzed. In a 129/Sv genetic background, TF null embryos do not survive beyond mid-gestation. In contrast, 14% of 129/Sv × C57BL/6 TF-deficient embryos escape this early mortality and survive to birth. On gross and microscopic inspection, these late gestation, TF-deficient embryos appear normal. The growth and vascularity of TF(+/+), TF(+/−), and TF(−/−) teratomas and teratocarcinomas are indistinguishable. Thus, tumor-derived TF is not required for tumor growth and angiogenesis and the combined data do not support an essential role for TF in embryonic vascular development.
Isolation and characterization of an amino acid-selective channel protein present in the chloroplastic outer envelope membrane
Authors: Pohlmeyer Kai , Soll Jürgen , Steinkamp Thomas , Hinnah Silke , Wagner Richard
Published: July 2002

The reconstituted pea chloroplastic outer envelope protein of 16 kDa (OEP16) forms a slightly cation-selective, high-conductance channel with a conductance of Λ = 1,2 nS (in 1 M KCl). The open probability of OEP16 channel is highest at 0 mV (P
open
= 0.8), decreasing exponentially with higher potentials. Transport studies using reconstituted recombinant OEP16 protein show that the OEP16 channel is selective for amino acids but excludes triosephosphates or uncharged sugars. Crosslinking indicates that OEP16 forms a homodimer in the membrane. According to its primary sequence and predicted secondary structure, OEP16 shows neither sequence nor structural homologies to classical porins. The results indicate that the intermembrane space between the two envelope membranes might not be as freely accessible as previously thought.
The product of ORF O located within the domain of herpes simplex virus 1 genome transcribed during latent infection binds to and inhibits
in vitro
binding of infected cell protein 4 to its cognate DNA site
Authors: Randall Glenn , Lagunoff Michael , Roizman Bernard
Published: July 2002

The partially overlapping ORF P and ORF O are located within the domains of the herpes simplex virus 1 genome transcribed during latency. Earlier studies have shown that ORF P is repressed by infected cell protein 4 (ICP4), the major viral regulatory protein, binding to its cognate site at the transcription initiation site of ORF P. The ORF P protein binds to p32, a component of the ASF/SF2 alternate splicing factors; in cells infected with a recombinant virus in which ORF P was derepressed there was a significant decrease in the expression of products of key regulatory genes containing introns. We report that (
i
) the expression of ORF O is repressed during productive infection by the same mechanism as that determining the expression of ORF P; (
ii
) in cells infected at the nonpermissive temperature for ICP4, ORF O protein is made in significantly lower amounts than the ORF P protein; (
iii
) the results of insertion of a sequence encoding 20 amino acids between the putative initiator methionine codons of ORF O and ORF P suggest that ORF O initiates at the methionine codon of ORF P and that the synthesis of ORF O results from frameshift or editing of its RNA; and (
iv
) glutathione
S
-transferase–ORF O fusion protein bound specifically ICP4 and precluded its binding to its cognate site on DNA
in vitro
. These and earlier results indicate that ORF P and ORF O together have the capacity to reduce the synthesis or block the expression of regulatory proteins essential for viral replication in productive infection.
Fluorescence energy transfer detection as a homogeneous DNA diagnostic method
Authors: Chen Xiangning , Zehnbauer Barbara , Gnirke Andreas , Kwok Pui-Yan
Published: July 2002

