bluuebunny/crossref_metadata_2025 huggingface dataset:
The catalytic domain of λ site-specific recombinase
Authors: Tirumalai Radhakrishna S. , Healey Erica , Landy Arthur
Published: July 2002

The
Escherichia coli
phage λ integrase protein (Int) belongs to the large Int family of site-specific recombinases. It is a heterobivalent DNA binding protein that makes use of a high energy covalent phosphotyrosine intermediate to catalyze integrative and excisive recombination at specific chromosomal sites (
att
sites). A 293-amino acid carboxy-terminal fragment of Int (C65) has been cloned, characterized, and used to further dissect the protein. From this we have cloned and characterized a 188-amino acid, protease-resistant, carboxy-terminal fragment (C170) that we believe is the minimal catalytically competent domain of Int. C170 has topoisomerase activity and converts
att
suicide substrates to the covalent phosphotyrosine complexes characteristic of recombination intermediates. However, it does not show efficient binding to
att
site DNA in a native gel shift assay. We propose that λ Int consists of three functional and structural domains: residues 1–64 specify recognition of “arm-type” DNA sequences distant from the region of strand exchange; residues 65–169 contribute to specific recognition of “core-type” sequences at the sites of strand exchange and possibly to protein–protein interactions; and residues 170–356 carry out the chemistry of DNA cleavage and ligation. The finding that the active site nucleophile Tyr-342 is in a uniquely protease-sensitive region complements and reinforces the recently solved C170 crystal structure, which places Tyr-342 at the center of a 17-amino acid flexible loop. It is proposed that C170 is likely to represent a generic Int family domain that thus affords a specific route to studying the chemistry of DNA cleavage and ligation in these recombinases.
Posttranscriptional modification of retroviral primers is required for late stages of DNA replication
Authors: Burnett Bruce P. , McHenry Charles S.
Published: July 2002

During reverse transcription of retroviral RNA, synthesis of (−) strand DNA is primed by a cellular tRNA that anneals to an 18-nt primer binding site within the 5′ long terminal repeat. For (+) strand synthesis using a (−) strand DNA template linked to the tRNA primer, only the first 18 nt of tRNA are replicated to regenerate the primer binding site, creating the (+) strand strong stop DNA intermediate and providing a 3′ terminus capable of strand transfer and further elongation. On model HIV templates that approximate the (−) strand linked to natural modified or synthetic unmodified tRNA
3
Lys
, we find that a (+) strand strong stop intermediate of the proper length is generated only on templates containing the natural, modified tRNA
3
Lys
, suggesting that a posttranscriptional modification provides the termination signal. In the presence of a recipient template, synthesis after strand transfer occurs only from intermediates generated from templates containing modified tRNA
3
Lys
. Reverse transcriptase from Moloney murine leukemia virus and avian myoblastosis virus shows the same requirement for a modified tRNA
3
Lys
template. Because all retroviral tRNA primers contain the same 1-methyl-A
58
modification, our results suggest that 1-methyl-A
58
is generally required for termination of replication 18 nt into the tRNA sequence, generating the (+) strand intermediate, strand transfer, and subsequent synthesis of the entire (+) strand. The possibility that the host methyl transferase responsible for methylating A
58
may provide a target for HIV chemotherapy is discussed.
Specific cleavage of chromosomal and plasmid DNA strands in gram-positive and gram-negative bacteria can be detected with nucleotide resolution
Authors: Zechner Ellen L. , Prüger Hermann , Grohmann Elisabeth , Espinosa Manuel , Högenauer Gregor
Published: July 2002

A sensitive and precise
in vitro
technique for detecting DNA strand discontinuities produced
in vivo
has been developed. The procedure, a form of runoff DNA synthesis on molecules released from lysed bacterial cells, mapped precisely the position of cleavage of the plasmid pMV158 leading strand origin in
Streptococcus pneumoniae
and the site of strand scission,
nic
, at the transfer origins of F and the F-like plasmid R1 in
Escherichia coli
. When high frequency of recombination strains of
E. coli
were examined, DNA strand discontinuities at the
nic
positions of the chromosomally integrated fertility factors were also observed. Detection of DNA strand scission at the
nic
position of F DNA in the high frequency of recombination strains, as well as in the episomal factors, was dependent on sexual expression from the transmissable element, but was independent of mating. These results imply that not only the transfer origins of extrachromosomal F and F-like fertility factors, but also the origins of stably integrated copies of these plasmids, are subject to an equilibrium of cleavage and ligation
in vivo
in the absence of DNA transfer.
Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells
Authors: Hiraoka Atsunobu , Sugimura Atsushi , Seki Tatsuya , Nagasawa Tetsuji , Ohta Naomi , Shimonishi Manabu , Hagiya Michio , Shimizu Shin
Published: July 2002

