bluuebunny/crossref_metadata_2025 huggingface dataset:
CD68+cells of monocyte/macrophage lineage in the environment of AIDS-associated and classic-sporadic Kaposi sarcoma are singly or doubly infected with human herpesviruses 7 and 6B
Authors: Kempf W. , Adams V. , Wey N. , Moos R. , Schmid M. , Avitabile E. , Campadelli-Fiume G.
Published: July 2002

Earlier studies have shown that Kaposi sarcomas contain cells infected with human herpesvirus (HHV) 6B, and in current studies we report that both AIDS-associated and classic-sporadic Kaposi sarcoma contain HHV-7 genome sequences detectable by PCR. To determine the distribution of HHV-7-infected cells relative to those infected with HHV-6, sections from paraffin-embedded tissues were allowed to react with antibodies to HHV-7 virion tegument phosphoprotein pp85 and to HHV-6B protein p101. The antibodies are specific for HHV-7 and HHV-6B, respectively, and they retained reactivity for antigens contained in formalin-fixed, paraffin-embedded tissue samples. We report that (i) HHV-7 pp85 was present in 9 of 32 AIDS-associated Kaposi sarcomas, and in 1 of 7 classical-sporadic HIV-negative Kaposi sarcomas; (ii) HHV-7 pp85 was detected primarily in cells bearing the CD68 marker characteristic of the monocyte/macrophage lineage present in or surrounding the Kaposi sarcoma lesions; and (iii) in a number of Kaposi sarcoma specimens, tumor-associated CD68+monocytes/macrophages expressed simultaneously antigens from both HHV-7 and HHV-6B, and therefore appeared to be doubly infected with the two viruses. CD68+monocytes/macrophages infected with HHV-7 were readily detectable in Kaposi sarcoma, but virtually absent from other normal or pathological tissues that harbor macrophages. Because all of the available data indicate that HHV-7 infects CD4+T lymphocytes, these results suggest that the environment of the Kaposi sarcoma (i) attracts circulating peripheral lymphocytes and monocytes, triggers the replication of latent viruses, and thereby increases the local concentration of viruses, (ii) renders CD68+monocytes/macrophages susceptible to infection with HHV-7, and (iii) the combination of both events enables double infections of cells with both HHV-6B and HHV-7.
Structure of the zero-pressure-gradient turbulent boundary layer
Authors: Barenblatt G. I. , Chorin A. J. , Hald O. H. , Prostokishin V. M.
Published: July 2002

A processing of recent experimental data by Nagib and Hites [Nagib, H. & Hites, M. (1995) AIAA paper 95-0786, Reno, NV) shows that the flow in a zero-pressure-gradient turbulent boundary layer, outside the viscous sublayer, consists of two self-similar regions, each described by a scaling law. The results concerning the Reynolds-number dependence of the coefficients of the wall-region scaling law are consistent with our previous results concerning pipe flow, if the proper definition of the boundary layer Reynolds number (or boundary layer thickness) is used.
Both apolipoprotein E and A-I genes are present in a nonmammalian vertebrate and are highly expressed during embryonic development
Authors: Babin Patrick J. , Thisse Christine , Durliat Michele , Andre Michele , Akimenko Marie-Andree , Thisse Bernard
Published: July 2002

Apolipoprotein E (apoE) is associated with several classes of plasma lipoproteins and mediates uptake of lipoproteins through its ability to interact with specific cell surface receptors. Besides its role in cardiovascular diseases, accumulating evidence has suggested that apoE could play a role in neurodegenerative diseases, such as Alzheimer disease. In vertebrates, apoA-I is the major protein of high-density lipoprotein. ApoA-I may play an important role in regulating the cholesterol content of peripheral tissues through the reverse cholesterol transport pathway. We have isolated cDNA clones that code for apoE and apoA-I from a zebrafish embryo library. Analysis of the deduced amino acid sequences showed the presence of a region enriched in basic amino acids in zebrafish apoE similar to the lipoprotein receptor-binding region of human apoE. We demonstrated by whole-mount
in situ
hybridization that
apoE
and
apoA-I
genes are highly expressed in the yolk syncytial layer, an extraembryonic structure implicated in embryonic and larval nutrition. ApoE transcripts were also observed in the deep cell layer during blastula stage, in numerous ectodermal derivatives after gastrulation, and after 3 days of development in a limited number of cells both in brain and in the eyes. Our data indicate that apoE can be found in a nonmammalian vertebrate and that the duplication events, from which
apoE
and
apoA-I
genes arose, occurred before the divergence of the tetrapod and teleost ancestors. Zebrafish can be used as a simple and useful model for studying the role of apolipoproteins in embryonic and larval nutrition and of apoE in brain morphogenesis and regeneration.
Mechanisms of spectral tuning in the mouse green cone pigment
Authors: Sun Hui , Macke Jennifer P. , Nathans Jeremy
Published: July 2002

Diversification of cone pigment spectral sensitivities during evolution is a prerequisite for the development of color vision. Previous studies have identified two naturally occurring mechanisms that produce variation among vertebrate pigments by red-shifting visual pigment absorbance: addition of hydroxyl groups to the putative chromophore binding pocket and binding of chloride to a putative extracellular loop. In this paper we describe the use of two blue-shifting mechanisms during the evolution of rodent long-wave cone pigments. The mouse green pigment belongs to the long-wave subfamily of cone pigments, but its absorption maximum is 508 nm, similar to that of the rhodopsin subfamily of visual pigments, but blue-shifted 44 nm relative to the human red pigment, its closest homologue. We show that acquisition of a hydroxyl group near the retinylidene Schiff base and loss of the chloride binding site mentioned above fully account for the observed blue shift. These data indicate that the chloride binding site is not a universal attribute of long-wave cone pigments as generally supposed, and that, depending upon location, hydroxyl groups can alter the environment of the chromophore to produce either red or blue shifts.
Immediate and simultaneous sensory reorganization at cortical and subcortical levels of the somatosensory system
Authors: Faggin Barbara M. , Nguyen Kevin Tri , Nicolelis Miguel A. L.
Published: July 2002