A homogeneous DNA diagnostic assay based on template-directed primer extension detected by fluorescence resonance energy transfer, named template-directed dye-terminator incorporation (TDI) assay, has been developed for mutation detection and high throughput genome analysis. Here, we report the successful application of the TDI assay to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the human leukocyte antigen H (HLA-H) gene, and the receptor tyrosin kinase (RET) protooncogene that are associated with cystic fibrosis, hemochromatosis, and multiple endocrine neoplasia, type 2, respectively. Starting with total human DNA, the samples are amplified by the PCR followed by enzymatic degradation of excess primers and deoxyribonucleoside triphosphates before the primer extension reaction is performed. All these standardized steps are performed in the same tube, and the fluorescence changes are monitored in real time, making it a useful clinical DNA diagnostic method.
Interaction between HLA-DM and HLA-DR involves regions that undergo conformational changes at lysosomal pH
Authors: Ullrich H. Joachim , Döring Klaus , Grüneberg Ulrike , Jähnig Fritz , Trowsdale John , van Ham S. Marieke
Published: July 2002

Antigenic peptide loading of major histocompatibility complex class II molecules is enhanced by lysosomal pH and catalyzed by the HLA-DM molecule. The physical mechanism behind the catalytic activity of DM was investigated by using time-resolved fluorescence anisotropy (TRFA) and fluorescence binding studies with the dye 8-anilino-1-naphthalenesulfonic acid (ANS). We demonstrate that the conformations of both HLA-DM and HLA-DR3, irrespective of the composition of bound peptide, are pH sensitive. Both complexes reversibly expose more nonpolar regions upon protonation. Interaction of DM with DR shields these hydrophobic domains from the aqueous environment, leading to stabilization of the DM and DR conformations. At lysosomal pH, the uncovering of additional hydrophobic patches leads to a more extensive DM–DR association. We propose that DM catalyzes class II peptide loading by stabilizing the low-pH conformation of DR, favoring peptide exchange. The DM–DR association involves a larger hydrophobic surface area with DR/class II-associated invariant chain peptides (CLIP) than with stable DR/peptide complexes, explaining the preferred association of DM with the former. The data support a release mechanism of DM from the DM–DR complex through reduction of the interactive surface, upon binding of class II molecules with antigenic peptide or upon neutralization of the DM–DR complex at the cell surface.
Genetic recombination of poliovirus in a cell-free system
Authors: Duggal Rohit , Cuconati Andrea , Gromeier Matthias , Wimmer Eckard
Published: July 2002

Genetic recombination of plus-strand RNA viruses is an important process for promoting genetic variation. By using genetically marked poliovirus RNAs, we have demonstrated that genetic recombination can occur in a cell-free system that generates infective virus from added poliovirus RNA. Recombinant polioviruses were isolated, and the region of crossing over was roughly mapped. Recombinants could be isolated even under conditions where the yield of viruses from one of the parental RNAs was depressed to levels comparable to or less than the yield of recombinant viruses, an observation suggesting that only one of the recombining RNAs needs to be replication-competent. The generation of poliovirus recombinants in a cell-free system offers new possibilities for studying recombination and evolution of RNA viruses.
In disperse solution, “osmotic stress” is a restricted case of preferential interactions
Authors: Timasheff Serge N.
Published: July 2002

In the practice of “osmotic stress,” the effect of excluded cosolvents on a biochemical equilibrium is interpreted as the number of water molecules participating in the reaction. This action is attributed to lowering of solvent water activity by the cosolvent. This concept of osmotic stress in disperse solution is erroneous: (
i
) A cosolvent cannot be both excluded and inert, i.e., noninteracting, because exclusion requires a positive free energy change; (
ii
) a decrease in water activity alone by addition of solute cannot affect an equilibrium when the reacting surface is in contact with the solvent; and (
iii
) osmotic stress in disperse solution is a restricted case of preferential interactions; the reaction is driven by the free energy of cosolvent exclusion, and the derived number of water molecules is solely a measure of the mutual perturbations of the chemical potentials of the cosolvent and the protein.
HD mice: A novel mouse mutant with a specific defect in the generation of CD4+T cells
Authors: Dave Vibhuti P. , Allman David , Keefe Robert , Hardy Richard R. , Kappes Dietmar J.
Published: July 2002