Multiple growth factors synergistically stimulate proliferation of primitive hematopoietic progenitor cells. A human myeloid cell line, KPB-M15, constitutively produces a novel hematopoietic cytokine, termed stem cell growth factor (SCGF), possessing species-specific proliferative activities. Here we report the molecular cloning, expression, and characterization of a cDNA encoding human SCGF using a newly developed λSHDM vector that is more efficient for differential and expression cloning. cDNA for SCGF encodes a 29-kDa polypeptide without N-linked glycosylation. SCGF transiently produced by COS-1 cells supports growth of hematopoietic progenitor cells through a short-term liquid culture of bone marrow cells and exhibits promoting activities on erythroid and granulocyte/macrophage progenitor cells in primary semisolid culture with erythropoietin and granulocyte/macrophage colony-stimulating factor, respectively. Expression of SCGF mRNA is restricted to myeloid cells and fibroblasts, suggesting that SCGF is a growth factor functioning within the hematopoietic microenvironment. SCGF could disclose some human-specific mechanisms as yet unidentified from studies on the murine hematopoietic system.
An upstream, DNase I hypersensitive region of the hematopoietic-expressed transcription factor GATA-1 gene confers developmental specificity in transgenic mice
Authors: McDevitt Michael A. , Fujiwara Yuko , Shivdasani Ramesh A. , Orkin Stuart H.
Published: July 2002

The transcription factor GATA-1, which is expressed in several hematopoietic lineages and multipotential progenitors, is required for the development of red blood cells and platelets. To identify control elements of the mouse
GATA-1
gene, we analyzed DNase I hypersensitivity of the locus in erythroid chromatin and the expression of
GATA-1
/
Escherichia coli
β-galactosidase (
lacZ
) transgenes in mice. Transgenes with 2.7 kb of promoter sequences are expressed infrequently and only within adult (definitive) erythroid cells. We show that inclusion of an upstream hypersensitive site (HS I) markedly enhances the frequency of expressing transgenic lines and activates expression in primitive erythroid cells. This pattern recapitulates the proper pattern of GATA-1 expression during development. By breeding a
GATA-1
/
lacZ
transgene into a GATA-1
−
background, we also have shown that the activation or maintenance of GATA-1 expression does not require the presence of GATA-1 itself, thereby excluding simple models of positive autoregulation. The transgene cassette reported here should be useful in directing expression of foreign sequences at the onset of hematopoiesis in the embryo and may assist in the identification of upstream regulators of the
GATA-1
gene.
Geochemistry of corals: Proxies of past ocean chemistry, ocean circulation, and climate
Authors: Druffel Ellen R. M.
Published: July 2002

This paper presents a discussion of the status of the field
of coral geochemistry as it relates to the recovery of past records of
ocean chemistry, ocean circulation, and climate. The first part is a
brief review of coral biology, density banding, and other important
factors involved in understanding corals as proxies of environmental
variables. The second part is a synthesis of the information available
to date on extracting records of the carbon cycle and climate change.
It is clear from these proxy records that decade time-scale variability
of mixing processes in the oceans is a dominant signal. That Western
and Eastern tropical Pacific El Niño-Southern Oscillation (ENSO)
records differ is an important piece of the puzzle for understanding
regional and global climate change. Input of anthropogenic
CO
2
to the oceans as observed by
13
C and
14
C isotopes in corals is partially obscured by natural
variability. Nonetheless, the general trend over time toward lower
δ
18
O values at numerous sites in the world’s tropical
oceans suggests a gradual warming and/or freshening of the surface
ocean over the past century.
A signaling organelle containing the nerve growth factor-activated receptor tyrosine kinase, TrkA
Authors: Grimes Mark L. , Beattie Eric , Mobley William C.
Published: July 2002

The topology of signal transduction is particularly important for neurons. Neurotrophic factors such as nerve growth factor (NGF) interact with receptors at distal axons and a signal is transduced by retrograde transport to the cell body to ensure survival of the neuron. We have discovered an organelle that may account for the retrograde transport of the neurotrophin signal. This organelle is derived from endocytosis of the receptor tyrosine kinase for NGF, TrkA.
In vitro
reactions containing semi-intact PC12 cells and ATP were used to enhance recovery of a novel organelle: small vesicles containing internalized NGF bound to activated TrkA. These vesicles were distinct from clathrin coated vesicles, uncoated primary endocytic vesicles, and synaptic vesicles, and resembled transport vesicles in their sedimentation velocity. They contained 10% of the total bound NGF and almost one-third of the total tyrosine phosphorylated TrkA. These small vesicles are compelling candidates for the organelles through which the neurotrophin signal is conveyed down the axon.
Selection and nuclear immobilization of exportable RNAs
Authors: Grimm Christian , Lund Elsebet , Dahlberg James E.
Published: July 2002

The intracellular distribution of RNAs depends on interactions of cis-acting nuclear export elements or nuclear retention elements with trans-acting nuclear transport or retention factors. To learn about the relationship between export and retention, we isolated RNAs that are exported from nuclei ofXenopus laevisoocytes even when most RNA export is blocked by an inhibitor of Ran-dependent nucleocytoplasmic transport, the Matrix protein of vesicular stomatitis virus. Export of the selected RNAs is saturable and specific. When present in chimeric RNAs, the selected sequences acted like nuclear export elements in promoting efficient export of RNAs that otherwise are not exported; the pathway used for export of these chimeric RNAs is that used for the selected RNAs alone. However, these chimeric RNAs, unlike the selected RNAs, were not exported in the presence of Matrix protein; thus, the nonselected sequences can cause retention of the selected RNA sequences under conditions of impaired nucleocytoplasmic transport. We propose that most RNAs are transiently immobilized in the nucleus and that release of these RNAs is an essential and early step in export. Release correlates with functional Ran-dependent transport, and the lack of export of chimeric RNAs may result from interference with the Ran system.
An interleukin 4 (IL-4)-independent pathway for CD4+T cell IL-4 production is revealed in IL-4 receptor-deficient mice
Authors: Noben-Trauth Nancy , Shultz Leonard D. , Brombacher Frank , Urban Joseph F. , Gu Hua , Paul William E.
Published: July 2002

