bluuebunny/crossref_metadata_2025 huggingface dataset:
Site-specific integration by adeno-associated virus.
Authors: Linden R M , Ward P , Giraud C , Winocour E , Berns K I
Published: July 2002

Adeno-associated virus (AAV) has attracted considerable interest as a potential vector for gene delivery. Wild-type virus is notable for the lack of association with any human disease and the ability to stably integrate its genome in a site-specific manner in a locus on human chromosome 19 (AAVS1). Use of a functional model system for AAV DNA integration into AAVS1 has allowed us to conclude that the recombination event is directed by cellular DNA sequences. Recombinant junctions isolated from our integration assay were analyzed and showed characteristics similar to those found in latently infected cell lines. The minimal DNA signals within AAVS1 required for targeted integration were identified and shown to contain functional motifs of the viral origin of replication. A replication mediated model of AAV DNA integration is proposed.
Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells
Authors: John Maliyakal E. , Keller Greg
Published: July 2002

Alcaligenes eutrophus
genes encoding the enzymes,
β-ketothiolase (
phaA
), acetoacetyl-CoA reductase
(
phaB
), and polyhydroxyalkanoate synthase
(
phaC
) catalyze the production of aliphatic polyester
poly-
d
-(−)-3-hydroxybutyrate (PHB) from acetyl-CoA. PHB is
a thermoplastic polymer that may modify fiber properties when
synthesized in cotton. Endogenous β-ketothiolase activity
is present in cotton fibers. Hence cotton was transformed with
engineered
phaB
and
phaC
genes by
particle bombardment, and transgenic plants were selected based on
marker gene, β-glucuronidase (
GUS
), expression. Fibers
of 10 transgenic plants expressed
phaB
gene, while eight
plants expressed both
phaB
and
phaC
genes. Electron microscopy examination of fibers expressing both genes
indicated the presence of electron-lucent granules in the cytoplasm.
High pressure liquid chromatography, gas chromatography, and mass
spectrometry evidence suggested that the new polymer produced in
transgenic fibers is PHB. Sixty-six percent of the PHB in fibers is in
the molecular mass range of 0.6 × 10
6
to 1.8 ×
10
6
Da. The presence of PHB granules in transgenic fibers
resulted in measurable changes of thermal properties. The fibers
exhibited better insulating characteristics. The rate of heat uptake
and cooling was slower in transgenic fibers, resulting in higher heat
capacity. These data show that metabolic pathway engineering in cotton
may enhance fiber properties by incorporating new traits from other
genetic sources. This is an important step toward producing new
generation fibers for the textile industry.
Molecular basis for the recognition of two structurally different major histocompatibility complex/peptide complexes by a single T-cell receptor
Authors: Brock Roland , Wiesmüller Karl-Heinz , Jung Günther , Walden Peter
Published: July 2002

2C is a typical alloreactive cytotoxic T lymphocyte clone that recognizes two different ligands. These ligands are adducts of the allo-major histocompatibility complex (MHC) molecule H-2Ldand an endogenous octapeptide, and of the self-MHC molecule H-2Kband another peptide. MHC-binding and T-cell assays with synthetic peptides in combination with molecular modeling studies were employed to analyze the structural basis for this crossreactivity. The molecular surfaces of the two complexes differ greatly in densities and distributions of positive and negative charges. However, modifications of the peptides that increase similarity decrease the capacities of the resulting MHC peptide complexes to induce T-cell responses. Moreover, the roles of the peptides in ligand recognition are different for self- and allo-MHC-restricted T-cell responses. The self-MHC-restricted T-cell responses were finely tuned to recognition of the peptide. The allo-MHC-restricted responses, on the other hand, largely ignore modifications of the peptide. The results strongly suggest that adaptation of the T-cell receptor to the different ligand structures, rather than molecular mimicry by the ligands, is the basis for the crossreactivity of 2C. This conclusion has important implications for T-cell immunology and for the understanding of immunological disorders.
Oxidative stress by tumor-derived macrophages suppresses the expression of CD3 ζ chain of T-cell receptor complex and antigen-specific T-cell responses
Authors: Otsuji Mizuto , Kimura Yoshimitsu , Aoe Tomohiko , Okamoto Yasuhiro , Saito Takashi
Published: July 2002

One of the important mechanisms of immunosuppression in the tumor-bearing status has been attributed to the down-modulation of the CD3 ζ chain and its associated signaling molecules in T cells. Thus, the mechanism of the disappearance of CD3ζ was investigated in tumor-bearing mice (TBM). The decrease of CD3ζ was observed both in the cell lysate and intact cells. Direct interaction of T cells with macrophages from TBM (TBM-macrophages) induced the decrease of CD3ζ, and depletion of macrophages rapidly restored the CD3ζ expression. We found that treatment of such macrophages withN-acetylcysteine, known as antioxidant compound, prevented the decrease of CD3ζ. Consistent with this result, the addition of oxidative reagents such as hydrogen peroxide and diamide induced the decrease of CD3ζ expression in T cells. Consequently, the loss of CD3ζ resulted in suppression of the antigen-specific T-cell response. These results demonstrate that oxidative stress by macrophages in tumor-bearing status induces abnormality of the T-cell receptor complex by cell interactions with T cells. Therefore, our findings suggest that oxidative stress contributes to the regulation of the expression and function of the T-cell receptor complex.
Molecular recognition of pathogen attack occurs inside of plant cells in plant disease resistance specified by the
Arabidopsis
genes
RPS2
and
RPM1
Authors: Leister R. Todd , Ausubel Frederick M. , Katagiri Fumiaki
Published: July 2002