We have identified a spontaneous mutation in mice, which we term HD for “helper T cell deficient.” This mouse is distinguished by the virtual absence of peripheral T cells of the CD4+8−major histocompatibility complex (MHC) class II-restricted T helper subset due to a specific block in thymic development. The developmental defect is selective for CD4+8−cells; the maturation of CD4−8+and γδ T cells is normal. The autosomal recessive mutation underlying the HD phenotype is unrelated to MHC class II, since it segregates independently of theMHC class IIlocus. Moreover, the HD phenotype is not caused by a defect of theCD4gene. Bone marrow transfer experiments demonstrate that the defect is intrinsic to cells of the hematopoietic lineage, i.e., most likely to developing thymocytes themselves. The frequency of CD4+8lowintermediate cells is markedly increased in HD mice, suggesting that class II-restricted thymocytes are arrested at this stage. This is the first genetic defect of its kind to be described in the mouse and may prove highly informative in understanding the molecular pathways underlying lineage commitment.
NADP, corepressor for the
Bacillus
catabolite control protein CcpA
Authors: Kim Jeong-Ho , Voskuil Martin I. , Chambliss Glenn H.
Published: July 2002

Expression of the α-amylase gene (
amyE
) of
Bacillus subtilis
is subject to CcpA (catabolite control protein A)-mediated catabolite repression, a global regulatory mechanism in
Bacillus
and other Gram-positive bacteria. To determine effectors of CcpA, we tested the ability of glycolytic metabolites, nucleotides, and cofactors to affect CcpA binding to the
amyE
operator,
amyO
. Those that stimulated the DNA-binding affinity of CcpA were tested for their effect on transcription. HPr-P (Ser-46), proposed as an effector of CcpA, also was tested. In DNase I footprint assays, the affinity of CcpA for
amyO
was stimulated 2-fold by fructose-1,6-diphosphate (FDP), 1.5-fold by oxidized or reduced forms of NADP, and 10-fold by HPr-P (Ser-46). However, the triple combinations, CcpA/NADP/HPr-P (Ser-46) and CcpA/FDP/HPr-P (Ser-46) synergistically stimulated DNA-binding affinity by 120- and 300-fold, respectively. NADP added to CcpA specifically stimulated transcription inhibition of the
amyE
promoter by 120-fold. CcpA combined with HPr (Ser-46) inhibited transcription from the
amyE
promoter, but it also inhibited several control promoters. FDP did not stimulate transcription inhibition by CcpA nor did the triple combinations. The finding that NADP had little effect on CcpA DNA binding but increased the ability of CcpA to inhibit transcription suggests that catabolite repression is not simply caused by CcpA binding
amyO
but rather a result of interactions with the transcription machinery enhanced by NADP.
The atomic model of the human protective protein/cathepsin A suggests a structural basis for galactosialidosis
Authors: Rudenko Gabby , Bonten Erik , Hol Wim. G. J. , d’Azzo Alessandra
Published: July 2002

Human protective protein/cathepsin A (PPCA), a serine carboxypeptidase, forms a multienzyme complex with β-galactosidase and neuraminidase and is required for the intralysosomal activity and stability of these two glycosidases. Genetic lesions in PPCA lead to a deficiency of β-galactosidase and neuraminidase that is manifest as the autosomal recessive lysosomal storage disorder galactosialidosis. Eleven amino acid substitutions identified in mutant PPCAs from clinically different galactosialidosis patients have now been modeled in the three-dimensional structure of the wild-type enzyme. Of these substitutions, 9 are located in positions likely to alter drastically the folding and stability of the variant protein. In contrast, the other 2 mutations that are associated with a more moderate clinical outcome and are characterized by residual mature protein appeared to have a milder effect on protein structure. Remarkably, none of the mutations occurred in the active site or at the protein surface, which would have disrupted the catalytic activity or protective function. Instead, analysis of the 11 mutations revealed a substantive correlation between the effect of the amino acid substitution on the integrity of protein structure and the general severity of the clinical phenotype. The high incidence of PPCA folding mutants in galactosialidosis reflects the fact that a single point mutation is unlikely to affect both the β-galactosidase and the neuraminidase binding sites of PPCA at the same time to produce the double glycosidase deficiency. Mutations in PPCA that result in defective folding, however, disrupt every function of PPCA simultaneously.
Chromosome translocation based on illegitimate recombination in human tumors
Authors: Zucman-Rossi Jessica , Legoix Patricia , Victor Jean-Marc , Lopez Bernard , Thomas Gilles
Published: July 2002