The occurrence of cortical plasticity during adulthood has been demonstrated using many experimental paradigms. Whether this phenomenon is generated exclusively by changes in intrinsic cortical circuitry, or whether it involves concomitant cortical and subcortical reorganization, remains controversial. Here, we addressed this issue by simultaneously recording the extracellular activity of up to 135 neurons in the primary somatosensory cortex, ventral posterior medial nucleus of the thalamus, and trigeminal brainstem complex of adult rats, before and after a reversible sensory deactivation was produced by subcutaneous injections of lidocaine. Following the onset of the deactivation, immediate and simultaneous sensory reorganization was observed at all levels of the somatosensory system. No statistical difference was observed when the overall spatial extent of the cortical (9.1 ± 1.2 whiskers, mean ± SE) and the thalamic (6.1 ± 1.6 whiskers) reorganization was compared. Likewise, no significant difference was found in the percentage of cortical (71.1 ± 5.2%) and thalamic (66.4 ± 10.7%) neurons exhibiting unmasked sensory responses. Although unmasked cortical responses occurred at significantly higher latencies (19.6 ± 0.3 ms, mean ± SE) than thalamic responses (13.1 ± 0.6 ms), variations in neuronal latency induced by the sensory deafferentation occurred as often in the thalamus as in the cortex. These data clearly demonstrate that peripheral sensory deafferentation triggers a system-wide reorganization, and strongly suggest that the spatiotemporal attributes of cortical plasticity are paralleled by subcortical reorganization.
Peripheral deletion of rheumatoid factor B cells after abortive activation by IgG
Authors: Tighe Helen , Warnatz Klaus , Brinson Diana , Corr Maripat , Weigle William O. , Baird Stephen M. , Carson Dennis A.
Published: July 2002

Rheumatoid factor (RF) B cells proliferate during secondary immune responses to immune complexed antigen and antigen specific T cells, but higher affinity RFs are not detected except in patients with rheumatoid arthritis and other autoimmune diseases. Consequently, there must exist highly efficient mechanisms for inactivation of these higher-affinity RF B cell clones under normal circumstances. Exposure of transgenic mice expressing a human IgM RF to soluble human IgG in the absence of T cell help causes antigen specific B cell deletion in 2–3 days. The deletion is independent of the Fas/Fas ligand (FasL) pathway of apoptosis and is preceded by a phase of partial activation involving increase in cell size and expression of B7 and ICAM-1, and transient release of low levels of immunoglobulin. Complete B cell activation involving the formation of germinal centers and sustained high level RF secretion only occurs if T cell help is provided simultaneously. RF B cells exposed to tolerogen remain competent to secrete RFin vitroif provided with an appropriate antigenic stimulus and T cell help. Consequently, death of these cells is not preceded by anergy. Abortive activation/deletion of B cells by antigen in the absence of T cell-derived survival signals may represent the major mechanism for maintaining peripheral tolerance in B cells expressing higher affinity RF. The lack of anergy, and the potential for reactivation before death, provide a means for maintaining RF production under pathologic circumstances, such as may occur in the inflamed rheumatoid synovium.
HOP-1, a
Caenorhabditis elegans
presenilin, appears to be functionally redundant with SEL-12 presenilin and to facilitate LIN-12 and GLP-1 signaling
Authors: Li Xiajun , Greenwald Iva
Published: July 2002

Mutant presenilins have been found to cause Alzheimer disease. Here, we describe the identification and characterization of HOP-1, a
Caenorhabditis elegans
presenilin that displays much more lower sequence identity with human presenilins than does the other
C. elegans
presenilin, SEL-12. Despite considerable divergence, HOP-1 appears to be a bona fide presenilin, because HOP-1 can rescue the egg-laying defect caused by mutations in
sel-12
when
hop-1
is expressed under the control of
sel-12
regulatory sequences. HOP-1 also has the essential topological characteristics of the other presenilins. Reducing
hop-1
activity in a
sel-12
mutant background causes synthetic lethality and terminal phenotypes associated with reducing the function of the
C. elegans lin-12
and
glp-1
genes. These observations suggest that
hop-1
is functionally redundant with
sel-12
and underscore the intimate connection between presenilin activity and LIN-12/Notch activity inferred from genetic studies in
C. elegans
and mammals.
Estrogenic responses in estrogen receptor-α deficient mice reveal a distinct estrogen signaling pathway
Authors: Das S. K. , Taylor Julia A. , Korach Kenneth S. , Paria B. C. , Dey S. K. , Lubahn Dennis B.
Published: July 2002