The
Arabidopsis thaliana
disease resistance genes
RPS2
and
RPM1
belong to a class of plant
disease resistance genes that encode proteins that contain an
N-terminal tripartite nucleotide binding site (NBS) and a C- terminal
tandem array of leucine-rich repeats.
RPS2
and
RPM1
confer resistance to strains of the bacterial
phytopathogen
Pseudomonas syringae
carrying the
avirulence genes
avrRpt2
and
avrB
,
respectively. In these gene-for-gene relationships, it has been
proposed that pathogen avirulence genes generate specific ligands that
are recognized by cognate receptors encoded by the corresponding plant
resistance genes. To test this hypothesis, it is crucial to know the
site of the potential molecular recognition. Mutational analysis of
RPS2 protein and
in vitro
translation/translocation
studies indicated that RPS2 protein is localized in the plant
cytoplasm. To determine whether avirulence gene products themselves are
the ligands for resistance proteins, we expressed the
avrRpt2
and
avrB
genes directly in plant
cells using a novel quantitative transient expression assay, and found
that expression of
avrRpt2
and
avrB
elicited a resistance response in plants carrying the corresponding
resistance genes. This observation indicates that no bacterial factors
other than the avirulence gene products are required for the specific
resistance response as long as the avirulence gene products are
correctly localized. We propose that molecular recognition of
P.
syringae
in
RPS2-
and
RPM1
-specified resistance occurs inside of plant cells.
Overexpression of a Rrp1 transgene reduces the somatic mutation and recombination frequency induced by oxidative DNA damage in Drosophila melanogaster.
Authors: Szakmary A , Huang S M , Chang D T , Beachy P A , Sander M
Published: July 2002

Recombination repair protein 1 (Rrp1) includes a C-terminal region homologous to several DNA repair proteins, including Escherichia coli exonuclease III and human APE, that repair oxidative and alkylation damage to DNA. The nuclease activities of Rrp1 include apurinic/apyrimidinic endonuclease, 3'-phosphodiesterase, 3'-phosphatase, and 3'-exonuclease. As shown previously, the C-terminal nuclease region of Rrp1 is sufficient to repair oxidative- and alkylation-induced DNA damage in repair-deficient E. coli mutants. DNA strand-transfer and single-stranded DNA renaturation activities are associated with the unique N-terminal region of Rrp1, which suggests possible additional functions that include recombinational repair or homologous recombination. By using the Drosophila w/w+ mosaic eye system, which detects loss of heterozygosity as changes in eye pigmentation, somatic mutation and recombination frequencies were determined in transgenic flies overexpressing wild-type Rrp1 protein from a heat-shock-inducible transgene. A large decrease in mosaic clone frequency is observed when Rrp1 overexpression precedes treatment with gamma-rays, bleomycin, or paraquat. In contrast, Rrp1 overexpression does not alter the spot frequency after treatment with the alkylating agents methyl methanesulfonate or methyl nitrosourea. A reduction in mosaic clone frequency depends on the expression of the Rrp1 transgene and on the nature of the induced DNA damage. These data suggest a lesion-specific involvement of Rrp1 in the repair of oxidative DNA damage.
The joining (J) chain is present in invertebrates that do not express immunoglobulins.
Authors: Takahashi T , Iwase T , Takenouchi N , Saito M , Kobayashi K , Moldoveanu Z , Mestecky J , Moro I
Published: July 2002

Joining (J) chain is a component of polymeric, but not monomeric, immunoglobulin (Ig) molecules and may play a role in their polymerization and transport across epithelial cells. To date, study of the J chain has been confined to vertebrates that produce Ig and in which the J chain displays a considerable degree of structural homology. The role of the J chain in Ig polymerization has been questioned and, since the J chain can be expressed in lymphoid cells that do not produce Ig, it is possible that the J chain may have other functions. To explore this possibility, we have surveyed J-chain gene, mRNA, and protein expression by using reverse transcriptase-coupled PCR, Northern blot analysis, and immunoblot analysis in invertebrate species that do not produce Ig. We report that the J-chain gene is expressed in invertebrates (Mollusca, Annelida, Arthropoda, Echinodermata, and Holothuroidea), as well as in representative vertebrates (Mammalia, Teleostei, Amphibia). Furthermore, J-chain cDNA from the earthworm has a high degree of homology (68-76%) to human, mouse, and bovine J chains. Immunohistochemical studies reveal that the J chain is localized in the mucous cells of body surfaces, intestinal epithelial cells, and macrophage-like cells of the earthworm and slug. This study suggests that the J chain is a primitive polypeptide that arose before the evolution of Ig molecules and remains highly conserved in extent invertebrates and vertebrates.
CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis.
Authors: Gerritse K , Laman J D , Noelle R J , Aruffo A , Ledbetter J A , Boersma W J , Claassen E
Published: July 2002