Recurrent chromosome translocations in nonhematological tumors are restricted to specific subtypes, and their mechanism is currently unknown. Analysis of the sequence data of 113 interchromosomal junctions derived from 77 Ewing’s tumors carrying the characteristic t(11;22) translocation indicate that, in this tumor, translocations are initiated independently on each chromosome in regions that lack site specific recombination signal. Local sequence duplications, deletions, and, most importantly, inversions that are diagnostic of DNA hairpin formation indicate that, at the breakpoint, single-stranded DNA ends are processed individually before interchromosomal joining. Taken together, these observations suggest that chromosome translocations in Ewing’s tumors are mediated through a genuine illegitimate recombination mechanism.
Mycobacterium aviumcomplex augments macrophage HIV-1 production and increases CCR5 expression
Authors: Wahl S. M. , Greenwell-Wild T. , Peng G. , Hale-Donze H. , Doherty T. M. , Mizel D. , Orenstein J. M.
Published: July 2002

Infection with HIV-1 results in pronounced immune suppression and susceptibility to opportunistic infections (OI). Reciprocally, OI augment HIV-1 replication. As we have shown forMycobacterium aviumcomplex (MAC) andPneumocystis carinii, macrophages infected with opportunistic pathogens and within lymphoid tissues containing OI, exhibit striking levels of viral replication. To explore potential underlying mechanisms for increased HIV-1 replication associated with coinfection, blood monocytes were exposed to MAC antigens (MAg) or viable MAC and their levels of tumor necrosis factor α (TNFα) and HIV-1 coreceptors monitored. MAC enhanced TNFα productionin vitro, consistent with its expression in coinfected lymph nodes. Using a polyclonal antibody to the CCR5 coreceptor that mediates viral entry of macrophage tropic HIV-1, a subset of unstimulated monocytes was shown to be CCR5-positive by fluorescence-activated cell sorter analysis. After stimulation with MAg or infection with MAC, CCR5 expression was increased at both the mRNA level and on the cell surface. Up-regulation of CCR5 by MAC was not paralleled by an increase in the T cell tropic coreceptor, CXCR4. Increases in NF-κB, TNFα, and CCR5 were consistent with the enhanced production of HIV-1 in MAg-treated adherent macrophage cultures as measured by HIV-1 p24 levels. Increased CCR5 was also detected in coinfected lymph nodes as compared with tissues with only HIV-1. The increased production of TNFα, together with elevated expression of CCR5, provide potential mechanisms for enhanced infection and replication of HIV-1 by macrophages in OI-infected cells and tissues. Consequently, treating OI may inhibit not only the OI-induced pathology, but also limit the viral burden.
Integration of the
Tetrahymena
group I intron into bacterial rRNA by reverse splicing
in vivo
Authors: Roman Judibelle , Woodson Sarah A.
Published: July 2002