IL-4 receptor α chain (IL-4Rα)-deficient mice were generated by gene-targeting in BALB/c embryonic stem cells. Mutant mice showed a loss of IL-4 signal transduction and functional activity. The lack of IL-4Rα resulted in markedly diminished, but not absent, TH2 responses after infection with the helminthic parasiteNippostrongylus brasiliensis. CD4+, CD62L-high, and CD62L-low T cell populations from uninfected IL-4Rα−/−mice were isolated by cell sorting. Upon primary stimulation by T cell receptor cross-linkage, the CD62L-low, but not the CD62L-high, cells secreted considerable amounts of IL-4, which was strikingly enhanced upon 4-day culture with anti-CD3 in the presence or absence of IL-4. CD62L-low cells isolated from IL-4Rα−/−, β2-microglobulin−/−double homozygous mice produced less IL-4 than did either IL-4Rα−/−or wild-type mice. These results indicate that an IL-4-independent, β2-microglobulin-dependent pathway exists through which the CD62L-low CD4+population has acquired IL-4-producing capacityin vivo, strongly suggesting that these cells are NK T cells.
Absence of a barrier to backwards rotation of the bacterial flagellar motor demonstrated with optical tweezers
Authors: Berry Richard M. , Berg Howard C.
Published: July 2002

A cell of the bacterium
Escherichia coli
was tethered covalently to a glass coverslip by a single flagellum, and its rotation was stopped by using optical tweezers. The tweezers acted directly on the cell body or indirectly, via a trapped polystyrene bead. The torque generated by the flagellar motor was determined by measuring the displacement of the laser beam on a quadrant photodiode. The coverslip was mounted on a computer-controlled piezo-electric stage that moved the tether point in a circle around the center of the trap so that the speed of rotation of the motor could be varied. The motor generated ≈4500 pN nm of torque at all angles, regardless of whether it was stalled, allowed to rotate very slowly forwards, or driven very slowly backwards. This argues against models of motor function in which rotation is tightly coupled to proton transit and back-transport of protons is severely limited.
Nucleosome packaging and nucleosome positioning of genomic DNA
Authors: Lowary P. T. , Widom J.
Published: July 2002

The goals of this study were to assess the extent to which bulk genomic DNA sequences contribute to their own packaging in nucleosomes and to reveal the relationship between nucleosome packaging and positioning. Using a competitive nucleosome reconstitution assay, we found that at least 95% of bulk DNA sequences have an affinity for histone octamer in nucleosomes that is similar to that of randomly synthesized DNA; they contribute little to their own packaging at the level of individual nucleosomes. An equation was developed that relates the measured free energy to the fractional occupancy of specific nucleosome positions. Evidently, the bulk of eukaryotic genomic DNA is also not evolved or constrained for significant sequence-directed nucleosome positioning at the level of individual nucleosomes. Implications for gene regulation
in vivo
are discussed.
The proliferation potential protein-related (P2P-R) gene with domains encoding heterogeneous nuclear ribonucleoprotein association and Rb1 binding shows repressed expression during terminal differentiation
Authors: Witte Michael M. , Scott Robert E.
Published: July 2002

Terminal differentiation is associated with repression in the expression of the proliferation potential proteins (P2P) subset of heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. We report here the cloning and characterization of a 5173-bp P2P-related (P2P-R) cDNA that contains a 4214-bp open reading frame. Probes to this cDNA detect a single 8-kb mRNA in multiple murine tissues and in proliferating 3T3T cells, but not in terminally differentiated 3T3T adipocytes. Evidence that this cDNA can encode peptides with domains for hnRNP association was established by showing that such peptides are recognized by two monoclonal antibodies known to detect core hnRNP proteins, and by showing that the C130 monoclonal antibody, produced against a cDNA-derived fusion protein, also selectively detects native P2P hnRNP proteins. In addition, P2P-R cDNA-derived fusion proteins bind single-stranded nucleic acids, and a P2P-R cDNA-derived antisense oligonucleotide selectively represses P2P expression. Because terminal differentiation is associated with modulation in Rb1 function, we assayed if products of this cDNA might interact with Rb1. Evidence that the P2P-R cDNA encodes a protein domain that binds Rb1 was established using a glutathione
S
-transferase fusion protein to selectively precipitate Rb1 from cellular extracts. Data also show that this binding is reduced by competition with the adenovirus E1a protein, indicating that binding occurs through the “pocket” domain of Rb1. These results establish that the P2P-R cDNA encodes protein domains involved in both hnRNP association and Rb1 binding and complement recent reports that localize Rb1 to sites of RNA processing in the nucleus.
Mitochondrial decay in hepatocytes from old rats: Membrane potential declines, heterogeneity and oxidants increase
Authors: Hagen Tory M. , Yowe David L. , Bartholomew James C. , Wehr Carol M. , Do Katherine L. , Park Jin-Y. , Ames Bruce N.
Published: July 2002

