bluuebunny/crossref_metadata_2025 huggingface dataset:
Chlorophyll
b
can serve as the major pigment in functional photosystem II complexes of cyanobacteria
Authors: Xu Hong , Vavilin Dmitrii , Vermaas Wim
Published: July 2002

An
Arabidopsis thaliana
chlorophyll(ide)
a
oxygenase gene (
cao
), which is responsible for chlorophyll
b
synthesis from chlorophyll
a
, was introduced and expressed in a photosystem I-less strain of the cyanobacterium
Synechocystis
sp
.
PCC 6803. In this strain, most chlorophyll is associated with the photosystem II complex. In line with observations by Satoh
et al.
[Satoh, S., Ikeuchi, M., Mimuro, M. & Tanaka, A. (2001)
J. Biol. Chem
. 276, 4293–4297], chlorophyll
b
was made but accounted for less than 10% of total chlorophyll. However, when
lhcb
encoding light-harvesting complex (LHC)II from pea was present in the same strain (
lhcb
+
/
cao
+
), chlorophyll
b
accumulated in the cell to levels exceeding those of chlorophyll
a
, although LHCII did not accumulate. In the
lhcb
+
/
cao
+
strain, the total amount of chlorophyll, the number of chlorophylls per photosystem II center, and the oxygen-evolving activity on a per-chlorophyll basis were similar to those in the photosystem I-less strain. Furthermore, the chlorophyll
a
/
b
ratio of photosystem II core particles (retaining CP47 and CP43) and of whole cells of the
lhcb
+
/
cao
+
strain was essentially identical, and PS II activity could be obtained efficiently by chlorophyll
b
excitation. These data indicate that chlorophyll
b
functionally substitutes for chlorophyll
a
in photosystem II. Therefore, the availability of chlorophylls, rather than their binding specificity, may determine which chlorophyll is incorporated at many positions of photosystem II. We propose that the transient presence of a LHCII/chlorophyll(ide)
a
oxygenase complex in the
lhcb
+
/
cao
+
strain leads to a high abundance of available chlorophyll
b
that is subsequently incorporated into photosystem II complexes. The apparent LHCII requirement for high chlorophyll(ide)
a
oxygenase activity may be instrumental to limit the occurrence of chlorophyll
b
in plants to LHC proteins.
Mapping the actin filament with myosin
Authors: Steffen Walter , Smith David , Simmons Robert , Sleep John
Published: July 2002

Structural studies have shown that the heads of the myosin motor
molecule bind preferentially to “target zones” of favorably
oriented sites on the helices of the actin filament. We present
direct evidence for target zones from the interactions of a single
myosin head with an actin filament held between two optically trapped
beads. With compliant traps, thermal motions of the filament allow the
fixed myosin-S1 to interact with at least two zones, observed as a
bi-modal distribution of filament displacements due to myosin binding,
whose spacing is near the 36-nm helix repeat distance. The number of
binding events and the “apparent working stroke” (mean
displacement with myosin bound), vary periodically as the filament is
moved past the fixed myosin by displacing the traps; observed periods
are close to 36 nm and the apparent stroke varies from 0–10 nm. We
also observe a strong modulation at the 5.5-nm actin monomer repeat
confirming that myosin interacts with a single strand and that the
actin is not free to rotate. Each interaction can be assigned to an
actin monomer and each active zone on the helix is made up of three
actin monomers.
Different responses of astrocytes and neurons to nitric oxide: The role of glycolytically generated ATP in astrocyte protection
Authors: Almeida Angeles , Almeida Julia , Bolaños Juan P. , Moncada Salvador
Published: July 2002

It was recently proposed that in Jurkat cells, after inhibition of respiration by NO, glycolytically generated ATP plays a critical role in preventing the collapse of mitochondrial membrane potential (Δψm) and thus apoptotic cell death. We have investigated this observation further in primary cultures of rat cortical neurons and astrocytes—cell types that differ greatly in their glycolytic capacity. Continuous and significant (≈85%) inhibition of respiration by NO (1.4 μM at 175 μM O2) generated by [(z)-1-[2-aminoethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2 diolate (DETA-NO) initially (10 min) depleted ATP concentrations by ≈25% in both cell types and increased the rate of glycolysis in astrocytes but not in neurons. Activation of glycolysis in astrocytes, as judged by lactate production, prevented further ATP depletion, whereas in neurons, which do not invoke this mechanism, there was a progressive decrease in ATP concentrations over the next 60 min. During this time, there was a persistent mitochondrial hyperpolarization and absence of apoptotic cell death in astrocytes, whereas in the neurons there was a progressive fall in Δψmand increased apoptosis. After glucose deprivation or treatment with inhibitors of the F1F0-ATPase and adenine nucleotide translocase, astrocytes responded to NO with a fall in Δψmand apoptotic cell death similar to the response in neurons. Finally, although treatment of astrocytes with NO partially prevented staurosporin-induced collapse in Δψmand cell death, NO and staurosporin synergized in decreasing Δψmand inducing apoptosis in neurons. These results demonstrate that although inhibition of cellular respiration by NO leads to neurotoxicity, it may also result in initial neuroprotection, depending on the glycolytic capacity of the particular cell.
Activation of the translational suppressor 4E-BP1 following infection with encephalomyocarditis virus and poliovirus.
Authors: Gingras A C , Svitkin Y , Belsham G J , Pause A , Sonenberg N
Published: July 2002