We investigated the role of CD40-CD40 ligand (CD40L) interactions in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). Activated helper T cells expressing CD40L (gp39) surface protein were found in MS patient brain sections, but not in brain tissue sections of normal controls or patients with other neurological disease. CD40L-positive cells were co-localized with CD40-bearing cells in active lesions (perivascular infiltrates). Most of these CD40-bearing cells proved to be of the monocytic lineage (macrophages or microglial cells), and relatively few were B cells. To functionally evaluate CD40-CD40L interactions, EAE was elicited in mice by means of proteolipid-peptide immunization. Treatment with anti-CD40L monoclonal antibody completely prevented the development of disease. Furthermore, administration of anti-CD40L monoclonal antibody, even after disease onset, shortly before maximum disability score was reached led to dramatic disease reduction. The presence of helper T cells expressing CD40L in brain tissue of MS patients and EAE animals, together with the functional evidence provided by successful experimental prevention and therapy in an animal model, indicates that blockade of CD40-CD40L-mediated cellular interactions may be a method for interference in active MS.
Tissue-specific knockout of the mouse
Pig-a
gene reveals important roles for GPI-anchored proteins in skin development
Authors: Tarutani Masahito , Itami Satoshi , Okabe Masaru , Ikawa Masahito , Tezuka Tadashi , Yoshikawa Kunihiko , Kinoshita Taroh , Takeda Junji
Published: July 2002

Glycosylphosphatidylinositol (GPI)-anchored proteins are widely distributed on plasma membranes of eukaryotes. More than 50 GPI-anchored proteins have been shown to be spatiotemporally expressed in mice with a deficiency of GPI-anchor biosynthesis that causes embryonic lethality. Here, we examine the functional roles of GPI-anchored proteins in mouse skin using the Cre-
loxP
recombination system. We disrupted the
Pig-a
gene, an X-linked gene essential for GPI-anchor biosynthesis, in skin. The Cre-mediated
Pig-a
disruption occurred in skin at almost 100% efficiency in male mice bearing two identically orientated
loxP
sites within the
Pig-a
gene. Expression of GPI-anchored proteins was completely absent in the skin of these mice. The skin of such mutants looked wrinkled and more scaly than that of wild-type mice. Furthermore, histological examination of mutant mice showed that the epidermal horny layer was tightly packed and thickened. Electron microscopy showed that the intercellular space was narrow and there were many small vesicles embedded in the intercellular space that were not observed in equivalent wild-type mouse skin preparations. Mutant mice died within a few days after birth, suggesting that
Pig-a
function is essential for proper skin differentiation and maintenance.
Specific T cell recognition of kinetic isomers in the binding of peptide to class II major histocompatibility complex
Authors: Rabinowitz Joshua D. , Liang Michael N. , Tate Keri , Lee Christopher , Beeson Craig , McConnell Harden M.
Published: July 2002

Helper T cells are triggered by molecular complexes of antigenic peptides and class II proteins of the major histocompatibility complex . The formation of stable complexes between class II major histocompatibility complex proteins and antigenic peptides is often accompanied by the formation of a short-lived complex. In this report, we describe T cell recognition of two distinct complexes, one short-lived and the other long-lived, formed during the binding of an altered myelin basic protein peptide to I-Ak. One myelin basic protein-specific T cell clone is triggered by only the short-lived complex, and another is triggered by only the stable complex. Thus, a single peptide bound to a particular class II molecule can activate different T cells depending on the conditions of the binding reaction.
Expression of adenoviral E3 transgenes in β cells prevents autoimmune diabetes
Authors: von Herrath Matthias G. , Efrat Shimon , Oldstone Michael B. A. , Horwitz Marshall S.
Published: July 2002

The adenovirus (Ad) genome contains immunoregulatory and cytokine inhibitory genes that are presumed to function in facilitating acute infection or in establishing persistencein vivo. Some of these genes are clustered in early region 3 (E3), which contains a 19-kDa glycoprotein (gp19) that inhibits the transport of selected class I major histocompatibility complex (MHC) molecules out of the endoplasmic reticulum. In addition, the E3 region contains three protein inhibitors of the cytolytic function of tumor necrosis factor α (TNF-α). Because type I autoimmune diabetes destroys islets by mechanisms that involve class I MHC and TNF-α, we investigated whether the entire cassette of Ad E3 genes might prevent the onset of diabetes in a well studied lymphocytic choriomeningitis viral (LCMV) murine model of virus-induced autoimmune diabetes. In this model, a LCMV polypeptide (either glycoprotein or nucleoprotein) expressed as a transgene in the islets is a target for autoimmune destruction of β cells after LCMV infection. In this scenario the LCMV-induced immune response is directed not only against the virus but also against the LCMV transgenes expressed in the β cells. Our experiments demonstrated a very efficient prevention of this LCMV-triggered diabetes by the Ad E3 genes. This resulted from the inhibition of target cell recognition by a fully competent and LCMV-primed immune system. Unlike the results from the β-2 microglobulin gene deletion experiments, our approach shows that selective regulation at the level of the target cell is sufficient to prevent autoimmune diabetes without disrupting the function of the systemic immune response. Although the Ad genes in these experiments were provided as transgenes, recent experiments may permit the introduction of such genes through the use of viral vectors. Although the decrease in class I MHC in islets by Ad genes was demonstrated in thesein vivostudies, the relative importance of this process and the control of TNF-α cytolysis must await further genetic dissection of the introduced Ad genes.
Substitution of a mutant α
2a
-adrenergic receptor via “hit and run” gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses
in vivo
Authors: Lakhlani Parul P. , MacMillan Leigh B. , Guo Tian Zhi , McCool Brian A. , Lovinger David M. , Maze Mervyn , Limbird Lee E.
Published: July 2002