Estrogens are thought to regulate female reproductive functions by altering gene transcription in target organs primarily via the nuclear estrogen receptor-α (ER-α). By using ER-α “knock-out” (ERKO) mice, we demonstrate herein that a catecholestrogen, 4-hydroxyestradiol-17β (4-OH-E
2
), and an environmental estrogen, chlordecone (kepone), up-regulate the uterine expression of an estrogen-responsive gene, lactoferrin (LF), independent of ER-α. A primary estrogen, estradiol-17β (E
2
), did not induce this LF response. An estrogen receptor antagonist, ICI-182,780, or E
2
failed to inhibit uterine LF gene expression induced by 4-OH-E
2
or kepone in ERKO mice, which suggests that this estrogen signaling pathway is independent of both ER-α and the recently cloned ER-β. 4-OH-E
2
, but not E
2
, also stimulated increases in uterine water imbibition and macromolecule uptake in ovariectomized ERKO mice. The results strongly imply the presence of a distinct estrogen-signaling pathway in the mouse uterus that mediates the effects of both physiological and environmental estrogens. This estrogen response pathway will have profound implications for our understanding of the physiology and pathophysiology of female sex steroid hormone actions in target organs.
P-glycoprotein function involves conformational transitions detectable by differential immunoreactivity
Authors: Mechetner Eugene B. , Schott Brigitte , Morse Brian S. , Stein Wilfred D. , Druley Todd , Davis Kenneth A. , Tsuruo Takashi , Roninson Igor B.
Published: July 2002

The MDR1 P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is a transmembrane ATPase efflux pump for various lipophilic compounds, including many anti-cancer drugs. mAb UIC2, reactive with the extracellular moiety of Pgp, inhibits Pgp-mediated efflux. UIC2 reactivity with Pgp was increased by the addition of several Pgp-transported compounds or ATP-depleting agents, and by mutational inactivation of both nucleotide-binding domains (NBDs) of Pgp. UIC2 binding to Pgp mutated in both NBDs was unaffected in the presence of Pgp transport substrates or in ATP-depleted cells, whereas the reactivities of the wild-type Pgp and Pgps mutated in a single NBD were increased by these treatments to the level of the double mutant. These results indicate the existence of different Pgp conformations associated with different stages of transport-associated ATP hydrolysis and suggest trapping in a transient conformation as a mechanism for antibody-mediated inhibition of Pgp.
Cloning and expression of rat 25-hydroxyvitamin D
3
-1α-hydroxylase cDNA
Authors: Shinki Toshimasa , Shimada Hiroko , Wakino Shu , Anazawa Hideharu , Hayashi Matsuhiko , Saruta Takao , DeLuca Hector F. , Suda Tatsuo
Published: July 2002

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D
3
-1α-hydroxylase (P4501α), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When P4501α cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D
3
-1α-hydroxylase activity. The sequence analysis showed that P4501α was of 2,469 bp long and contained an ORF encoding 501 amino acids. The deduced amino acid sequence showed a 53% similarity and 44% identity to the vitamin D
3
-25-hydroxylase (CYP27), whereas it has 42.6% similarity and 34% identity with the 25-hydroxyvitamin D
3
-24-hydroxylase (CYP24). Thus, it composes a new subfamily of the CYP27 family. Further, it is more closely related to the CYP27 than to the CYP24. The expression of P4501α mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1α,25-dihydroxyvitamin D
3
(rats fed a low Ca diet), P4501α mRNA was greatly increased in the renal proximal convoluted tubules.
CD19 and CD22 expression reciprocally regulates tyrosine phosphorylation of Vav protein during B lymphocyte signaling
Authors: Sato Shinichi , Jansen Paul J. , Tedder Thomas F.
Published: July 2002

B cell development and humoral immune responses are controlled by signaling thresholds established through the B lymphocyte antigen receptor (BCR) complex. BCR signaling thresholds are differentially regulated by the CD22 and CD19 cell surface receptorsin vivo. B cells from CD22-deficient mice exhibit characteristics of chronic stimulation and are hyper-responsive to BCR crosslinking with augmented intracellular Ca2+responses. By contrast, B cells from CD19-deficient mice are hypo-responsive to transmembrane signals. To identify signaling molecules involved in the positive and negative regulation of signaling thresholds, the signal transduction pathways activated after BCR crosslinking were examined in CD22- and CD19-deficient B cells. These comparisons revealed that tyrosine phosphorylation of Vav protein was uniquely augmented after BCR or CD19 crosslinking in CD22-deficient B cells, yet was modest and transient after BCR crosslinking in CD19-deficient B cells. Ligation of CD19 and CD22in vivois likely to positively and negatively regulate BCR signaling, respectively, because CD19 crosslinking was more efficient than BCR crosslinking at inducing Vav phosphorylation. However, simultaneous crosslinking of CD19 with the BCR resulted in a substantial decrease in Vav phosphorylation when CD22 was expressed. Thus, the differential regulation of Vav tyrosine phosphorylation by CD19 and CD22 may provide a molecular mechanism for adjusting BCR signaling thresholds.
Targeted disruption of the murine dihydrolipoamide dehydrogenase gene (
Dld
) results in perigastrulation lethality
Authors: Johnson Mark T. , Yang Hsin-Sheng , Magnuson Terry , Patel Mulchand S.
Published: July 2002