Infection of cells with picornaviruses, such as poliovirus and encephalomyocarditis virus (EMCV), causes a shutoff of host protein synthesis. The molecular mechanism of the shutoff has been partly elucidated for poliovirus but not for EMCV. Translation initiation in eukaryotes is facilitated by the mRNA 5' cap structure to which the multisubunit translation initiation factor eIF4F binds to promote ribosome binding. Picornaviruses use a mechanism for the translation of their RNA that is independent of the cap structure. Poliovirus infection engenders the cleavage of the eIF4G (formerly p220) component of eIF4F and renders this complex inactive for cap-dependent translation. In contrast, EMCV infection does not result in eIF4G cleavage. Here, we report that both EMCV and poliovirus activate a translational repressor, 4E-BP1, that inhibits cap-dependent translation by binding to the cap-binding subunit eIF4E. Binding of eIF4E occurs only to the underphosphorylated form of 4E-BP1, and this interaction is highly regulated in cells. We show that 4E-BP1 becomes dephosphorylated upon infection with both EMCV and poliovirus. Dephosphorylation of 4E-BP1 temporally coincides with the shutoff of protein synthesis by EMCV but lags behind the shutoff and eIF4G cleavage in poliovirus-infected cells. Dephosphorylation of 4E-BP1 by specifically inhibiting cap-dependent translation may be the major cause of the shutoff phenomenon in EMCV-infected cells.
Independent determination of symmetry and polarity in the Drosophila eye.
Authors: Choi K W , Mozer B , Benzer S
Published: July 2002

In each facet of the Drosophila compound eye, a cluster of photoreceptor cells assumes an asymmetric trapezoidal pattern. These clusters have opposite orientations above and below an equator, showing global dorsoventral mirror symmetry. However, in the mutant spiny legs, the polarization of each cluster appears to be random, so that no equator is evident. The apparent lack of an equator suggests that spiny legs+ may be involved in the establishment of global dorsoventral identity that might be essential for proper polarization of the photoreceptor clusters. Alternatively, a global dorsoventral pattern could be present, but spiny legs+ may be required for local polarization of individual clusters. Using an enhancer trap strain in which white+ gene expression is restricted to the dorsal field, we show that white+ expression in spiny legs correctly respects dorsoventral position even in facets with inappropriate polarizations; the dorsoventral boundary is indeed present, whereas the mechanism for polarization is perturbed. It is suggested that the boundary is established before the action of spiny legs+ by an independent mechanism.
Cell-type and promoter-context dependent retinoic acid receptor (RAR) redundancies for RAR beta 2 and Hoxa-1 activation in F9 and P19 cells can be artefactually generated by gene knockouts.
Authors: Taneja R , Roy B , Plassat J L , Zusi C F , Ostrowski J , Reczek P R , Chambon P
Published: July 2002

By using RAR type (alpha, beta, or gamma)-specific synthetic retinoids and a pan-retinoic X receptor (RXR)-specific ligand, we have investigated the contribution of RARs and RXRs in the activation of RA target genes and the differentiation of embryonal carcinoma cells. We demonstrate cell-type- and promoter context-dependent functional redundancies that differ between the three RAR types for mediating the induction of RARbeta2 and Hoxa-1 in wild-type, RARgamma-/- and RARalpha-/- F9 cells and in P19 cells. The extent of redundancy between RARs is further modulated by the synergistic activation of RXRs with a pan-RXR agonist. We also demonstrate that the expression of RARbeta2 is auto-inducible in RARgamma-/- but not in wild-type F9 cells, indicating that the functional redundancies observed between RARs in gene disruption studies can be artefactually generated. Thus, even though all three RARs can functionally substitute each other for inducing the expression of RA target genes and cell differentiation, one RAR can cell-specifically override the activity of the other RARs. Interestingly, only RARgamma can mediate the retinoic acid-induced differentiation of wild-type F9 cells, whereas the differentiation of P19 cells can be mediated by either RARalpha or RARgamma.
RETRACTED: ATP-dependent uptake of natural product cytotoxic drugs by membrane vesicles establishes MRP as a broad specificity transporter.
Authors: Paul S , Breuninger L M , Tew K D , Shen H , Kruh G D
Published: July 2002

MRP is a recently isolated ATP-binding cassette family transporter. We previously reported transfection studies that established that MRP confers multidrug resistance [Kruh, G. D., Chan, A., Myers, K., Gaughan, K., Miki, T. & Aaronson, S. A. (1994) Cancer Res. 54, 1649-1652] and that expression of MRP is associated with enhanced cellular efflux of lipophilic cytotoxic agents [Breuninger, L. M., Paul, S., Gaughan, K., Miki, T., Chan, A., Aaronson, S. A. & Kruh, G. D. (1995) Cancer Res. 55, 5342-5347]. To examine the biochemical mechanism by which MRP confers multidrug resistance, drug uptake experiments were performed using inside-out membrane vesicles prepared from NIH 3T3 cells transfected with an MRP expression vector. ATP-dependent transport was observed for several lipophilic cytotoxic agents including daunorubicin, etoposide, and vincristine, as well as for the glutathione conjugate leukotriene C4 (LTC4). However, only marginally increased uptake was observed for vinblastine and Taxol. Drug uptake was osmotically sensitive and saturable with regard to substrate concentration, with Km values of 6.3 microM, 4.4 microM, 4.2 microM, 35 nM, and 38 microM, for daunorubicin, etoposide, vincristine, LTC4, and ATP, respectively. The broad substrate specificity of MRP was confirmed by the observation that daunorubicin transport was competitively inhibited by reduced and oxidized glutathione, the glutathione conjugates S-(p-azidophenacyl)-glutathione (APA-SG) and S-(2,4-dinitrophenyl)glutathione (DNP-SG), arsenate, and the LTD4 antagonist MK571. This study establishes that MRP pumps unaltered lipophilic cytotoxic drugs, and suggests that this activity is an important mechanism by which the transporter confers multidrug resistance. The present study also indicates that the substrate specificity of MRP is overlapping but distinct from that of P-glycoprotein, and includes both the neutral or mildly cationic natural product cytotoxic drugs and the anionic products of glutathione conjugation. The widespread expression of MRP in tissues, combined with its ability to transport both lipophilic xenobiotics and the products of phase II detoxification, indicates that the transporter represents a widespread and remarkably versatile cellular defense mechanism.
Ciliary neurotrophic factor protects striatal output neurons in an animal model of Huntington disease.
Authors: Anderson K D , Panayotatos N , Corcoran T L , Lindsay R M , Wiegand S J
Published: July 2002