Horizontal gene transfer is thought to contribute to the wide distribution of group I introns among organisms. Integration of an intron into foreign RNA or DNA by reverse self-splicing, followed by reverse transcription and recombination, could lead to its transposition. Reverse self-splicing of group I introns has been demonstrated
in vitro
, but not
in vivo
. Here we report RNA-dependent integration of the
Tetrahymena
intron into the 23S rRNA in
Escherichia coli
. Analysis of products by Northern blot and reverse transcription–PCR amplification revealed precise intron insertion into a site homologous to the natural splice junction. Products are sensitive to treatment with RNase but not DNase and depend on the splicing activity of the intron. Partial reaction with 11 novel sites in the 23S RNA that are complementary to the guide sequence of the intron illustrates lower specificity than intron homing. Reverse splicing of the
Tetrahymena
intron in bacteria demonstrates the possibility of RNA-catalyzed transposition of group I introns in foreign hosts.
The remarkably high excitation planetary nebula GC 6537
Authors: Aller Lawrence H. , Hung Siek , Feibelman Walter A.
Published: July 2002

NGC 6537 is an unusually high excitation point symmetric planetary nebula with a rich spectrum. Its kinematical structures are of special interest. We are here primarily concerned with the high resolution spectrum as revealed by the Hamilton echelle Spectrograph at Lick Observatory (resolution ≈ 0.2 Å) and supplemented by UV and near-UV data. These extensive data permit a determination of interstellar extinction, plasma diagnostics, and ionic concentrations. The photoionization models that have been used successfully for many planetary nebulae are not entirely satisfactory here. The plasma electron temperature of a photoionization model cannot much exceed 20,000 K, but plasma diagnostics show that regions emitting radiation of highly ionized atoms such as [Ne
iv
] and [Ne
v
] are much hotter, showing that shock excitation must be important, as suggested by the remarkable kinematics of this object. Hence, instead of employing a strict photoionization model, we are guided by the nebular diagnostics, which reveal how electron temperature varies with ionization potential and accommodates density effects. The predictions of the photoionization model may be useful in estimating ionization correction factor. In effect, we have estimated the chemical composition by using both photoionization and shock considerations.
Induction of differentiation of pre-NKT cells to mature Vα14 NKT cells by granulocyte/macrophage colony-stimulating factor
Authors: Sato Hiroshi , Nakayama Toshinori , Tanaka Yujiro , Yamashita Masakatsu , Shibata Yoichi , Kondo Eisuke , Saito Yasushi , Taniguchi Masaru
Published: July 2002

Vα14 NKT cells express an invariant antigen receptor encoded by Vα14 and Jα281 gene segments as well as natural killer (NK) markers, including NK1.1. Here, we describe a precursor population of NKT cells (pre-NKT) that expresses NK1.1, T cell antigen receptor β, pTα, and RAG1/2 but not Vα14 and surface CD3ɛ. Such pre-NKT cells were differentiated successfullyin vitrointo mature CD3ɛ+Vα14+NKT cells by IL-15 and granulocyte/macrophage colony-stimulating factor (GM-CSF) in conjunction with stroma cells. Interestingly, only GM-CSF without stroma cells induced the Vα14-Jα281 gene rearrangement in the pre-NKT cells. This also was confirmed by the findings that the number of mature Vα14 NKT cells and the frequency of Vα14-Jα281 rearrangements were decreased significantly in the mice lacking a GM-CSF receptor component, common β-chain. These results suggest a crucial role of GM-CSF in the development of Vα14 NKT cellsin vivo.
Potent selective nonpeptidic inhibitors of human lung tryptase
Authors: Burgess Laurence E. , Newhouse Bradley J. , Ibrahim Prabha , Rizzi James , Kashem Mohammed A. , Hartman Ann , Brandhuber Barbara J. , Wright Clifford D. , Thomson David S. , Vigers Guy P. A. , Koch Kevin
Published: July 2002

Human lung tryptase, a homotetrameric serine protease unique to mast cell secretory granules, has been implicated in the pathogenesis of asthma. A hypothesis that tethered symmetrical inhibitors might bridge two adjacent active sites was explored via a rationally designed series of bisbenzamidines. These compounds demonstrated a remarkable distanced-defined structure–activity relationship against human tryptase with one series possessing subnanomolar potencies. Additional evidence supporting the concept of active-site bridging is also presented.