Norepinephrine contributes to antinociceptive, sedative, and
sympatholytic responses
in vivo
, and α
2
adrenergic receptor (α
2
AR) agonists are used clinically
to mimic these effects. Lack of subtype-specific agonists has prevented
elucidation of the role that each α
2
AR subtype
(α
2A
, α
2B
, and α
2C
) plays in
these central effects. Here we demonstrate that α
2
AR
agonist-elicited sedative, anesthetic-sparing, and analgesic responses
are lost in a mouse line expressing a subtly mutated
α
2A
AR, D79N α
2A
AR, created by two-step
homologous recombination. These functional changes are accompanied by
failure of the D79N α
2A
AR to inhibit voltage-gated
Ca
2+
currents and spontaneous neuronal firing, a measure of
K
+
current activation. These results provide definitive
evidence that the α
2A
AR subtype is the primary mediator
of clinically important central actions of α
2
AR agonists
and suggest that the D79N α
2A
AR mouse may serve as a
model for exploring other possible α
2A
AR functions
in vivo
.
Structure of the imprinted mouse
Snrpn
gene and establishment of its parental-specific methylation pattern
Authors: Shemer Ruth , Birger Yehudit , Riggs Arthur D. , Razin Aharon
Published: July 2002

The mouse
Snrpn
gene encodes the Smn protein, which is involved in RNA splicing. The gene maps to a region in the central part of chromosome 7 that is syntenic to the Prader–Willi/Angelman syndromes (PWS-AS) region on human chromosome 15q11-q13. The mouse gene, like its human counterpart, is imprinted and paternally expressed, primarily in brain and heart. We provide here a detailed description of the structural features and differential methylation pattern of the gene. We have identified a maternally methylated region at the 5′ end (DMR1), which correlates inversely with the
Snrpn
paternal expression. We also describe a region at the 3′ end of the gene (DMR2) that is preferentially methylated on the paternal allele. Analysis of
Snrpn
mRNA levels in a methylase-deficient mouse embryo revealed that maternal methylation of DMR1 may play a role in silencing the maternal allele. Yet both regions, DMR1 and DMR2, inherit the parental-specific methylation profile from the gametes. This methylation pattern is erased in 12.5-days postcoitum (dpc) primordial germ cells and reestablished during gametogenesis. DMR1 is remethylated during oogenesis, whereas DMR2 is remethylated during spermatogenesis. Once established, these methylation patterns are transmitted to the embryo and maintained, protected from methylation changes during embryogenesis and cell differentiation. Transfections of DMR1 and DMR2 into embryonic stem cells and injection into pronuclei of fertilized eggs reveal that embryonic cells lack the capacity to establish anew the differential methylation pattern of
Snrpn
. That all PWS patients lack DMR1, together with the overall high resemblance of the mouse gene to the human
SNRPN
, offers an excellent experimental tool to study the regional control of this imprinted chromosomal domain.
The exaptive excellence of spandrels as a term and prototype
Authors: Gould Stephen Jay
Published: July 2002

In 1979, Lewontin and I borrowed the architectural term “spandrel” (using the pendentives of San Marco in Venice as an example) to designate the class of forms and spaces that arise as necessary byproducts of another decision in design, and not as adaptations for direct utility in themselves. This proposal has generated a large literature featuring two critiques: (i) the terminological claim that the spandrels of San Marco are not true spandrels at all and (ii) the conceptual claim that they are adaptations and not byproducts. The features of the San Marco pendentives that we explicitly defined as spandrel-properties—their necessary number (four) and shape (roughly triangular)—are inevitable architectural byproducts, whatever the structural attributes of the pendentives themselves. The term spandrel may be extended from its particular architectural use for two-dimensional byproducts to the generality of “spaces left over,” a definition that properly includes the San Marco pendentives. Evolutionary biology needs such an explicit term for features arising as byproducts, rather than adaptations, whatever their subsequent exaptive utility. The concept of biological spandrels—including the examples here given of masculinized genitalia in female hyenas, exaptive use of an umbilicus as a brooding chamber by snails, the shoulder hump of the giant Irish deer, and several key features of human mentality—anchors the critique of overreliance upon adaptive scenarios in evolutionary explanation. Causes of historical origin must always be separated from current utilities; their conflation has seriously hampered the evolutionary analysis of form in the history of life.
Noninfectious virus-like particles produced by Moloney murine leukemia virus-based retrovirus packaging cells deficient in viral envelope become infectious in the presence of lipofection reagents
Authors: Sharma Sanjai , Murai Fukashi , Miyanohara Atsushi , Friedmann Theodore
Published: July 2002

Retrovirus packaging cell lines expressing the Moloney murine leukemia virus
gag
and
pol
genes but lacking virus envelope genes produce virus-like particles constitutively, whether or not they express a transcript from an integrated retroviral provirus. In the absence of a proviral transcript, the assembled particles contain processed
gag
and reverse transcriptase, and particles made by cells expressing an integrated
lacZ
provirus also contain viral RNA. The virus-like particles from both cell types are enveloped and are secreted/budded into the extracellular space but are noninfectious. Their physicochemical properties are similar to those of mature retroviral particles. The noninfectious
gag pol RNA
particles can readily be made infectious by the addition of lipofection reagents to produce preparations with titers of up to 10
5
colony-forming units per ml.
Synergistic inhibition of human cancer cell growth by cytotoxic drugs and mixed backbone antisense oligonucleotide targeting protein kinase A
Authors: Tortora Giampaolo , Caputo Rosa , Damiano Vincenzo , Bianco Roberto , Pepe Stefano , Bianco A. Raffaele , Jiang Zhiwei , Agrawal Sudhir , Ciardiello Fortunato
Published: July 2002