Huntington disease is a dominantly inherited, untreatable neurological disorder featuring a progressive loss of striatal output neurons that results in dyskinesia, cognitive decline, and, ultimately, death. Neurotrophic factors have recently been shown to be protective in several animal models of neurodegenerative disease, raising the possibility that such substances might also sustain the survival of compromised striatal output neurons. We determined whether intracerebral administration of brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3, or ciliary neurotrophic factor could protect striatal output neurons in a rodent model of Huntington disease. Whereas treatment with brain-derived neurotrophic factor, nerve growth factor, or neurotrophin-3 provided no protection of striatal output neurons from death induced by intrastriatal injection of quinolinic acid, an N-methyl-D-aspartate glutamate receptor agonist, treatment with ciliary neurotrophic factor afforded marked protection against this neurodegenerative insult.
Deletion of the mouse T-cell receptor beta gene enhancer blocks alphabeta T-cell development.
Authors: Bouvier G , Watrin F , Naspetti M , Verthuy C , Naquet P , Ferrier P
Published: July 2002

Intrathymic T-cell development requires temporally regulated rearrangement and expression of T-cell receptor (TCR) genes. To assess the role of the TCR beta gene transcriptional enhancer (Ebeta) in this process, mouse strains in which Ebeta is deleted were generated using homologous recombination techniques. We report that mice homozygous for the Ebeta deletion, whether a selectable marker gene is present or not, show a block in alphabeta T-cell development at the CD4-CD8- double-negative cell stage, whereas the number of gammadelta+ T cells is normal, few CD4+CD8+ double-positive thymocytes and no alphabeta+ T cells are produced. DNA-PCR and RNA-PCR analyses of thymic cells from homozygous mutants showed no evidence of TCR beta gene rearrangement although germ-line Vbeta transcripts were detected at a low level, in heterozygous T cells, the targeted allele is not rearranged. Thus, deletion of Ebeta totally prevents rearrangement, but not transcription, of the targeted beta locus. These data formally establish the critical role played by Ebeta in cis-activation of the TCR beta locus for V(D)J recombination during alphabeta T-cell development.
Comparison of circumsporozoite proteins from avian and mammalian malarias: biological and phylogenetic implications.
Authors: McCutchan T F , Kissinger J C , Touray M G , Rogers M J , Li J , Sullivan M , Braga E M , Krettli A U , Miller L H
Published: July 2002

The circumsporozoite (CS) protein of malaria parasites (Plasmodium) covers the surface of sporozoites that invade hepatocytes in mammalian hosts and macrophages in avian hosts. CS genes have been characterized from many Plasmodium that infect mammals; two domains of the corresponding proteins, identified initially by their conservation (region I and region II), have been implicated in binding to hepatocytes. The CS gene from the avian parasite Plasmodium gallinaceum was characterized to compare these functional domains to those of mammalian Plasmodium and for the study of Plasmodium evolution. The P. gallinaceum protein has the characteristics of CS proteins, including a secretory signal sequence, central repeat region, regions of charged amino acids, and an anchor sequence. Comparison with CS signal sequences reveals four distinct groupings, with P. gallinaceum most closely related to the human malaria Plasmodium falciparum. The 5-amino acid sequence designated region I, which is identical in all mammalian CS and implicated in hepatocyte invasion, is different in the avian protein. The P. gallinaceum repeat region consists of 9-amino acid repeats with the consensus sequence QP(A/V)GGNGG(A/V). The conserved motif designated region II-plus, which is associated with targeting the invasion of liver cells, is also conserved in the avian protein. Phylogenetic analysis of the aligned Plasmodium CS sequences yields a tree with a topology similar to the one obtained using sequence data from the small subunit rRNA gene. The phylogeny using the CS gene supports the proposal that the human malaria P. falciparum is significantly more related to avian parasites than to other parasites infecting mammals, although the biology of sporozoite invasion is different between the avian and mammalian species.
Redox transitions between oxygen intermediates in cytochrome-c oxidase.
Authors: Verkhovsky M I , Morgan J E , Wikström M
Published: July 2002