Mitochondrial function during aging was assessed in isolated rat hepatocytes to avoid the problem of differential lysis when old, fragile mitochondria are isolated. Rhodamine 123, a fluorescent dye that accumulates in mitochondria on the basis of their membrane potential, was used as a probe to determine whether this key function is affected by aging. A marked fluorescent heterogeneity was observed in hepatocytes from old (20–28 months) but not young (3–5 months) rats, suggesting age-associated alterations in mitochondrial membrane potential, the driving force for ATP synthesis. Three distinct cell subpopulations were separated by centrifugal elutriation; each exhibited a unique rhodamine 123 fluorescence pattern, with the largest population from old rats having significantly lower fluorescence than that seen in young rats. This apparent age-associated alteration in mitochondrial membrane potential was confirmed by measurements with radioactive tetraphenylphosphonium bromide. Cells from young rats had a calculated membrane potential of −154 mV, in contrast to that of the three subpopulations from old rats of −70 mV (the largest population), −93 mV, and −154 mV. Production of oxidants was examined using 2′,7′dichlorofluorescin, a dye that forms a fluorescent product upon oxidation. The largest cell subpopulation and a minor one from old animals produced significantly more oxidants than cells from young rats. To investigate the molecular cause(s) for the heterogeneity, we determined the levels of an age-associated mtDNA deletion. No significant differences were seen in the three subpopulations, indicating that the mitochondrial decay is due to other mutations, epigenetic changes, or both.
Multi-functional acetyl-CoA carboxylase from
Brassica napus
is encoded by a multi-gene family: Indication for plastidic localization of at least one isoform
Authors: Schulte Wolfgang , Töpfer Reinhard , Stracke Ralf , Schell Jeff , Martini Norbert
Published: July 2002

Three genes coding for different multi-functional acetyl-CoA carboxylase (ACCase; EC
6.4.1.2
) isoenzymes from
Brassica napus
were isolated and divided into two major classes according to structural features in their 5′ regions: class I comprises two genes with an additional coding exon of approximately 300 bp at the 5′ end, and class II is represented by one gene carrying an intron of 586 bp in its 5′ untranslated region. Fusion of the peptide sequence encoded by the additional first exon of a class I ACCase gene to the jellyfish
Aequorea victoria
green fluorescent protein (GFP) and transient expression in tobacco protoplasts targeted GFP to the chloroplasts. In contrast to the deduced primary structure of the biotin carboxylase domain encoded by the class I gene, the corresponding amino acid sequence of the class II ACCase shows higher identity with that of the
Arabidopsis
ACCase, both lacking a transit peptide. The
Arabidopsis
ACCase has been proposed to be a cytosolic isoenzyme. These observations indicate that the two classes of ACCase genes encode plastidic and cytosolic isoforms of multi-functional, eukaryotic type, respectively, and that
B. napus
contains at least one multi-functional ACCase besides the multi-subunit, prokaryotic type located in plastids. Southern blot analysis of genomic DNA from
B. napus
,
Brassica rapa
, and
Brassica oleracea
, the ancestors of amphidiploid rapeseed, using a fragment of a multi-functional ACCase gene as a probe revealed that ACCase is encoded by a multi-gene family of at least five members.
Nitric oxide is the mediator of both endothelium-dependent relaxation and hyperpolarization of the rabbit carotid artery
Authors: Cohen Richard A. , Plane Frances , Najibi Soheil , Huk Igor , Malinski Tadeusz , Garland Chris J.
Published: July 2002

It is controversial whether the endothelial cell release of nitric oxide (NO) or a different factor(s) accounts for endothelium-dependent hyperpolarization, because in many arteries endothelium-dependent relaxation and hyperpolarization resists inhibitors of NO synthase. The contribution of NO to acetylcholine-induced endothelium-dependent hyperpolarization and relaxation of the rabbit carotid artery was determined by measuring NO with electrochemical and chemiluminescence techniques. In the presence of phenylephrine to depolarize and contract the smooth muscle cells, acetylcholine caused concentration-dependent hyperpolarization and relaxation which were closely correlated to the release of NO.
N
ω
-nitro-
l
-arginine methyl ester (30 μM) partially reduced the release of NO and caused a similar reduction in smooth muscle cell relaxation and hyperpolarization. To determine if the residual responses were mediated by another endothelium-derived mediator or NO released despite treatment with
N
ω
-nitro-
l
-arginine methyl ester,
N
ω
-nitro-
l
-arginine (300 μM) was added. The combined inhibitors further reduced, but did not eliminate, NO release, smooth muscle relaxation, and hyperpolarization. Hyperpolarization and relaxation to acetylcholine remained closely correlated with the release of NO in the presence of the inhibitors. In addition, the NO donor, SIN-1, caused hyperpolarization and relaxation which correlated with the concentrations of NO that it released. These studies indicate that (
i
) the release of NO by acetylcholine is only partially inhibited by these inhibitors of NO synthase when used even at high concentrations, and (
ii
) NO rather than another factor accounts fully for endothelium-dependent responses of the rabbit carotid artery.
Visualization of elongation factor G on the
Escherichia coli
70S ribosome: The mechanism of translocation
Authors: Agrawal Rajendra K. , Penczek Pawel , Grassucci Robert A. , Frank Joachim
Published: July 2002

During protein synthesis, elongation factor G (EF-G) binds to the ribosome and promotes the step of translocation, a process in which tRNA moves from the A to the P site of the ribosome and the mRNA is advanced by one codon. By using three-dimensional cryo-electron microscopy, we have visualized EF-G in a ribosome–EF-G–GDP–fusidic acid complex. Fitting the crystal structure of EF-G–GDP into the cryo density map reveals a large conformational change mainly associated with domain IV, the domain that mimics the shape of the anticodon arm of the tRNA in the structurally homologous ternary complex of Phe-tRNA
Phe
, EF-Tu, and a GTP analog. The tip portion of this domain is found in a position that overlaps the anticodon arm of the A-site tRNA, whose position in the ribosome is known from a study of the pretranslocational complex, implying that EF-G displaces the A-site tRNA to the P site by physical interaction with the anticodon arm.
B7–CTLA4 interaction enhances both production of antitumor cytotoxic T lymphocytes and resistance to tumor challenge
Authors: Zheng Pan , Wu Yan , Guo Yong , Lee Cheng , Liu Yang
Published: July 2002