The
Dld
gene product, known as dihydrolipoamide dehydrogenase or the E3 component, catalyzes the oxidation of dihydrolipoyl moieties of four mitochondrial multienzyme complexes: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched-chain α-ketoacid dehydrogenase, and the glycine cleavage system. Deficiency of E3 activity in humans results in various degrees of neurological dysfunction and organic acidosis caused by accumulation of branched-chain amino acids and lactic acid. In this study, we have introduced a null mutation into the murine
Dld
gene (
Dld
tm1mjp
). The heterozygous animals are shown to have approximately half of wild-type activity levels for E3 and all affected multienzyme complexes but are phenotypically normal. In contrast, the
Dld
−/−
class dies prenatally with apparent developmental delay at 7.5 days postcoitum followed by resorption by 9.5 days postcoitum. The
Dld
−/−
embryos cease to develop at a time shortly after implantation into the uterine wall when most of the embryos have begun to gastrulate. This null phenotype provides
in vivo
evidence for the requirement of a mitochondrial oxidative pathway during the perigastrulation period. Furthermore, the early prenatal lethal condition of the complete deficiency state may explain the low incidence of detectable cases of E3 deficiency in humans.
CD4-independent, CCR5-dependent infection of brain capillary endothelial cells by a neurovirulent simian immunodeficiency virus strain
Authors: Edinger Aimee L. , Mankowski Joseph L. , Doranz Benjamin J. , Margulies Barry J. , Lee Benhur , Rucker Joseph , Sharron Matthew , Hoffman Trevor L. , Berson Joanne F. , Zink M. Christine , Hirsch Vanessa M. , Clements Janice E. , Doms Robert W.
Published: July 2002

Brain capillary endothelial cells (BCECs) are targets of CD4-independent infection by HIV-1 and simian immunodeficiency virus (SIV) strains
in vitro
and
in vivo
. Infection of BCECs may provide a portal of entry for the virus into the central nervous system and could disrupt blood–brain barrier function, contributing to the development of AIDS dementia. We found that rhesus macaque BCECs express chemokine receptors involved in HIV and SIV entry including CCR5, CCR3, CXCR4, and STRL33, but not CCR2b, GPR1, or GPR15. Infection of BCECs by the neurovirulent strain SIV/17E-Fr was completely inhibited by aminooxypentane regulation upon activation, normal T cell expression and secretion in the presence or absence of ligands, but not by eotaxin or antibodies to CD4. We found that the envelope (env) proteins from SIV/17E-Fr and several additional SIV strains mediated cell–cell fusion and virus infection with CD4-negative, CCR5-positive cells. In contrast, fusion with cells expressing the coreceptors STRL33, GPR1, and GPR15 was CD4-dependent. These results show that CCR5 can serve as a primary receptor for SIV in BCECs and suggest a possible CD4-independent mechanism for blood–brain barrier disruption and viral entry into the central nervous system.
Two active states of the Ras-related Bud1/Rsr1 protein bind to different effectors to determine yeast cell polarity
Authors: Park Hay-Oak , Bi Erfei , Pringle John R. , Herskowitz Ira
Published: July 2002

Cells of budding yeast organize their cytoskeleton in a highly polarized manner during vegetative growth. Selection of a site for polarization requires a group of proteins including a Ras-like GTPase, Bud1, and its regulators. Another group of proteins, which includes a Rho-like GTPase (Cdc42), its guanine nucleotide exchange factor (Cdc24), and Bem1, is necessary for organization of the actin cytoskeleton and for cell polarization. We have proposed previously that the Bud1 protein, through its GTPase cycle, determines the localization of one or more of the cell polarity proteins to the bud site. Herein we demonstrate that Bud1 directly interacts with Cdc24 and Bem1: Bud1 in its GTP-bound form associates preferentially with Cdc24, whereas the GDP-bound form of Bud1 associates with Bem1. We also present subcellular fractionation data for Bud1 that is consistent with the idea that Bud1 can travel between the site for budding on the plasma membrane and the cytosol. We propose that Bud1 can exist in two active states for association with different partners and that the switch from Bud1–GTP to Bud1–GDP provides a regulatory device for ordered assembly of a macromolecular complex at the bud site.
A membrane-anchored E-type endo-1,4-β-glucanase is localized on Golgi and plasma membranes of higher plants
Authors: Brummell David A. , Catala Carmen , Lashbrook Coralie C. , Bennett Alan B.
Published: July 2002

Endo-1,4-β-
d
-glucanases (EGases, EC
3.2.1.4
) are enzymes produced in bacteria, fungi, and plants that hydrolyze polysaccharides possessing a 1,4-β-
d
-glucan backbone. All previously identified plant EGases are E-type endoglucanases that possess signal sequences for endoplasmic reticulum entry and are secreted to the cell wall. Here we report the characterization of a novel E-type plant EGase (tomato Cel3) with a hydrophobic transmembrane domain and structure typical of type II integral membrane proteins. The predicted protein is composed of 617 amino acids and possesses seven potential sites for N-glycosylation. Cel3 mRNA accumulates in young vegetative tissues with highest abundance during periods of rapid cell expansion, but is not hormonally regulated. Antibodies raised to a recombinant Cel3 protein specifically recognized three proteins, with apparent molecular masses of 93, 88, and 53 kDa, in tomato root microsomal membranes separated by sucrose density centrifugation. The 53-kDa protein comigrated in the gradient with plasma membrane markers, the 88-kDa protein with Golgi membrane markers, and the 93-kDa protein with markers for both Golgi and plasma membranes. EGase enzyme activity was also found in regions of the density gradient corresponding to both Golgi and plasma membranes, suggesting that Cel3 EGase resides in both membrane systems, the sites of cell wall polymer biosynthesis. The
in vivo
function of Cel3 is not known, but the only other known membrane-anchored EGase is present in
Agrobacterium tumefaciens
where it is required for cellulose biosynthesis.
CCR5 coreceptor utilization involves a highly conserved arginine residue of HIV type 1 gp120
Authors: Wang Wei-Kung , Dudek Tim , Zhao Yun-Juan , Brumblay Hunter G. , Essex Max , Lee Tun-Hou
Published: July 2002