Some intermediates in the reduction of O2 to water by cytochrome-c oxidase have been characterized by optical, Raman, and magnetic circular dichroism spectroscopy. The so-called "peroxy" (P) and "ferryl" (F) forms of the enzyme, which have been considered to be intermediates of the oxygen reaction, can be generated when the oxidized enzyme reacts with H2O2, or when the two-electron reduced ("CO mixed-valence") enzyme reacts with O2. The structures as well as the overall redox states of P and F have recently been controversial. We show here, using tris(2,2'-bipyridyl)ruthenium(II) as a photoinducible reductant, that one-electron reduction of P yields F, and that one-electron reduction of F yields the oxidized enzyme. This confirms that the overall redox states of P and F differ from the oxidized enzyme by two and one electron equivalents, respectively. The structures of the P and F states are discussed.
Lipopolysaccharide and interleukin 1 augment the effects of hypoxia and inflammation in human pulmonary arterial tissue.
Authors: Ziesche R , Petkov V , Williams J , Zakeri S M , Mosgöller W , Knöfler M , Block L H
Published: July 2002

The combined effects of hypoxia and interleukin 1, lipopolysaccharide, or tumor necrosis factor alpha on the expression of genes encoding endothelial constitutive and inducible nitric oxide synthases, endothelin 1, interleukin 6, and interleukin 8 were investigated in human primary pulmonary endothelial cells and whole pulmonary artery organoid cultures. Hypoxia decreased the expression of constitutive endothelial nitric oxide synthase (NOS-3) mRNA and NOS-3 protein as compared with normoxic conditions. The inhibition of expression of NOS-3 corresponded with a reduced production of NO. A combination of hypoxia with bacterial lipopolysaccharide, interleukin 1 beta, or tumor necrosis factor alpha augmented both effects. In contrast, the combination of hypoxia and the inflammatory mediators superinduced the expression of endothelin 1, interleukin 6, and interleukin 8. Here, we have shown that inflammatory mediators aggravate the effect of hypoxia on the down-regulation of NOS-3 and increase the expression of proinflammatory cytokines in human pulmonary endothelial cells and whole pulmonary artery organoid cultures.
Three-dimensional structure of human electron transfer flavoprotein to 2.1-Å resolution
Authors: Roberts David L. , Frerman Frank E. , Kim Jung-Ja P.
Published: July 2002

Mammalian electron transfer flavoproteins (ETF) are heterodimers
containing a single equivalent of flavin adenine dinucleotide (FAD).
They function as electron shuttles between primary flavoprotein
dehydrogenases involved in mitochondrial fatty acid and amino acid
catabolism and the membrane-bound electron transfer flavoprotein
ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-Å
resolution reveals that the ETF molecule is comprised of three distinct
domains: two domains are contributed by the α subunit and the third
domain is made up entirely by the β subunit. The N-terminal portion
of the α subunit and the majority of the β subunit have identical
polypeptide folds, in the absence of any sequence homology. FAD lies in
a cleft between the two subunits, with most of the FAD molecule
residing in the C-terminal portion of the α subunit. Alignment of all
the known sequences for the ETF α subunits together with the putative
FixB gene product shows that the residues directly involved in FAD
binding are conserved. A hydrogen bond is formed between the N5 of the
FAD isoalloxazine ring and the hydroxyl side chain of αT266,
suggesting why the pathogenic mutation, αT266M, affects ETF activity
in patients with glutaric acidemia type II. Hydrogen bonds between the
4′-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine
ring, and between αH286 and the C2-carbonyl oxygen of the
isoalloxazine ring, may play a role in the stabilization of the anionic
semiquinone. With the known structure of medium chain acyl-CoA
dehydrogenase, we hypothesize a possible structure for docking the two
proteins.
Identification of surface residues mediating tissue factor binding and catalytic function of the serine protease factor VIIa
Authors: Dickinson Craig D. , Kelly Curtis R. , Ruf Wolfram
Published: July 2002

Factor VIIa (VIIa), the serine protease that initiates the
coagulation pathways, is catalytically activated upon binding to its
cell surface receptor and cofactor tissue factor (TF). This study
provides a comprehensive analysis of the functional surface of VIIa by
alanine scanning mutagenesis of 112 residues. Residue side chains were
defined which contribute to TF binding and factor X hydrolysis.
Energetically important binding contacts at the interface with TF were
identified in the first epidermal growth factor domain of VIIa (Gln-64,
Ile-69, Phe-71, Arg-79) and in the protease domain (Arg-277, Met-306,
Asp-309). The observed energetic defects are in good agreement with the
corresponding residues in TF, suggesting that the VIIa light chain
plays a prominent role in high affinity binding of cofactor. Mutation
of protease domain interface residues indicated that TF allosterically
influences the active site of VIIa. Stabilization of a labile zymogen
to enzyme transition could explain the activating effect of TF on VIIa
catalytic function. Residues important for factor X hydrolysis were
found in three regions of the protease domain: (
i
)
specificity determinants in the catalytic cleft and adjacent loops,
(
ii
) an exosite near the TF binding site, and
(
iii
) a large electronegative exosite which is in a
position analogous to the basic exosite I of thrombin. TF regions
involved in factor X activation are positioned on the same face of the
TF·VIIa complex as the two exosites identified on the protease
domain surface, providing evidence for an extended interaction of
TF·VIIa with macromolecular substrate.
Distinct ligand preferences of Src homology 3 domains from Src, Yes, Abl, Cortactin, p53bp2, PLCgamma, Crk, and Grb2.
Authors: Sparks A B , Rider J E , Hoffman N G , Fowlkes D M , Quillam L A , Kay B K
Published: July 2002