Protein kinase A type I plays a key role in neoplastic transformation, conveying mitogenic signals of different growth factors and oncogenes. Inhibition of protein kinase A type I by antisense oligonucleotides targeting its RIα regulatory subunit results in cancer cell growth inhibition
in vitro
and
in vivo
. A novel mixed backbone oligonucleotide HYB 190 and its mismatched control HYB 239 were tested on soft agar growth of several human cancer cell types. HYB 190 demonstrated a dose-dependent inhibition of colony formation in all cell lines whereas the HYB 239 at the same doses caused a modest or no growth inhibition. A noninhibitory dose of each mixed backbone oligonucleotide was used in OVCAR-3 ovarian and GEO colon cancer cells to study whether any cooperative effect may occur between the antisense and a series of cytotoxic drugs acting by different mechanisms. Treatment with HYB 190 resulted in an additive growth inhibitory effect with several cytotoxic drugs when measured by soft agar colony formation. A synergistic growth inhibition, which correlated with increased apoptosis, was observed when HYB 190 was added to cancer cells treated with taxanes, platinum-based compounds, and topoisomerase II selective drugs. This synergistic effect was also observed in breast cancer cells and was obtained with other related drugs such as docetaxel and carboplatin. Combination of HYB 190 and paclitaxel resulted in an accumulation of cells in late S-G
2
phases of cell cycle and marked induction of apoptosis. A cooperative effect of HYB 190 and paclitaxel was also obtained
in vivo
in nude mice bearing human GEO colon cancer xenografts. These results are the first report of a cooperative growth inhibitory effect obtained in a variety of human cancer cell lines by antisense mixed backbone oligonucleotide targeting protein kinase A type I-mediated mitogenic signals and specific cytotoxic drugs.
Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein–Barr virus nuclear antigen 1
Authors: Levitskaya Jelena , Sharipo Anatoly , Leonchiks Ainars , Ciechanover Aaron , Masucci Maria G.
Published: July 2002

The Epstein–Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.
Widespread expression and estrogen regulation of PPEIA-3′ nuclear RNA in the rat brain
Authors: Brooks P. J. , Kleopoulos Steven P. , Funabashi Toshiya , Mobbs Charles V. , Pfaff Donald W.
Published: July 2002

We previously identified a novel nuclear RNA species derived from
the preproenkephalin (PPE) gene. This transcript, which we have named
PPEIA-3′ RNA, hybridizes with probes directed at a region of PPE intron
A downstream of an alternative germ-cell transcription start site, but
does not contain PPE protein coding sequences. We now report that
estrogen treatment of ovariectomized rats increases the expression of
conventional PPE heteronuclear RNA, and also induces the expression of
PPEIA-3′ RNA, apparently in separate cell populations within the
ventromedial nucleus of the hypothalamus. Further, we show that cells
expressing PPEIA-3′ are found in several neuronal groups in the rat
forebrain and brainstem, with a distinct topographical distribution.
High densities of PPEIA-3′ containing cells are found in the reticular
thalamic nucleus, the basal forebrain, the vestibular complex, the deep
cerebellar nuclei, and the trapezoid body, a pattern that parallels the
distribution of atypical nuclear RNAs described by other groups. These
results suggest that this diverse neuronal population shares a common
set of nuclear factors responsible for the expression and retention of
this atypical RNA transcript. The implication of these results for
cell-specific gene transcription and regulation in the brain and the
possible relationship of PPEIA-3′ RNA and other atypical nuclear RNAs
is discussed.
Expression of lipocalin-type prostaglandin D synthase (β-trace) in human heart and its accumulation in the coronary circulation of angina patients
Authors: Eguchi Yutaka , Eguchi Naomi , Oda Hiroshi , Seiki Kousuke , Kijima Yoshiyuki , Matsu-ura Yasuhiko , Urade Yoshihiro , Hayaishi Osamu
Published: July 2002

Lipocalin-type prostaglandin D synthase (L-PGDS) is localized in the central nervous system and male genital organs of various mammals and is secreted as β-trace into the closed compartment of these tissues separated from the systemic circulation. In this study, we found that the mRNA for the human enzyme was expressed most intensely in the heart among various tissues examined. In human autopsy specimens, the enzyme was localized immunocytochemically in myocardial cells, atrial endocardial cells, and a synthetic phenotype of smooth muscle cells in the arteriosclerotic intima, and accumulated in the atherosclerotic plaque of coronary arteries with severe stenosis. In patients with stable angina (75–99% stenosis), the plasma level of L-PGDS was significantly (
P
< 0.05) higher in the great cardiac vein (0.694 ± 0.054 μg/ml,
n
= 7) than in the coronary artery (0.545 ± 0.034 μg/ml), as determined by a sandwich enzyme immunoassay. However, the veno-arterial difference in the plasma L-PGDS concentration was not observed in normal subjects without stenosis. After a percutaneous transluminal coronary angioplasty was performed to compress the stenotic atherosclerotic plaques, the L-PGDS concentration in the cardiac vein decreased significantly (
P
< 0.05) to 0.610 ± 0.051 μg/ml at 20 min and reached the arterial level within 1 h. These findings suggest that L-PGDS is present in both endocardium and myocardium of normal subjects and the stenotic site of patients with stable angina and is secreted into the coronary circulation.
Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses
Authors: Schiavo Giampietro , Stenbeck Gudrun , Rothman James E. , Söllner Thomas H.
Published: July 2002