The seven-transmembrane CCR5 was recently found to double as a coreceptor for a genetically diverse family of human and nonhuman primate lentiviruses. Paradoxically, the main region of the envelope protein believed to be involved in CCR5 utilization was mapped to hypervariable region 3, or V3, of the envelope glycoprotein gp120. In this study, we addressed the question of whether functional convergence in CCR5 utilization is mediated by certain V3 residues that are highly conserved among HIV type 1 (HIV-1), HIV type 2, and simian immunodeficiency virus. Site-directed mutagenesis carried out on three such V3 residues revealed that the Arg-298 of HIV-1 gp120 has an important role in CCR5 utilization. In contrast, no effect was observed for the other residues we tested. The inability of Arg-298 mutants to use CCR5 was not attributed to global alteration of gp120 conformation. Neither the expression, processing, and incorporation of mutant envelope proteins into virions, nor CD4 binding were significantly affected by the mutations. This interpretation is further supported by the finding that alanine substitutions of five residues immediately adjacent to the arginine residue had no effect on CCR5 utilization. Taken together, our data strongly suggests that the highly conserved Arg-298 residue identified in the V3 of HIV-1 has a significant role in CCR5 utilization, and may represent an unusually conserved target for future anti-viral designs.
Free energy of burying hydrophobic residues in the interface between protein subunits
Authors: Vallone Beatrice , Miele Adriana E. , Vecchini Paola , Chiancone Emilia , Brunori Maurizio
Published: July 2002

We have obtained an experimental estimate of the free energy change associated with variations at the interface between protein subunits, a subject that has raised considerable interest since the concept of accessible surface area was introduced by Lee and Richards [Lee, B. & Richards, F. M. (1971)
J. Mol. Biol.
55, 379–400]. We determined by analytical ultracentrifugation the dimer–tetramer equilibrium constant of five single and three double mutants of human Hb. One mutation is at the stationary α
1
β
1
interface, and all of the others are at the sliding α
1
β
2
interface where cleavage of the tetramer into dimers and ligand-linked allosteric changes are known to occur. A surprisingly good linear correlation between the change in the free energy of association of the mutants and the change in buried hydrophobic surface area was obtained, after corrections for the energetic cost of losing steric complementarity at the αβ dimer interface. The slope yields an interface stabilization free energy of −15 ± 1.2 cal/mol upon burial of 1 Å
2
of hydrophobic surface, in very good agreement with the theoretical estimate given by Eisenberg and McLachlan [Eisenberg, D. & McLachlan, A. D. (1986)
Nature (London)
319, 199–203].
The 3′ untranslated region of a rice α-amylase gene functions as a sugar-dependent mRNA stability determinant
Authors: Chan Ming-Tsair , Yu Su-May
Published: July 2002

In plants, sugar feedback regulation provides a mechanism for control of carbohydrate allocation and utilization among tissues and organs. The sugar repression of α-amylase gene expression in rice provides an ideal model for studying the mechanism of sugar feedback regulation. We have shown previously that sugar repression of α-amylase gene expression in rice suspension cells involves control of both transcription rate and mRNA stability. The α-amylase mRNA is significantly more stable in sucrose-starved cells than in sucrose-provided cells. To elucidate the mechanism of sugar-dependent mRNA turnover, we have examined the effect of α
Amy3
3′ untranslated region (UTR) on mRNA stability by functional analyses in transformed rice suspension cells. We found that the entire α
Amy3
3′ UTR and two of its subdomains can independently mediate sugar-dependent repression of reporter mRNA accumulation. Analysis of reporter mRNA half-lives demonstrated that the entire α
Amy3
3′ UTR and the two subdomains each functioned as a sugar-dependent destabilizing determinant in the turnover of mRNA. Nuclear run-on transcription analysis further confirmed that the α
Amy3
3′ UTR and the two subdomains did not affect the transcription rate of promoter. The identification of sequence elements in the α-amylase mRNA that dictate the differential stability has very important implications for the study of sugar-dependent mRNA decay mechanisms.
The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates
in vivo
Authors: MacAlpine David M. , Perlman Philip S. , Butow Ronald A.
Published: July 2002

Abf2p is a high mobility group (HMG) protein found in yeast mitochondria that is required for the maintenance of wild-type (ρ
+
) mtDNA in cells grown on fermentable carbon sources, and for efficient recombination of mtDNA markers in crosses. Here, we show by two-dimensional gel electrophoresis that Abf2p promotes or stabilizes Holliday recombination junction intermediates in ρ
+
mtDNA
in vivo
but does not influence the high levels of recombination intermediates readily detected in the mtDNA of petite mutants (ρ
−
). mtDNA recombination junctions are not observed in ρ
+
mtDNA of wild-type cells but are elevated to detectable levels in cells with a null allele of the
MGT1
gene (Δ
mgt1
), which codes for a mitochondrial cruciform-cutting endonuclease. The level of recombination intermediates in ρ
+
mtDNA of Δ
mgt1
cells is decreased about 10-fold if those cells contain a null allele of the
ABF2
gene. Overproduction of Abf2p by ≥ 10-fold in wild-type ρ
+
cells, which leads to mtDNA instability, results in a dramatic increase in mtDNA recombination intermediates. Specific mutations in the two Abf2p HMG boxes required for DNA binding diminishes these responses. We conclude that Abf2p functions in the recombination of ρ
+
mtDNA.
Cocaine reward models: Conditioned place preference can be established in dopamine- and in serotonin-transporter knockout mice
Authors: Sora Ichiro , Wichems Christine , Takahashi Nobuyuki , Li Xiao-Fei , Zeng Zhizhen , Revay Randal , Lesch Klaus-Peter , Murphy Dennis L. , Uhl George R.
Published: July 2002