Src homology 3 (SH3) domains are conserved protein modules 50-70 amino acids long found in a variety of proteins with important roles in signal transduction. These domains have been shown to mediate protein-protein interactions by binding short proline-rich regions in ligand proteins. However, the ligand preferences of most SH3 domains and the role of these preferences in regulating SH3-mediated protein-protein interactions remain poorly defined. We have used a phage-displayed library of peptides of the form X6PXXPX6 to identify ligands for eight different SH3 domains. Using this approach, we have determined that each SH3 domain prefers peptide ligands with distinct sequence characteristics. Specifically, we have found that the Src SH3 domain selects peptides sharing the consensus motif LXXRPLPXpsiP, whereas Yes SH3 selects psiXXRPLPXLP, Abl SH3 selects PPXthetaXPPPpsiP, Cortactin SH3 selects +PPpsiPXKPXWL, p53bp2 SH3 selects RPXpsiPpsiR+SXP, PLCgamma SH3 selects PPVPPRPXXTL, Crk N-terminal SH3 selects psiPpsiLPpsiK, and Grb2 N-terminal SH3 selects +thetaDXPLPXLP (where psi, theta, and + represent aliphatic, aromatic, and basic residues, respectively). Furthermore, we have compared the binding of phage expressing peptides related to each consensus motif to a panel of 12 SH3 domains. Results from these experiments support the ligand preferences identified in the peptide library screen and evince the ability of SH3 domains to discern subtle differences in the primary structure of potential ligands. Finally, we have found that most known SH3-binding proteins contain proline-rich regions conforming to the ligand preferences of their respective SH3 targets.
Transgenic studies with a keratin promoter-driven growth hormone transgene: Prospects for gene therapy
Authors: Wang Xiaoming , Zinkel Sandra , Polonsky Kenneth , Fuchs Elaine
Published: July 2002

Keratinocytes are potentially appealing vehicles for the delivery of secreted gene products because they can be transferred to human skin by the relatively simple procedure of grafting. Adult human keratinocytes can be efficiently propagated in culture with sufficient proliferative capacity to produce enough epidermis to cover the body surface of an average adult. However, the feasibility of delivering secreted proteins through skin grafting rests upon (i) the strength of the promoter in keratinocytes and (ii) the efficiency of protein transport through the basement membrane of the stratified epithelium and into the bloodstream. In this paper, we use transgenic technology to demonstrate that the activity of the human keratin 14 promoter remains high in adult skin and that keratinocyte-derived human growth hormone (hGH) can be produced, secreted, and transported to the bloodstream of mice with efficiency that is sufficient to exceed by an order of magnitude the circulating hGH concentration in growing children. Transgenic skin grafts from these adults continue to produce and secrete hGH stably, at ≈1/10 physiological levels in the bloodstream of nontransgenic recipient mice. These studies underscore the utility of the keratin 14 promoter for expressing foreign transgenes in keratinocytes and demonstrate that keratinocytes can be used as effective vehicles for transporting factors to the bloodstream and for eliciting metabolic changes. These findings have important implications for considering the keratinocyte as a possible vehicle for gene therapy.
The amino terminus of JAK3 is necessary and sufficient for binding to the common γ chain and confers the ability to transmit interleukin 2-mediated signals
Authors: Chen Min , Cheng Alan , Chen Yi-Qing , Hymel Anka , Hanson Eric P. , Kimmel Lida , Minami Yasuhiro , Taniguchi Tadatsugu , Changelian Paul S. , O’Shea John J.
Published: July 2002

JAK3 is a protein tyrosine kinase that specifically associates with the common γ chain (γc), a shared subunit of receptors for interleukin (IL) 2, 4, 7, 9, and 15. Patients deficient in either JAK3 or γcpresented with virtually identical forms of severe combined immunodeficiency (SCID), underscoring the importance of the JAK3–γcinteraction. Despite the key roles of JAK3 and γcin lymphocytic development and function, the molecular basis of this interaction remains poorly understood. In this study, we have characterized the regions of JAK3 involved in γcassociation. By developing a number of chimeric JAK3–JAK2 constructs, we show that the binding specificity to γccan be conferred to JAK2 by transferring the N-terminal domains of JAK3. Moreover, those JAK3–JAK2 chimeras capable of binding γcwere also capable of reconstituting IL-2 signaling as measured by inducible phosphorylation of the chimeric JAK3–JAK2 protein, JAK1, the IL-2 receptor β chain, and signal transducer and activator of transcription 5A. Subsequent deletion analyses of JAK3 have identified the N-terminal JH7-6 domains as a minimal region sufficient for γcassociation. Furthermore, expression of the mutant containing only the JH7-6 domains effectively competed with full-length JAK3 for binding to γc. We conclude that the JH7-6 domains of JAK3 are necessary and sufficient for γcassociation. These studies offer clues toward a broader understanding of JAK-mediated cytokine signaling and may provide a target for the development of novel therapeutic modalities in immunologically mediated diseases.
Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages
Authors: Xia Yong , Zweier Jay L.
Published: July 2002