Neurotransmitter release requires the specific docking of synaptic vesicles to the presynaptic plasma membrane followed by a calcium-triggered fusion event. Herein we report a previously unsuspected interaction of the synaptic vesicle protein and likely calcium sensor synaptotagmin with the plasma membrane t-SNARE SNAP-25. This interaction appears to resolve the apparent paradox that synaptic vesicles are capable of docking even when VAMP (vesicle-associated membrane protein) or syntaxin is cleaved or deleted and suggests that two species of v-SNAREs (VAMP and synaptotagmin) and two species of t-SNAREs (SNAP-25 and syntaxin) interact to functionally dock synaptic vesicles.
Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice
Authors: Eglitis Martin A. , Mezey Éva
Published: July 2002

Glial cells are thought to derive embryologically from either myeloid cells of the hematopoietic system (microglia) or neuroepithelial progenitor cells (astroglia and oligodendrocytes). However, it is unclear whether the glia in adult brains free of disease or injury originate solely from cells present in the brain since the fetal stage of development, or if there is further input into such adult brains from cells originating outside the central nervous system. To test the ability of hematopoietic cells to contribute to the central nervous system, we have transplanted adult female mice with donor bone marrow cells genetically marked either with a retroviral tag or by using male donor cells. Using
in situ
hybridization histochemistry, a continuing influx of hematopoietic cells into the brain was detected. Marrow-derived cells were already detected in the brains of mice 3 days after transplant, and their numbers increased over the next several weeks, exceeding 14,000 cells per brain in several animals. Marrow-derived cells were widely distributed throughout the brain, including the cortex, hippocampus, thalamus, brain stem, and cerebellum. When
in situ
hybridization histochemistry was combined with immunohistochemical staining using lineage-specific markers, some bone marrow-derived cells were positive for the microglial antigenic marker F4/80. Other marrow-derived cells surprisingly expressed the astroglial marker glial fibrillary acidic protein. These results indicate that some microglia and astroglia arise from a precursor that is a normal constituent of adult bone marrow.
Regulation of the pAD1 sex pheromone response of
Enterococcus faecalis
by direct interaction between the cAD1 peptide mating signal and the negatively regulating, DNA-binding TraA protein
Authors: Fujimoto Shuhei , Clewell Don B.
Published: July 2002

The
Enterococcus faecalis
conjugative plasmid pAD1 (60 kb) encodes a mating response to the recipient-produced peptide sex pheromone cAD1. The response involves two key plasmid-encoded regulatory proteins: TraE1, which positively regulates all or most structural genes relating to conjugation, and TraA, which binds DNA and negatively regulates expression of
traE1. In vitro
studies that included development of a DNA-associated protein-tag affinity chromatography technique showed that TraA (37.9 kDa) binds directly to cAD1 near its carboxyl-terminal end and, as a consequence, loses its affinity for DNA. Analyses of genetically modified TraA proteins indicated that truncations within the carboxyl-terminal 9 residues significantly affected the specificity of peptide-directed association/dissociation of DNA. The data support earlier observations that transposon insertions near the 3′ end of traA eliminated the ability of cells to respond to cAD1.
Patterns of brain angiogenesis after vascular endothelial growth factor administration
in vitro
and
in vivo
Authors: Rosenstein Jeffrey M. , Mani Nina , Silverman William F. , Krum Janette M.
Published: July 2002

Vascular endothelial growth factor (VEGF) is a secreted endothelial cell mitogen that has been shown to induce vasculogenesis and angiogenesis in many organ systems and tumors. Considering the importance of VEGF to embryonic vascularization and survival, the effects of administered VEGF on developing or adult cerebrovasculature are unknown: can VEGF alter brain angiogenesis or mature cerebrovascular patterns? To examine these questions we exposed fetal, newborn, and adult rat cortical slice explants to graduated doses of recombinant VEGF. The effects of another known angiogenic factor, basic fibroblast growth factor (bFGF), were evaluated in a comparable manner. In addition, we infused VEGF via minipump into the adult cortex. Significant angiogenic effects were found in all VEGF experiments in a dose-responsive manner that were abolished by the addition of VEGF neutralizing antibody. Fetal and newborn explants had a highly complex network of branched vessels that immunoexpressed the
flt-1
VEGF receptor, and
flk-1
VEGF receptor expression was determined by reverse transcription–PCR. Adult explants had enlarged, dilated vessels that appeared to be an expansion of the existing network. All bFGF-treated explants had substantially fewer vascular profiles. VEGF infusions produced both a remarkable localized neovascularization and, unexpectedly, the expression of
flt-1
on reactive astrocytes but not on endothelial cells. The preponderance of neovascularization
in vitro
and
in vivo
, however, lacked the blood–brain barrier (BBB) phenotype marker, GLUT-1, suggesting that in brain the angiogenic role of VEGF may differ from a potential BBB functional role, i.e., transport and permeability. VEGF may serve an important capacity in neovascularization or BBB alterations after brain injury.
Resonating circadian clocks enhance fitness in cyanobacteria
Authors: Ouyang Yan , Andersson Carol R. , Kondo Takao , Golden Susan S. , Johnson Carl Hirschie
Published: July 2002