Expression of B7-family costimulatory molecules CD80 (B7–1) and CD86 (B7–2) on tumor cells enhances host immunity. However, the role of the two B7 receptors, CD28 and CTLA4 (CD152), on T cells in antitumor immune response has not been clearly elucidated. Based on the effects of anti-CD28 and anti-CTLA4 mAbs on T cell response, it was proposed that CD28-B7 interaction promotes antitumor immunity, whereas B7-CTLA4 interaction down-regulates it. A critical test for the hypothesis is whether selective engagement of CTLA4 receptors by their natural ligands CD80 and CD86 enhances or reduces antitumor immunity. Here we used tumors expressing wild-type and mutant CD80, as well as mice with targeted mutation of CD28, to address this issue. We report that in syngeneic wild-type mice, B7W (W88>A), a CD80 mutant that has lost binding to CD28 but retained binding to CTLA4, can enhance the induction of antitumor cytotoxic T lymphocytes (CTL); B7Y (Y201>A), which binds neither CD28 nor CTLA4, fails to do so. Consistent with these observations, B7W-transfected J558 plasmocytoma and EL4 thymoma grow significantly more slowly than those transfected with either vector alone or with B7Y. Optimal tumor rejection requires wild-type CD80. Moreover, expression of a high level of CD80 on thymoma EL4 cells conveys immunity in mice with a targeted mutation of CD28 gene. Taken together, our results demonstrate that B7-CTLA4 interaction enhances production of antitumor CTL and resistance to tumor challenge and that optimal enhancement of antitumor immunity by CD80 requires its engagement of both CD28 and CTLA4.
trans
/
cis
(
Z
/
E
) photoisomerization of the chromophore of photoactive yellow protein is not a prerequisite for the initiation of the photocycle of this photoreceptor protein
Authors: Cordfunke Robert , Kort Remco , Pierik Antonio , Gobets Bas , Koomen Gert-Jan , Verhoeven Jan W. , Hellingwerf Klaas J.
Published: July 2002

The chromophore of photoactive yellow protein (PYP) (i.e., 4-hydroxycinnamic acid) has been replaced by an analogue with a triple bond, rather than a double bond (by using 4-hydroxyphenylpropiolic acid in the reconstitution, yielding hybrid I) and by a “locked” chromophore (through reconstitution with 7-hydroxycoumarin-3-carboxylic acid, in which a covalent bridge is present across the vinyl bond, resulting in hybrid II). These hybrids absorb maximally at 464 and 443 nm, respectively, which indicates that in both hybrids the deprotonated chromophore does fit into the chromophore-binding pocket. Because the triple bond cannot undergo
cis
/
trans
(or
E
/
Z
) photoisomerization and because of the presence of the lock across the vinyl double bond in hybrid II, it was predicted that these two hybrids would not be able to photocycle. Surprisingly, both are able. We have demonstrated this ability by making use of transient absorption, low-temperature absorption, and Fourier-transform infrared (FTIR) spectroscopy. Both hybrids, upon photoexcitation, display authentic photocycle signals in terms of a red-shifted intermediate; hybrid I, in addition, goes through a blue-shifted-like intermediate state, with very slow kinetics. We interpret these results as further evidence that rotation of the carbonyl group of the thioester-linked chromophore of PYP, proposed in a previous FTIR study and visualized in recent time-resolved x-ray diffraction experiments, is of critical importance for photoactivation of PYP.
Targeted expansion of genetically modified bone marrow cells
Authors: Jin Liqing , Siritanaratkul Noppadol , Emery David W. , Richard Robert E. , Kaushansky Kenneth , Papayannopoulou Thalia , Blau C. Anthony
Published: July 2002

The ability to specifically target a mitogenic signal to a population of genetically modified primary cells would have potential applications both for gene and cell therapy. Toward this end, a gene encoding a fusion protein containing the FK506-binding protein FKBP12, fused to the intracellular portion of the receptor for thrombopoietin (mpl), was introduced into primary murine bone marrow cells. Dimerization of this fusion protein through the addition of a dimeric form of the drug FK506, called FK1012, resulted in a marked proliferative expansion of marrow cells that was restricted to the genetically modified population. FK1012’s proliferative effect was sustained and reversible. An apparent preference for differentiation along the megakaryocytic lineage was observed. This approach allows for the specific delivery of a mitogenic signal to a population of genetically modified primary cells and may have applications for studies in hematopoiesis and receptor biology, and for gene and cell therapy.
Kinetics of conformational fluctuations in DNA hairpin-loops
Authors: Bonnet Grégoire , Krichevsky Oleg , Libchaber Albert
Published: July 2002