Cocaine and methylphenidate block uptake by neuronal plasma membrane transporters for dopamine, serotonin, and norepinephrine. Cocaine also blocks voltage-gated sodium channels, a property not shared by methylphenidate. Several lines of evidence have suggested that cocaine blockade of the dopamine transporter (DAT), perhaps with additional contributions from serotonin transporter (5-HTT) recognition, was key to its rewarding actions. We now report that knockout mice without DAT and mice without 5-HTT establish cocaine-conditioned place preferences. Each strain displays cocaine-conditioned place preference in this major mouse model for assessing drug reward, while methylphenidate-conditioned place preference is also maintained in DAT knockout mice. These results have substantial implications for understanding cocaine actions and for strategies to produce anticocaine medications.
The endogenous cannabinoid anandamide inhibits human breast cancer cell proliferation
Authors: De Petrocellis Luciano , Melck Dominique , Palmisano Antonella , Bisogno Tiziana , Laezza Chiara , Bifulco Maurizio , Di Marzo Vincenzo
Published: July 2002

Anandamide was the first brain metabolite shown to act as a ligand of “central” CB1 cannabinoid receptors. Here we report that the endogenous cannabinoid potently and selectively inhibits the proliferation of human breast cancer cells
in vitro
. Anandamide dose-dependently inhibited the proliferation of MCF-7 and EFM-19 cells with IC
50
values between 0.5 and 1.5 μM and 83–92% maximal inhibition at 5–10 μM. The proliferation of several other nonmammary tumoral cell lines was not affected by 10 μM anandamide. The anti-proliferative effect of anandamide was not due to toxicity or to apoptosis of cells but was accompanied by a reduction of cells in the S phase of the cell cycle. A stable analogue of anandamide (
R
)-methanandamide, another endogenous cannabinoid, 2-arachidonoylglycerol, and the synthetic cannabinoid HU-210 also inhibited EFM-19 cell proliferation, whereas arachidonic acid was much less effective. These cannabimimetic substances displaced the binding of the selective cannabinoid agonist [
3
H]CP 55,940 to EFM-19 membranes with an order of potency identical to that observed for the inhibition of EFM-19 cell proliferation. Moreover, anandamide cytostatic effect was inhibited by the selective CB1 receptor antagonist SR 141716A. Cell proliferation was arrested by a prolactin mAb and enhanced by exogenous human prolactin, whose mitogenic action was reverted by very low (0.1–0.5 μM) doses of anandamide. Anandamide suppressed the levels of the long form of the prolactin receptor in both EFM-19 and MCF-7 cells, as well as a typical prolactin-induced response, i.e., the expression of the breast cancer cell susceptibility gene
brca1
. These data suggest that anandamide blocks human breast cancer cell proliferation through CB1-like receptor-mediated inhibition of endogenous prolactin action at the level of prolactin receptor.
Concerted formation of macromolecular
Suppressor–mutator
transposition complexes
Authors: Raina Ramesh , Schläppi Michael , Karunanandaa Balasulojini , Elhofy Adam , Fedoroff Nina
Published: July 2002

Transposition of the maize
Suppressor–mutator
(
Spm
) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA–DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein–protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range.
Signaling pathways in Ras-mediated tumorigenicity and metastasis
Authors: Webb Craig P. , Van Aelst Linda , Wigler Michael H. , Vande Woude George F.
Published: July 2002

The effector domain mutants of oncogenic Ras, V12S35 Ras, V12G37 Ras, and V12C40 Ras were tested for their abilities to mediate tumorigenic and metastatic phenotypes in athymic nude mice when expressed in NIH 3T3 fibroblasts. All mutants displayed comparable tumorigenic properties, but only the mutant that activates the Raf-mitogen-activated protein kinase kinase (MEK)-extracellular regulated kinase (ERK) 1/2 pathway, V12S35 Ras, induced tumors in the experimental metastasis assay. Furthermore, direct activation of the MEK-ERK1/2 pathway in NIH 3T3 cells by
mos
or a constitutively active form of MEK was sufficient to induce metastasis whereas R-Ras, which fails to activate the ERK1/2 pathway, is tumorigenic but nonmetastatic. The subcutaneous tumors and lung metastases derived from V12S35 Ras-transformed NIH 3T3 cells expressed higher levels of activated ERK1/2 in culture when compared with the parental cellular pool before injection, indicating that selection for cells with higher levels of activated ERK1/2 occurred during tumor growth and metastasis. By contrast, cells explanted from V12G37-Ras or V12C40-Ras-induced tumors did not show changes in the level of ERK1/2 activation when compared with the parental cells. When tumor-explanted cell lines derived from each of the effector domain mutants were passaged one additional time
in vivo
, all mediated rapid tumor growth, but, again, only cells derived from V12S35 Ras-tumors formed numerous metastatic lesions within the lung. These results show that the metastatic properties of the Ras effector domain mutants segregate, and that, whereas Ras-mediated tumorigenicity can arise independently of ERK1/2 activation, experimental metastasis appears to require constitutive activation of the ERK1/2 pathway.
Crystal structure of the simian immunodeficiency virus (SIV) gp41 core: Conserved helical interactions underlie the broad inhibitory activity of gp41 peptides
Authors: Malashkevich Vladimir N. , Chan David C. , Chutkowski Christine T. , Kim Peter S.
Published: July 2002