In some organisms longevity, growth, and developmental rate are improved when they are maintained on a light/dark cycle, the period of which “resonates” optimally with the period of the endogenous circadian clock. However, to our knowledge no studies have demonstrated that reproductive fitness
per se
is improved by resonance between the endogenous clock and the environmental cycle. We tested the adaptive significance of circadian programming by measuring the relative fitness under competition between various strains of cyanobacteria expressing different circadian periods. Strains that had a circadian period similar to that of the light/dark cycle were favored under competition in a manner that indicates the action of soft selection.
Cloning of the
Arabidopsis
ent
-kaurene oxidase gene

GA
3
Authors: Helliwell Chris A. , Sheldon Candice C. , Olive Mark R. , Walker Amanda R. , Zeevaart Jan A. D. , Peacock W. James , Dennis Elizabeth S.
Published: July 2002

The
ga3
mutant of
Arabidopsis
is a gibberellin-responsive dwarf. We present data showing that the
ga3-1
mutant is deficient in
ent-
kaurene oxidase activity, the first cytochrome P450-mediated step in the gibberellin biosynthetic pathway. By using a combination of conventional map-based cloning and random sequencing we identified a putative cytochrome P450 gene mapping to the same location as
GA3
. Relative to the progenitor line, two
ga3
mutant alleles contained single base changes generating in-frame stop codons in the predicted amino acid sequence of the P450. A genomic clone spanning the P450 locus complemented the
ga3-2
mutant. The deduced GA3 protein defines an additional class of cytochrome P450 enzymes. The
GA3
gene was expressed in all tissues examined, RNA abundance being highest in inflorescence tissue.
Different requirements for
EDS1
and
NDR1
by disease resistance genes define at least two
R
gene-mediated signaling pathways in
Arabidopsis
Authors: Aarts Nicole , Metz Matthew , Holub Eric , Staskawicz Brian J. , Daniels Michael J. , Parker Jane E.
Published: July 2002

The
Arabidopsis
genes
EDS1
and
NDR1
were shown previously by mutational analysis to encode essential components of race-specific disease resistance. Here, we examined the relative requirements for
EDS1
and
NDR1
by a broad spectrum of Resistance (
R
) genes present in three
Arabidopsis
accessions (Columbia, Landsberg-
erecta
, and Wassilewskija). We show that there is a strong requirement for
EDS1
by a subset of
R
loci (
RPP2
,
RPP4
,
RPP5
,
RPP21
, and
RPS4
), conferring resistance to the biotrophic oomycete
Peronospora parasitica
, and to
Pseudomonas
bacteria expressing the avirulence gene
avrRps4
. The requirement for
NDR1
by these
EDS1
-dependent
R
loci is either weak or not measurable. Conversely, three
NDR1
-dependent
R
loci,
RPS2
,
RPM1
, and
RPS5
, operate independently of
EDS1
. Another
RPP
locus,
RPP8
, exhibits no strong exclusive requirement for
EDS1
or
NDR1
in isolate-specific resistance to
P. parasitica
, although resistance is compromised weakly by
eds1
. Similarly, resistance conditioned by two
EDS1
-dependent
RPP
genes,
RPP4
and
RPP5
, is impaired partially by
ndr1
, implicating a degree of pathway cross-talk. Our results provide compelling evidence for the preferential utilization of either signaling component by particular
R
genes and thus define at least two disease resistance pathways. The data also suggest that strong dependence on
EDS1
or
NDR1
is governed by R protein structural type rather than pathogen class.
Expression of a dopamine D2 receptor-activated K
+
channel on identified striatopallidal and striatonigral neurons
Authors: Waszczak Barbara L. , Martin Lynn P. , Greif Gabriela J. , Freedman Jonathan E.
Published: July 2002

One view of the efferent circuitry of the basal ganglia holds that dopamine D1 and D2 receptors are segregated to striatonigral and striatopallidal neurons, respectively. The present studies investigated whether functional D2-like receptors are, in fact, restricted to striatopallidal neurons. Single, freshly dissociated cells from rat striatum were identified as either striatonigral or striatopallidal projection neurons by fluorescence retrograde labeling. By using cell-attached patch-clamp recordings, neurons of each efferent group were evaluated for the presence of a D2-like receptor-activated 85-pS K
+
channel as a measure of receptor function. We now report the presence of this D2 receptor-mediated response on both striatal efferent populations, but we observed an approximately 2-fold higher likelihood of encountering the channel on pallidal- versus nigral-projecting neurons. The channel’s conductance properties appeared identical in both groups of neurons, but there was a significantly greater open probability for channels detected on striatopallidal neurons. These results indicate that functional D2 receptors are not segregated to striatopallidal neurons, but may be expressed in a higher proportion of, or at a higher density and/or efficiency of coupling on, pallidal- versus nigral-projecting striatal efferents.
Compartmentation of phosphoglycerate kinase in
Trypanosoma brucei
plays a critical role in parasite energy metabolism
Authors: Blattner Judith , Helfert Sandra , Michels Paul , Clayton Christine
Published: July 2002