The kinetics of DNA hairpin-loop fluctuations has been investigated by using a combination of fluorescence energy transfer and fluorescence correlation spectroscopy. We measure the chemical rates and the activation energies associated with the opening and the closing of the hairpin for different sizes and sequences of the loop and for various salt concentrations. The rate of unzipping of the hairpin stem is essentially independent of the characteristics of the loop, whereas the rate of closing varies greatly with the loop length and sequence. The closing rate scales with the loop length, with an exponent 2.6 ± 0.3. The closing rate is increased at higher salt concentrations. For hairpin closing, a loop of adenosine repeats leads to smaller rates and higher activation energies than a loop with thymine repeats.
The Epstein–Barr virus nuclear protein SM is both a post-transcriptional inhibitor and activator of gene expression
Authors: Ruvolo Vivian , Wang Eryu , Boyle Sarah , Swaminathan Sankar
Published: July 2002

The Epstein–Barr virus (EBV) nuclear protein BS-MLF1 (SM) is expressed early after entry of EBV into the lytic cycle. SM transactivates reporter gene constructs driven by a wide variety of promoters, but the mechanism of SM action is poorly understood. In this study, we demonstrate that the SM protein inhibits expression of intron-containing genes and activates expression of intron-less genes. We demonstrate that SM has the predicted inhibitory effect on expression of a spliced EBV gene but activates an unspliced early EBV gene. SM inhibited gene expression at the post-transcriptional level by preventing the accumulation of nuclear and cytoplasmic RNA transcripts. Conversely, SM led to increased accumulation of nuclear mRNA from intron-less genes without affecting the rate of transcription, indicating that SM enhances nuclear RNA stability. The ratio of cytoplasmic to nuclear polyadenylated mRNA was increased in the presence of SM, suggesting that SM also enhances nucleo-cytoplasmic mRNA transport. The degree of transactivation by SM was dependent on the sequence of the 3′-untranslated region of the target mRNA. Finally, we demonstrate that the amino-terminal portion of SM fused to glutathione-
S
-transferase binds radioactively labeled RNA
in vitro
, indicating that SM is a single-stranded RNA binding protein. Importantly, the latent and immediate-early genes of EBV contain introns whereas many early and late genes do not. Thus, SM may down-regulate synthesis of host cell proteins and latent EBV proteins while simultaneously enhancing expression of specific lytic EBV genes by binding to mRNA and modulating its stability and transport.
Increase in the 64-kDa subunit of the polyadenylation/cleavage stimulatory factor during the G0to S phase transition
Authors: Martincic Kathleen , Campbell Ronna , Edwalds-Gilbert Gretchen , Souan Lina , Lotze Michael T. , Milcarek Christine
Published: July 2002

The amount of the 64-kDa subunit of polyadenylation/cleavage stimulatory factor (CstF-64) increases 5-fold during the G0to S phase transition and concomitant proliferation induced by serum in 3T6 fibroblasts. Higher levels of CstF-64 result in an increase in CstF trimer. The rise in CstF-64 occurs at a time when the amount of poly(A)-containing RNA rose at least 5–8 fold in the cytoplasm. Primary human splenic B cells, resting in G0, show a similar 5-fold increase in CstF-64 when cultured under conditions inducing proliferation (CD40 ligand exposure). Therefore, the increase in CstF-64 is associated with the G0to S phase transition. As B cell development progresses, RNA processing changes occur at the Ig heavy chain locus resulting in a switch from the membrane- to the upstream secretory-specific poly(A) site. Treating resting B cells with agents triggering this switch in Ig mRNA production along with proliferation (CD40 ligand plus lymphokines orStapylococcus aureusprotein A) induces no further increase in CstF-64 above that seen for proliferation alone. The rise in CstF-64 is therefore insufficient to induce secretion. After stimulation of a continuously growing B cell line with lymphokines, a switch to Ig μ secretory mRNA and protein occurs but without a change in the CstF-64 level. Therefore, an increase in CstF-64 levels is not necessary to mediate the differentiation-induced switch to secreted forms of Ig-μ heavy chain. Because augmentation of CstF-64 levels is neither necessary nor sufficient for Ig secretory mRNA production, we conclude that other lymphokine-induced factors play a role.
Activation of nicotinic receptor-induced postsynaptic responses to luteinizing hormone-releasing hormone in bullfrog sympathetic ganglia via a Na
+
-dependent mechanism
Authors: Cao Ying-Jun , Peng Yan-Yi
Published: July 2002

Nicotine at very low doses (5–30 nM) induced large amounts of luteinizing hormone-releasing hormone (LHRH) release, which was monitored as slow membrane depolarizations in the ganglionic neurons of bullfrog sympathetic ganglia. A nicotinic antagonist,
d
-tubocurarine chloride, completely and reversibly blocked the nicotine-induced LHRH release, but it did not block the nerve-firing-evoked LHRH release. Thus, nicotine activated nicotinic acetylcholine receptors and produced LHRH release via a mechanism that is different from the mechanism for evoked release. Moreover, this release was not caused by Ca
2+
influx through either the nicotinic receptors or the voltage-gated Ca
2+
channels because the release was increased moderately when the extracellular solution was changed into a Ca
2+
-free solution that also contained Mg
2+
(4 mM) and Cd
2+
(200 μM). The release did not depend on Ca
2+
release from the intraterminal Ca
2+
stores either because fura-2 fluorimetry showed extremely low Ca
2+
elevation (≈30 nM) in response to nicotine (30 nM). Moreover, nicotine evoked LHRH release when [Ca
2+
] elevation in the terminals was prevented by loading the terminals with 1,2-bis(2-aminophenoxy)ethane-
N,N,N′,N′
-tetraacetic acid and fura-2. Instead, the nicotine-induced release required extracellular Na
+
because substitution of extracellular NaCl with
N
-methyl-
d
-glucamine chloride completely blocked the release. The Na
+
-dependent mechanism was not via Na
+
influx through the voltage-gated Na
+
channels because the release was not affected by tetrodotoxin (1–50 μM) plus Cd
2+
(200 μM). Thus, nicotine at very low concentrations induced LHRH release via a Na
+
-dependent, Ca
2+
-independent mechanism.
Inhibition of RNA polymerase II transcription in human cells by synthetic DNA-binding ligands
Authors: Dickinson Liliane A. , Gulizia Richard J. , Trauger John W. , Baird Eldon E. , Mosier Donald E. , Gottesfeld Joel M. , Dervan Peter B.
Published: July 2002

Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole–imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located within RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.
Identification of two distinct human SMC protein complexes involved in mitotic chromosome dynamics
Authors: Schmiesing John A. , Ball Alexander R. , Gregson Heather C. , Alderton Janet M. , Zhou Sharleen , Yokomori Kyoko
Published: July 2002

The
s
tructural
m
aintenance of
c
hromosomes (SMC) family member proteins previously were shown to play a critical role in mitotic chromosome condensation and segregation in yeast and
Xenopus
. Other family members were demonstrated to be required for DNA repair in yeast and mammals. Although several different SMC proteins were identified in different organisms, little is known about the SMC proteins in humans. Here, we report the identification of four human SMC proteins that form two distinct heterodimeric complexes in the cell, the human chromosome-associated protein (hCAP)-C and hCAP-E protein complex (hCAP-C/hCAP-E), and the human SMC1 (hSMC1) and hSMC3 protein complex (hSMC1/hSMC3). The hCAP-C/hCAP-E complex is the human ortholog of the
Xenopus
chromosome-associated protein (XCAP)-C/XCAP-E complex required for mitotic chromosome condensation. We found that a second complex, hSMC1/hSMC3, is required for metaphase progression in mitotic cells. Punctate vs. diffuse distribution patterns of the hCAP-C/hCAP-E and hSMC1/hSMC3 complexes in the interphase nucleus indicate independent behaviors of the two complexes during the cell cycle. These results suggest that two distinct classes of SMC protein complexes are involved in different aspects of mitotic chromosome organization in human cells.
Three distinct domains of SSI-1/SOCS-1/JAB protein are required for its suppression of interleukin 6 signaling
Authors: Narazaki Masashi , Fujimoto Minoru , Matsumoto Tomoshige , Morita Yoshiaki , Saito Hiroshi , Kajita Tadahiro , Yoshizaki Kazuyuki , Naka Tetsuji , Kishimoto Tadamitsu
Published: July 2002

Cytokine-inducible protein SSI-1 [signal transducers and activators of transcription (STAT)-induced STAT inhibitor 1, also referred to as SOCS-1 (suppressor of cytokine signaling 1) or JAB (Janus kinase-binding protein)] negatively regulates cytokine receptor signaling by inhibition of JAK kinases. The SSI family of proteins includes eight members that are structurally characterized by an SH2 domain and a C-terminal conserved region that we have called the SC-motif. In this study, we investigated the roles of these domains in the function of SSI-1. Results of reporter assays demonstrated that the pre-SH2 domain (24 aa in front of the SH2 domain) and the SH2 domain of SSI-1 were required for the suppression by SSI-1 of interleukin 6 signaling. Coexpression studies of COS7 cells revealed that these domains also were required for inhibition of three JAKs (JAK1, JAK2, and TYK2). Furthermore, deletion of the SH2 domain, but not the pre-SH2 domain, resulted in loss of association of SSI-1 with TYK2. Thus, SSI-1 associates with JAK family kinase via its SH2 domain, and the pre-SH2 domain is required for the function of SSI-1. Deletion of the SC-motif markedly reduced expression of SSI-1 protein in M1 cells, and this reduction was reversed by treatment with proteasome inhibitors, suggesting that this motif is required to protect the SSI-1 molecule from proteolytic degradation. Based on these findings, we concluded that three distinct domains of SSI-1 (the pre-SH2 domain, the SH2 domain, and the SC-motif) cooperate in the suppression of interleukin 6 signaling.
Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte–macrophage colony-stimulating factor generates potent antitumor immunity in patients with metastatic melanoma
Authors: Soiffer Robert , Lynch Thomas , Mihm Martin , Jung Ken , Rhuda Catherine , Schmollinger Jan C. , Hodi F. Stephen , Liebster Laura , Lam Prudence , Mentzer Steven , Singer Samuel , Tanabe Kenneth K. , Cosimi A. Benedict , Duda Rosemary , Sober Arthur , Bhan Atul , Daley John , Neuberg Donna , Parry Gordon , Rokovich Joseph , Richards Laurie , Drayer Jan , Berns Anton , Clift Shirley , Cohen Lawrence K. , Mulligan Richard C. , Dranoff Glenn
Published: July 2002

We conducted a Phase I clinical trial investigating the biologic activity of vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte–macrophage colony-stimulating factor in patients with metastatic melanoma. Immunization sites were intensely infiltrated with T lymphocytes, dendritic cells, macrophages, and eosinophils in all 21 evaluable patients. Although metastatic lesions resected before vaccination were minimally infiltrated with cells of the immune system in all patients, metastatic lesions resected after vaccination were densely infiltrated with T lymphocytes and plasma cells and showed extensive tumor destruction (at least 80%), fibrosis, and edema in 11 of 16 patients examined. Antimelanoma cytotoxic T cell and antibody responses were associated with tumor destruction. These results demonstrate that vaccination with irradiated autologous melanoma cells engineered to secrete granulocyte–macrophage colony-stimulating factor stimulates potent antitumor immunity in humans with metastatic melanoma.
Primary CD8+cells from HIV-infected individuals can suppress productive infection of macrophages independent of β-chemokines
Authors: Barker Edward , Bossart Katharine N. , Levy Jay A.
Published: July 2002