Superoxide (O2⨪) and nitric oxide (NO) act to kill invading microbes in phagocytes. In macrophages NO is synthesized by inducible nitric oxide synthase (iNOS, NOS 2) froml-arginine (l-Arg) and oxygen; however, O2⨪was thought to be produced mainly by NADPH oxidase. Electron paramagnetic resonance (EPR) spin trapping experiments performed in murine macrophages demonstrate a novel pathway of O2⨪generation. It was observed that depletion of cytosolicl-Arg triggers O2⨪generation from iNOS. This iNOS-mediated O2⨪generation was blocked by the NOS inhibitorN-nitro-l-arginine methyl ester or byl-Arg, but not by the noninhibitory enantiomerN-nitro-d-arginine methyl ester. Inl-Arg-depleted macrophages iNOS generates both O2⨪and NO that interact to form the potent oxidant peroxynitrite (ONOO−), which was detected by luminol luminescence and whose formation was blocked by superoxide dismutase, urate, orl-Arg. This iNOS-derived ONOO−resulted in nitrotyrosine formation, and this was inhibited by iNOS blockade. iNOS-mediated O2⨪and ONOO−increased the antibacterial activity of macrophages. Thus, with reducedl-Arg availability iNOS produces O2⨪and ONOO−that modulate macrophage function. Due to the existence ofl-Arg depletion in inflammation, iNOS-mediated O2⨪and ONOO−may occur and contribute to cytostatic/cytotoxic actions of macrophages.
Transcription initiation is controlled by three core promoter elements in the
hgl5
gene of the protozoan parasite
Entamoeba histolytica
Authors: Singh Upinder , Rogers Joshua B. , Mann Barbara J. , Petri William A.
Published: July 2002

Entamoeba histolytica
is a single cell eukaryote that is the etiologic agent of amoebic colitis. Core promoter elements of
E. histolytica
protein encoding genes include a TATA-like sequence (GTATTTAAA
G
/
C
) at −30, a novel element designated GAAC (GAACT) that has a variable location between TATA and the site of transcription initiation, and a putative initiator (Inr) element (AAAAATTCA) overlying the site of transcription initiation. The presence of three separate conserved sequences in a eukaryotic core promoter is unprecedented and prompted examination of their roles in regulating transcription initiation. Alterations of all three regions in the
hgl5
gene decreased reporter gene activity with the greatest effect seen by mutation of the GAAC element. Positional analysis of the TATA box demonstrated that transcription initiated consistently 30–31 bases downstream of the TATA region. Mutation of either the TATA or GAAC elements resulted in the appearance of new transcription start sites upstream of +1 in the promoter of the
hgl5
gene. Mutation of the Inr element resulted in no change in the site of transcription initiation; however, in the presence of a mutated TATA and GAAC regions, the Inr element controlled the site of transcription initiation. We conclude that all three elements play a role in determining the site of transcription initiation. The variable position of the GAAC element relative to the site of transcription initiation, and the multiple transcription initiations that resulted from its mutation, indicate that the GAAC element has an important and apparently novel role in transcriptional control in
E. histolytica
.
Role of
Saccharomyces cerevisiae
Msh2 and Msh3 repair proteins in double-strand break-induced recombination
Authors: Sugawara Neal , Pâques Frédéric , Colaiácovo Mónica , Haber James E.
Published: July 2002

When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In
Saccharomyces cerevisiae
, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1/Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1/Rad10 and Msh2/Msh3, but reveals a difference in their roles. When the flanking homologous regions that anneal are 205 bp, the requirement for Msh2/Msh3 is as great as for Rad1/Rad10; but when the annealing partners are 1,170 bp, Msh2/Msh3 have little effect, while Rad1/Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3′ tail.
Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA
Authors: Li Hsin-Pai , Zhang Xuming , Duncan Roger , Comai Lucio , Lai Michael M. C.
Published: July 2002

A cellular protein, previously described as p35/38, binds to the complementary (−)-strand of the leader RNA and intergenic (IG) sequence of mouse hepatitis virus (MHV) RNA. The extent of the binding of this protein to IG sites correlates with the efficiency of the subgenomic mRNA transcription from that IG site, suggesting that it is a requisite transcription factor. We have purified this protein and determined by partial peptide sequencing that it is heterogeneous nuclear ribonucleoprotein (hnRNP) A1, an abundant, primarily nuclear protein. hnRNP A1 shuttles between the nucleus and cytoplasm and plays a role in the regulation of alternative RNA splicing. The MHV(−)-strand leader and IG sequences conform to the consensus binding motifs of hnRNP A1. Recombinant hnRNP A1 bound to these two RNA regions
in vitro
in a sequence-specific manner. During MHV infection, hnRNP A1 relocalizes from the nucleus to the cytoplasm, where viral replication occurs. These data suggest that hnRNP A1 is a cellular factor that regulates the RNA-dependent RNA transcription of the virus.
Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress
Authors: Yakes F. Michael , Van Houten Bennett
Published: July 2002

A significant amount of reactive oxygen species (ROS) is generated during mitochondrial oxidative phosphorylation. Several studies have suggested that mtDNA may accumulate more oxidative DNA damage relative to nuclear DNA. This study used quantitative PCR to examine the formation and repair of hydrogen peroxide-induced DNA damage in a 16.2-kb mitochondrial fragment and a 17.7-kb fragment flanking the β-globin gene. Simian virus 40-transformed fibroblasts treated with 200 μM hydrogen peroxide for 15 or 60 min exhibited 3-fold more damage to the mitochondrial genome compared with the nuclear fragment. Following a 60-min treatment, damage to the nuclear fragment was completely repaired within 1.5 hr, whereas no DNA repair in the mitochondrion was observed. Mitochondrial function, as assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, also showed a sharp decline. These cells displayed arrested-cell growth, large increases in p21 protein levels, and morphological changes consistent with apoptosis. In contrast, when hydrogen peroxide treatments were limited to 15 min, mtDNA damage was repaired with similar kinetics as the nuclear fragment, mitochondrial function was restored, and cells resumed division within 12 hr. These results indicate that mtDNA is a critical cellular target for ROS. A model is presented in which chronic ROS exposure, found in several degenerative diseases associated with aging, leads to decreased mitochondrial function, increased mitochondrial-generated ROS, and persistent mitochondrial DNA damage. Thus persistent mitochondrial DNA damage may serve as a useful biomarker for ROS-associated diseases.
Myelin basic protein kinase activity in tomato leaves is induced systemically by wounding and increases in response to systemin and oligosaccharide elicitors
Authors: Stratmann Johannes W. , Ryan Clarence A.
Published: July 2002