The gp41 subunit of the envelope protein complex from human and simian immunodeficiency viruses (HIV and SIV) mediates membrane fusion during viral entry. The crystal structure of the HIV-1 gp41 ectodomain core in its proposed fusion-active state is a six-helix bundle. Here we have reconstituted the core of the SIV gp41 ectodomain with two synthetic peptides called SIV N36 and SIV C34, which form a highly helical trimer of heterodimers. The 2.2 Å resolution crystal structure of this SIV N36/C34 complex is very similar to the analogous structure in HIV-1 gp41. In both structures, three N36 helices form a central trimeric coiled coil. Three C34 helices pack in an antiparallel orientation into highly conserved, hydrophobic grooves along the surface of this coiled coil. The conserved nature of the N36-C34 interface suggests that the HIV-1 and SIV peptides are functionally interchangeable. Indeed, a heterotypic complex between HIV-1 N36 and SIV C34 peptides is highly helical and stable. Moreover, as with HIV-1 C34, the SIV C34 peptide is a potent inhibitor of HIV-1 infection. These results identify conserved packing interactions between the N and C helices of gp41 and have implications for the development of C peptide analogs with broad inhibitory activity.
Subtle conformational changes induced in major histocompatibility complex class II molecules by binding peptides
Authors: Chervonsky Alexander V. , Medzhitov Ruslan M. , Denzin Lisa K. , Barlow Avlin K. , Rudensky Alexander Yu. , Janeway Charles A.
Published: July 2002

Intracellular trafficking of major histocompatibility complex (MHC) class II molecules is characterized by passage through specialized endocytic compartment(s) where antigenic peptides replace invariant chain fragments in the presence of the DM protein. These changes are accompanied by structural transitions of the MHC molecules that can be visualized by formation of compact SDS-resistant dimers, by changes in binding of mAbs, and by changes in T cell responses. We have observed that a mAb (25-9-17) that is capable of staining I-Abon the surface of normal B cells failed to interact with I-Abcomplexes with a peptide derived from the Eαchain of the I-E molecule but bound a similar covalent complex of I-Abwith the class II binding fragment (class II-associated invariant chain peptides) of the invariant chain. Moreover, 25-9-17 blocked activation of several I-Ab-reactive T cell hybridomas but failed to block others, suggesting that numerous I-Ab-peptide complexes acquire the 25-9-17+or 25-9-17−conformation. Alloreactive T cells were also able to discriminate peptide-dependent variants of MHC class II molecules. Thus, peptides impose subtle structural transitions upon MHC class II molecules that affect T cell recognition and may thus be critical for T cell selection and autiommunity.
The promoter of
LE-ACS7
, an early flooding-induced 1-aminocyclopropane-1-carboxylate synthase gene of the tomato, is tagged by a
Sol3
transposon
Authors: Shiu Oi Yin , Oetiker Jürg H. , Yip Wing Kin , Yang Shang Fa
Published: July 2002

Many terrestrial plants respond to flooding with enhanced ethylene production. The roots of flooded plants produce 1-aminocyclopropane-1-carboxylic acid (ACC), which is transported from the root to the shoot, where it is converted to ethylene. In the roots, ACC is synthesized by ACC synthase, which is encoded by a multigene family. Previously, we identified two ACC synthase genes of tomato that are involved in flooding-induced ethylene production. Here, we report the cloning of
LE-ACS7
, a new tomato ACC synthase with a role early during flooding but also in the early wound response of leaves. The promoter of
LE-ACS7
is tagged by a
Sol3
transposon. A
Sol3
transposon is also present in the tomato polygalacturonase promoter to which it conferred regulatory elements. Thus,
Sol3
transposons may affect the regulation of
LE-ACS7
and may be involved in the communication between the root and the shoot of waterlogged tomato plants.
Crystal structures of eukaryotic translation initiation factor 5A fromMethanococcus jannaschiiat 1.8 Å resolution
Authors: Kim Kyeong Kyu , Hung Li-Wei , Yokota Hisao , Kim Rosalind , Kim Sung-Hou
Published: July 2002

Eukaryotic translation initiation factor 5A (eIF-5A) is a ubiquitous protein found in all eukaryotic cells. The protein is closely associated with cell proliferation in the G1–S stage of the cell cycle. Recent findings show that the eIF-5A proteins are highly expressed in tumor cells and act as a cofactor of the Rev protein in HIV-1-infected cells. The mature eIF is the only protein known to have the unusual amino acid hypusine, a post-translationally modified lysine. The crystal structure of eIF-5A fromMethanococcus jannaschii(MJ eIF-5A) has been determined at 1.9 Å and 1.8 Å resolution in two crystal forms by using the multiple isomorphous replacement method and the multiwavelength anomalous diffraction method for the first crystal form and the molecular replacement method for the second crystal form. The structure consists of two folding domains, one of which is similar to the oligonucleotide-binding domain found in the prokaryotic cold shock protein and the translation initiation factor IF1 despite the absence of any significant sequence similarities. The 12 highly conserved amino acid residues found among eIF-5As include the hypusine site and form a long protruding loop at one end of the elongated molecule.
Mouse embryonic stem cells carrying one or two defective
Msh2
alleles respond abnormally to oxidative stress inflicted by low-level radiation
Authors: DeWeese Theodore L. , Shipman Jennifer M. , Larrier Nicole A. , Buckley Nicola M. , Kidd LaCreis R. , Groopman John D. , Cutler Richard G. , te Riele Hein , Nelson William G.
Published: July 2002