African trypanosomes compartmentalize glycolysis in a microbody, the glycosome. When growing in the mammalian bloodstream, trypanosomes contain only a rudimentary mitochondrion, and the first seven glycolytic enzymes, including phosphoglycerate kinase, are located in the glycosome. Procyclic trypanosomes, growing in the gut of tsetse flies, possess a fully developed mitochondrion that is active in oxidative phosphorylation. The first six glycolytic enzymes are still glycosomal, but phosphoglycerate kinase is now found in the cytosol. We demonstrate here that bloodstream trypanosomes are killed by expression of cytosolic phosphoglycerate kinase. The toxicity depends on both enzyme activity and cytosolic location. One possible explanation is that cytosolic phosphoglycerate kinase creates an ATP-generating shunt in the cytosol, thus preventing full ATP regeneration in the glycosome and ultimately inhibiting the first, ATP-consuming, steps of glycolysis.
Synthetic amino acid copolymers that bind to HLA-DR proteins and inhibit type II collagen-reactive T cell clones
Authors: Fridkis-Hareli Masha , Rosloniec Edward F. , Fugger Lars , Strominger Jack L.
Published: July 2002

Copolymer 1 [poly(Y,E,A,K)] is a random synthetic amino acid copolymer ofl-tyrosine,l-glutamic acid,l-alanine, andl-lysine that is effective both in suppression of experimental allergic encephalomyelitis and in the treatment of relapsing forms of multiple sclerosis. Copolymer 1 binds promiscuously and very efficiently to purified HLA-DR molecules within the peptide-binding groove. In the present study, YEAK and YEAK-related copolymers and type II collagen (CII) peptide 261–273, a candidate autoantigen in rheumatoid arthritis (RA), competed for binding to RA-associated HLA-DR molecules encoded by DRB1*0101 and DRB1*0401. Moreover, these copolymers (particularly YEAK, YAK, and YEK) inhibited the response of DR1- and DR4-restricted T cell clones to the CII epitope 261–273 by >50%. This direct evidence both for competitive interactions of these copolymers and CII peptide with RA-associated HLA-DR molecules and for inhibition of CII-specific T cell responses suggests that these compounds should be evaluated in animal models for rheumatoid arthritis.
Influenza C virus CM2 integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence
Authors: Pekosz Andrew , Lamb Robert A.
Published: July 2002

The influenza C virus CM2 protein is a small glycosylated integral membrane protein (115 residues) that spans the membrane once and contains a cleavable signal sequence at its N terminus. The coding region for CM2 (CM2 ORF) is located at the C terminus of the 342-amino acid (aa) ORF of a colinear mRNA transcript derived from influenza C virus RNA segment 6. Splicing of the colinear transcript introduces a translational stop codon into the ORF and the spliced mRNA encodes the viral matrix protein (CM1) (242 aa). The mechanism of CM2 translation was investigated by usingin vitroandin vivotranslation of RNA transcripts. It was found that the colinear mRNA derived from influenza C virus RNA segment 6 serves as the mRNA for CM2. Furthermore, CM2 translation does not depend on any of the three in-frame methionine residues located at the beginning of CM2 ORF. Rather, CM2 is a proteolytic cleavage product of the p42 protein product encoded by the colinear mRNA: a cleavage event that involves the recognition and cleavage of an internal signal peptide presumably by signal peptidase resident in the endoplasmic reticulum. Alteration of the predicted signal peptidase cleavage site by mutagenesis blocked generation of CM2. The other polypeptide species resulting from the cleavage of p42, designated p31, contains the CM1 coding region and an additional C-terminal 17 aa (formerly the CM2 signal peptide). Protein p31, in comparison to CM1, displays characteristics of an integral membrane protein.
The cyclin-dependent kinase inhibitor p27
Kip1
induces N-terminal proteolytic cleavage of cyclin A
Authors: Bastians Holger , Townsley Fiona M. , Ruderman Joan V.
Published: July 2002

Progression through the cell cycle is regulated in part by the sequential activation and inactivation of cyclin-dependent kinases (CDKs). Many signals arrest the cell cycle through inhibition of CDKs by CDK inhibitors (CKIs). p27
Kip1
(p27) was first identified as a CKI that binds and inhibits cyclin A/CDK2 and cyclin E/CDK2 complexes in G
1
. Here we report that p27 has an additional property, the ability to induce a proteolytic activity that cleaves cyclin A, yielding a truncated cyclin A lacking the mitotic destruction box. Other CKIs (p15
Ink4b
, p16
Ink4a
, p21
Cip1
, and p57
Kip2
) do not induce cleavage of cyclin A; other cyclins (cyclin B, D1, and E) are not cleaved by the p27-induced protease activity. The C-terminal half of p27, which is dispensable for its kinase inhibitory activity, is required to induce cleavage. Mechanistically, p27 does not appear to cause cleavage through direct interaction with cyclin/CDK complexes. Instead, it activates a latent protease that, once activated, does not require the continuing presence of p27. Mutation of cyclin A at R70 or R71, residues at or very close to the cleavage site, blocks cleavage. Noncleavable mutants are still recognized by the anaphase-promoting complex/cyclosome pathway responsible for ubiquitin-dependent proteolysis of mitotic cyclins, indicating that the p27-induced cleavage of cyclin A is part of a separate pathway. We refer to this protease as Tsap (p
T
wenty-
s
even-
a
ctivated
p
rotease).