In response to wounding, a 48-kDa myelin basic protein (MBP) kinase is activated within 2 min, both locally and systemically, in leaves of young tomato plants. The activating signal is able to pass through a steam girdle on the stem, indicating that it moves through the xylem and does not require intact phloem tissue. A 48-kDa MBP kinase is also activated by the 18-amino acid polypeptide systemin, a potent wound signal for the synthesis of systemic wound response proteins (swrps). The kinase activation by systemin is strongly inhibited by a systemin analog having a Thr-17 → Ala-17 substitution, which is a powerful antagonist of systemin activation of swrp genes. A 48-kDa MBP kinase activity also increases in response to polygalacturonic acid and chitosan but not in response to jasmonic acid or phytodienoic acid. In
def1
, a mutant tomato line having a defective octadecanoid pathway, the 48-kDa MBP kinase is activated by wounding and systemin as in the wild-type plants. This indicates that MBP kinase functions between the perception of primary signals and the
DEF1
gene product. In response to wounding, the MBP kinase is phosphorylated on phosphotyrosine residues, indicating a relationship to the mitogen-activated protein kinase family of protein kinases.
Direct evidence to support the role of HLA-G in protecting the fetus from maternal uterine natural killer cytolysis
Authors: Rouas-Freiss Nathalie , Gonçalves Rachel Marchal-Bras , Menier Catherine , Dausset Jean , Carosella Edgardo D.
Published: July 2002

HLA-G is a nonclassical major histocompatibility complex class I molecule selectively expressed on cytotrophoblasts at the feto–maternal interface, where it may play an important role in maternal tolerance of the fetus. We provide direct evidence under physiological conditions that supports the role of HLA-G in protecting cytotrophoblasts against natural killer (NK) cytolysis in 6 semiallogenic combinations of maternal uterine NK cells and their own trophoblast counterparts, as well as in 20 allogenic combinations of maternal uterine NK cells and trophoblasts from different mothers. We show that, in all cases studied, this HLA-G-mediated protection was abolished by treatment of cytotrophoblasts with an HLA-G-specific mAb. The HLA class I-negative K562 cell line transfected with the predominant HLA-G1 isoform results in similar protection and abolition from maternal uterine NK lysis. Because maternal uterine NK cells express killer inhibitory receptors for HLA-G, we conclude that their interactions contribute to the survival of the fetal semiallograft by confering immunological tolerance to its tissues.
Alternative genetic pathways in colorectal carcinogenesis
Authors: Olschwang Sylviane , Hamelin Richard , Laurent-Puig Pierre , Thuille Bénédicte , De Rycke Yann , Li You-Jun , Muzeau Françoise , Girodet Jacques , Salmon Rémy-Jacques , Thomas Gilles
Published: July 2002

The comparative typing of matched tumor and blood DNAs at dinucleotide repeat (microsatellite) loci has revealed in tumor DNA the presence of alleles that are not observed in normal DNA. The occurrence of these additional alleles is possibly due to replication errors (RERs). Although this observation has led to the recognition of a subtype of colorectal cancer with a high incidence of RERs (caused by a deficiency in DNA mismatch repair), a thorough analysis of the RER frequency in a consecutive series of colorectal cancers had not been reported. It is shown here that the extensive typing of 88 colorectal tumors reveals a bimodal distribution for the frequency of RER at microsatellite loci. Within the major mode (75 tumors, RER− subtype), the probability that a locus exhibited instability did not differ significantly among loci and tumors, being 0.02. The subsequent development of a statistical test for an operational discrimination between the RER− and RER+ subtypes indicated that the probability of misclassification did not exceed 0.001 in this series. The frequency of K-
ras
mutation was found to be equivalent in the two subtypes. However, in the RER+ tumors, the
p53
gene mutation was less frequently detected, the adenomatous polyposis coli (
APC
) mutation was rare, and the biallelic inactivation of either of these genes was not observed. Furthermore, the concomitant occurrence of
APC
and tumor growth factor β receptor type II gene alterations was found only once. These data suggest that the repertoires of genes that are frequently altered in RER+ and RER− tumors may be more different than previously thought.
Genetic control of abscisic acid biosynthesis in maize
Authors: Tan Bao Cai , Schwartz Steven H. , Zeevaart Jan A. D. , McCarty Donald R.
Published: July 2002