Chronic oxidative stress may play a critical role in the pathogenesis of many human cancers. Here, we report that mouse embryonic stem (ES) cells deficient in DNA mismatch repair responded abnormally when exposed to low levels of ionizing radiation, a stress known to generate oxidative DNA damage. ES cells derived from mice carrying either one or two disrupted
Msh2
alleles displayed an increased survival following protracted exposures to low-level ionizing radiation as compared with wild-type ES cells. The increases in survival exhibited by ES cells deficient in DNA mismatch repair appeared to have resulted from a failure to efficiently execute cell death (apoptosis) in response to radiation exposure. For each of the ES cell types, prolonged low-level radiation treatment generated oxidative genome damage that manifested as an accumulation of oxidized bases in genomic DNA. However, ES cells from
Msh2
+/−
and
Msh2
−/−
mice accumulated more oxidized bases as a consequence of low-level radiation exposure than ES cells from
Msh2
+/+
mice. The propensity for normal cells with mismatch repair enzyme deficiencies, including cells heterozygous for inactivating mismatch repair enzyme gene mutations, to survive promutagenic genome insults accompanying oxidative stresses may contribute to the increased cancer risk characteristic of the hereditary nonpolyposis colorectal cancer syndrome.
Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy
Authors: Fritz Jürgen , Katopodis Andreas G. , Kolbinger Frank , Anselmetti Dario
Published: July 2002

Leukocytes roll along the endothelium of postcapillary venules in response to inflammatory signals. Rolling under the hydrodynamic drag forces of blood flow is mediated by the interaction between selectins and their ligands across the leukocyte and endothelial cell surfaces. Here we present force-spectroscopy experiments on single complexes of P-selectin and P-selectin glycoprotein ligand-1 by atomic force microscopy to determine the intrinsic molecular properties of this dynamic adhesion process. By modeling intermolecular and intramolecular forces as well as the adhesion probability in atomic force microscopy experiments we gain information on rupture forces, elasticity, and kinetics of the P-selectin/P-selectin glycoprotein ligand-1 interaction. The complexes are able to withstand forces up to 165 pN and show a chain-like elasticity with a molecular spring constant of 5.3 pN nm
−1
and a persistence length of 0.35 nm. The dissociation constant (off-rate) varies over three orders of magnitude from 0.02 s
−1
under zero force up to 15 s
−1
under external applied forces. Rupture force and lifetime of the complexes are not constant, but directly depend on the applied force per unit time, which is a product of the intrinsic molecular elasticity and the external pulling velocity. The high strength of binding combined with force-dependent rate constants and high molecular elasticity are tailored to support physiological leukocyte rolling.
In vitro
evolution of horse heart myoglobin to increase peroxidase activity
Authors: Wan Lianglu , Twitchett Mark B. , Eltis Lindsay D. , Mauk A. Grant , Smith Michael
Published: July 2002

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in
Escherichia coli
LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with
k
1
(for H
2
O
2
oxidation of metmyoglobin) of 1.34 × 10
4
M
−1
s
−1
(≈25-fold that of wild-type myoglobin) and
k
3
[for reducing the substrate (2, 2′-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 × 10
6
M
−1
s
−1
(1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the
T
m
of the quadruple variant is decreased only slightly compared with wild-type (74.1°C vs. 76.5°C). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (
k
on
, 22.2 × 10
−6
M
−1
s
−1
vs. 22.3 × 10
−6
M
−1
s
−1
;
k
off
, 24.3 s
−1
vs. 24.2 s
−1
;
K
O2
, 0.91 × 10
−6
M
−1
vs. 0.92 × 10
−6
M
−1
). The affinity of the quadruple variant for CO is increased slightly (
k
on
, 0.90 × 10
−6
M
−1
s
−1
vs. 0.51 × 10
−6
M
−1
s
−1
;
k
off
, 5.08 s
−1
vs. 3.51 s
−1
;
K
CO
, 1.77 × 10
−7
M
−1
vs. 1.45 × 10
−7
M
−1
). All four substitutions are in the heme pocket and within 5 Å of the heme group.
Imaging of H
2
17
O distribution in the brain of a live rat by using proton-detected
17
O MRI
Authors: Ronen Itamar , Merkle Hellmut , Ugurbil Kamil , Navon Gil
Published: July 2002

Imaging of H
2
17
O has a number of important applications. Mapping the distribution of H
2
17
O produced by oxidative metabolism of
17
O-enriched oxygen gas may lead to a new method of metabolic functional imaging; regional cerebral blood flow also can be measured by measuring the H
2
17
O distribution after the injection of
17
O-enriched physiological saline solution. Previous studies have proposed a method for indirect detection of
17
O. The method is based on the shortening of the proton T
2
in H
2
17
O solutions, caused by the residual
17
O-
1
H scalar coupling and transferred to the bulk water via fast chemical exchange. It has been shown that the proton T
2
of H
2
17
O solutions can be restored to that of H
2
16
O by irradiating the resonance frequency of the
17
O nucleus. The indirect
17
O image thus is obtained by taking the difference between two T
2
-weighted spin-echo images: one acquired after irradiation of the
17
O resonance and one acquired without irradiation. It also has been established that, at relatively low concentrations of H
2
17
O, the indirect method yields an image that quantitatively reflects the H
2
17
O distribution in the sample. The method is referred to as PRIMO (proton imaging of oxygen). In this work, we show
in vivo
proton images of the H
2
17
O distribution in a rat brain after an i.v. injection of H
2
17
O-enriched physiological saline solution. Implementing the indirect detection method in an echo-planar imaging sequence enabled obtaining H
2
17
O images with good spatial and temporal resolution of few seconds.