The productive infection of human monocyte-derived macrophages (Mφ) by HIV was suppressed by primary CD8+cells from asymptomatic HIV-infected individuals. This anti-HIV response was noncytotoxic; removal of the CD8+cells from the infected Mφ leads to virus production. CD8+cells inhibited HIV replication when separated from the infected Mφ by a transwell filter insert, indicating a diffusible factor made by the CD8+cells suppressed productive infection of Mφ. Three β-chemokines, which can be secreted by activated CD8+cells, RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1α and MIP-1β prevented HIV replication in the Mφ cultures. In addition, incubation of acutely infected Mφ with a mixture of neutralizing antibodies to RANTES, MIP-1α, and MIP-1β enhanced virus replication. Nevertheless, neutralization of β-chemokines with specific antibodies did not abolish the suppression by CD8+cells of HIV replication in Mφ. Thus, even though β-chemokines decrease HIV replication in Mφ, these cytokines are not responsible for the ability of CD8+cells to inhibit HIV production in these cells.
Toward a successful multinational crop plant genome initiative
Authors: Cook R. James
Published: July 2002

Plant genome research is needed as the foundation for an entirely
new level of efficiency and success in the application of genetics and
breeding to crop plants and products from crop plants. Genetic
improvements in crop plants beyond current capabilities are needed to
meet the growing world demand not only for more food, but also a
greater diversity of food, higher-quality food, and safer food,
produced on less land, while conserving soil, water, and genetic
resources. Plant biology research, which is poised for dramatic
advances, also depends fundamentally on plant genome research. The
current
Arabidopsis
Genome Project has proved of
immediate value to plant biology research, but a much greater effort is
needed to ensure the full benefits of plant biology and especially
plant genome research to agriculture. International cooperation is
critical, both because genome projects are too large for any one
country and the information forthcoming is of benefit to the world and
not just the countries that do the work. Recent research on grass
genomes has revealed that, because of extensive senteny and colinearity
within linkage groups that make up the chromosomes, new information on
the genome of one grass can be used to understand the genomes and
predict the location of genes on chromosomes of the other grasses.
Genome research applied to grasses as a group thereby can increase the
efficiency and effectiveness of breeding for improvement of each member
of this group, which includes wheat, corn, and rice, the world’s three
most important sources of food.
Identification of an activation region in the proteasome activator REGα
Authors: Zhang Zhiguo , Clawson Andrew , Realini Claudio , Jensen Christopher C. , Knowlton J. Randolph , Hill Christopher P. , Rechsteiner Martin
Published: July 2002

Proteasomes can be markedly activated by associating with 19S regulatory complexes to form the 26S protease or by binding 11S protein complexes known as REG or PA28. Three REG subunits, α, β, and γ, have been expressed inEscherichia coli, and each recombinant protein can activate human proteasomes. Combining PCR mutagenesis with anin vitroactivity assay, we have isolated and characterized 36 inactive, single-site mutants of recombinant REGα. Most are monomers that produce functional proteasome activators when mixed with REGβ subunits. Five REGα mutants that remain inactive in the mixing assay contain amino acid substitutions clustered between Arg-141 and Gly-149. The crystal structure of the REGα heptamer shows that this region forms a loop at the base of each REGα subunit. One mutation in this loop (N146Y) yields a REGα heptamer that binds the proteasome as tightly as wild-type REGα but does not activate peptide hydrolysis. Corresponding amino acid substitutions in REGβ (N135Y) and REGγ (N151Y) produce inactive proteins that also bind the proteasome and inhibit proteasome activation by their normal counterparts. Our studies clearly demonstrate that REG binding to the proteasome can be separated from activation of the enzyme. Moreover, the dominant negative REGs identified here should prove valuable for elucidating the role(s) of these proteins in antigen presentation.
Functional activation of p53 via phosphorylation following DNA damage by UV but not γ radiation
Authors: Kapoor Mini , Lozano Guillermina
Published: July 2002

The tumor suppressor p53 is a nuclear phosphoprotein in which DNA-binding activity is increased on exposure to DNA-damaging agents such as UV or γ radiation by unknown mechanisms. Because phosphorylation of p53 at the casein kinase (CK) II site activates p53 for DNA-binding function
in vitro
, we sought to determine the
in vivo
relevance of phosphorylation at this site after UV and γ radiation. A polyclonal antibody was generated that binds to bacterially expressed p53 only when phosphorylated
in vitro
by CK II. Using this antibody, we showed that p53 is phosphorylated at the CK II site upon UV treatment of early passage rat embryo fibroblasts and RKO cells. In addition, DNA-binding assays indicated that phosphorylated p53 bound to a p53-responsive element, suggesting functional activation. However, γ radiation, which also stabilizes p53, did not result in phosphorylation at the CK II site. These results indicate that phosphorylation at the CK II site is one of the post-translational mechanisms through which p53 is activated in response to UV radiation and that different mechanisms activate p53 after DNA damage by γ radiation.