Abscisic acid (ABA), an apocarotenoid synthesized from cleavage of carotenoids, regulates seed maturation and stress responses in plants. The viviparous seed mutants of maize identify genes involved in synthesis and perception of ABA. Two alleles of a new mutant,viviparous14 (vp14),were identified by transposon mutagenesis. Mutant embryos had normal sensitivity to ABA, and detached leaves of mutant seedlings showed markedly higher rates of water loss than those of wild type. The ABA content of developing mutant embryos was 70% lower than that of wild type, indicating a defect in ABA biosynthesis.vp14 embryos were not deficient in epoxy-carotenoids, and extracts ofvp14embryos efficiently converted the carotenoid cleavage product, xanthoxin, to ABA, suggesting a lesion in the cleavage reaction.vp14was cloned by transposon tagging. The VP14 protein sequence is similar to bacterial lignostilbene dioxygenases (LSD). LSD catalyzes a double-bond cleavage reaction that is closely analogous to the carotenoid cleavage reaction of ABA biosynthesis. Southern blots indicated a family of four to six related genes in maize. TheVp14mRNA is expressed in embryos and roots and is strongly induced in leaves by water stress. A family ofVp14-related genes evidently controls the first committed step of ABA biosynthesis. These genes are likely to play a key role in the developmental and environmental control of ABA synthesis in plants.
Acoustic and neural bases for innate recognition of song
Authors: Whaling C. S. , Solis M. M. , Doupe A. J. , Soha J. A. , Marler P.
Published: July 2002

In behavior reminiscent of the responsiveness of human infants to speech, young songbirds innately recognize and prefer to learn the songs of their own species. The acoustic and physiological bases for innate recognition were investigated in fledgling white-crowned sparrows lacking song experience. A behavioral test revealed that the complete conspecific song was not essential for innate recognition: songs composed of single white-crowned sparrow phrases and songs played in reverse elicited vocal responses as strongly as did normal song. In all cases, these responses surpassed those to other species’ songs. Although auditory neurons in the song nucleus HVc and the underlying neostriatum of fledglings did not prefer conspecific song over foreign song, some neurons responded strongly to particular phrase types characteristic of white-crowned sparrows and, thus, could contribute to innate song recognition.
Identification of specific Rp-phosphate oxygens in the tRNA anticodon loop required for ribosomal P-site binding
Authors: Schnitzer Wolfgang , von Ahsen Uwe
Published: July 2002

tRNA binding to the ribosomal P site is dependent not only on correct codon–anticodon interaction but also involves identification of structural elements of tRNA by the ribosome. By using a phosphorothioate substitution–interference approach, we identified specific nonbridging Rp-phosphate oxygens in the anticodon loop of tRNA
Phe
from
Escherichia coli
which are required for P-site binding. Stereo-specific involvement of phosphate oxygens at these positions was confirmed by using synthetic anticodon arm analogues at which single Rp- or Sp-phosphorothioates were incorporated. Identical interference results with yeast tRNA
Phe
and
E. coli
tRNA
f
Met
indicate a common backbone conformation or common recognition elements in the anticodon loop of tRNAs.
N-
ethyl-
N
-nitrosourea modification–interference experiments with natural tRNAs point to the importance of the same phosphates in the loop. Guided by the crystal structure of tRNA
Phe
, we propose that specific Rp-phosphate oxygens are required for anticodon loop (“U-turn”) stabilization or are involved in interactions with the ribosome on correct tRNA–mRNA complex formation.
Use of model plant hosts to identify
Pseudomonas aeruginosa
virulence factors
Authors: Rahme Laurence G. , Tan Man-Wah , Le Long , Wong Sandy M. , Tompkins Ronald G. , Calderwood Stephen B. , Ausubel Frederick M.
Published: July 2002

We used plants as an
in vivo
pathogenesis model for the identification of virulence factors of the human opportunistic pathogen
Pseudomonas aeruginosa
. Nine of nine Tn
phoA
mutant derivatives of
P. aeruginosa
strain UCBPP-PA14 that were identified in a plant leaf assay for less pathogenic mutants also exhibited significantly reduced pathogenicity in a burned mouse pathogenicity model, suggesting that
P. aeruginosa
utilizes common strategies to infect both hosts. Seven of these nine mutants contain Tn
phoA
insertions in previously unknown genes. These results demonstrate that an alternative nonvertebrate host of a human bacterial pathogen can be used in an
in vivo
high throughput screen to identify novel bacterial virulence factors involved in mammalian pathogenesis.
Synthesis and characterization of a novel retinylamine analog inhibitor of constitutively active rhodopsin mutants found in patients with autosomal dominant retinitis pigmentosa
Authors: Yang Tong , Snider Barry B. , Oprian Daniel D.
Published: July 2002

Two different mutations of the active-site Lys-296 in rhodopsin, K296E and K296M, have been found to cause autosomal dominant retinitis pigmentosa (ADRP).
In vitro
studies have shown that both mutations result in constitutive activation of the protein, suggesting that the activated state of the receptor may be responsible for retinal degeneration in patients with these mutations. Previous work has highlighted the potential of retinylamine analogs as active-site directed inactivators of constitutively active mutants of rhodopsin with the idea that these or related compounds might be used therapeutically for cases of ADRP involving mutations of the active-site Lys. Unfortunately, however, amine derivatives of 11-
cis
-retinal, although highly effective against a K296G mutant of rhodopsin, were without affect on the two naturally occurring ADRP mutants, presumably because of the greater steric bulk of Glu and Met side chains in comparison to Gly. For this reason we synthesized a retinylamine analog one carbon shorter than the parent 11-
cis
-retinal and show that this compound is indeed an effective inhibitor of both the K296E and K296M mutants. The 11-
cis
C19 retinylamine analog 1 inhibits constitutive activation of transducin by these mutants and their constitutive phosphorylation by rhodopsin kinase, and it does so in the presence of continuous illumination from room lights.