bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear punctate distribution of ALL-1 is conferred by distinct elements at the N terminus of the protein
Authors: Yano Takahiro , Nakamura Tatsuya , Blechman Janna , Sorio Claudio , Dang Chi V. , Geiger Benjamin , Canaani Eli
Published: July 2002

The ALL-1 gene positioned at 11q23 is directly involved in human acute leukemia either through a variety of chromosome translocations or by partial tandem duplications. ALL-1 is the human homologue of
Drosophila trithorax
which plays a critical role in maintaining proper spatial and temporal expression of the
Antennapedia
-
bithorax
homeotic genes determining the fruit fly’s body pattern. Utilizing specific antibodies, we found that the ALL-1 protein distributes in cultured cells in a nuclear punctate pattern. Several chimeric ALL-1 proteins encoded by products of the chromosome translocations and expressed in transfected cells showed similar speckles. Dissection of the ALL-1 protein identified within its ≈1,100 N-terminal residues three polypeptides directing nuclear localization and at least two main domains conferring distribution in dots. The latter spanned two short sequences conserved with TRITHORAX. Enforced nuclear expression of other domains of ALL-1, such as the PHD (zinc) fingers and the SET motif, resulted in uniform nonpunctate patterns. This indicates that positioning of the ALL-1 protein in subnuclear structures is mediated via interactions of ALL-1 N-terminal elements. We suggest that the speckles represent protein complexes which contain multiple copies of the ALL-1 protein and are positioned at ALL-1 target sites on the chromatin. Therefore, the role of the N-terminal portion of ALL-1 is to direct the protein to its target genes.
Manipulation of T cell costimulatory and inhibitory signals for immunotherapy of prostate cancer
Authors: Kwon Eugene D. , Hurwitz Arthur A. , Foster Barbara A. , Madias Christopher , Feldhaus Andrew L. , Greenberg Norman M. , Burg Maurice B. , Allison James P.
Published: July 2002

The identification of potentially useful immune-based treatments for prostate cancer has been severely constrained by the scarcity of relevant animal research models for this disease. Moreover, some of the most critical mechanisms involved in complete and proper antitumoral T cell activation have only recently been identified for experimental manipulation, namely, components involved in the costimulatory pathway for T cell activation. Thus, we have established a novel syngeneic murine prostate cancer model that permits us to examine two distinct manipulations intended to elicit an antiprostate cancer response through enhanced T cell costimulation: (i) provision of direct costimulation by prostate cancer cells transduced to express the B7.1 ligand and (ii)in vivoantibody-mediated blockade of the T cell CTLA-4, which prevents T cell down-regulation. In the present study we found that a tumorigenic prostate cancer cell line, TRAMPC1 (pTC1), derived from transgenic mice, is rejected by syngeneic C57BL/6 mice, but not athymic mice, after this cell line is transduced to express the costimulatory ligand B7.1. Also, we demonstrated thatin vivoantibody-mediated blockade of CTLA-4 enhances antiprostate cancer immune responses. The response raised by anti-CTLA-4 administration ranges from marked reductions in wild-type pTC1 growth to complete rejection of these cells. Collectively, these experiments suggest that appropriate manipulation of T cell costimulatory and inhibitory signals may provide a fundamental and highly adaptable basis for prostate cancer immunotherapy. Additionally, the syngeneic murine model that we introduce provides a comprehensive system for further testing of immune-based treatments for prostate cancer.
Intrastriatal injection of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminergic neuron degeneration and behavioral impairment in a rat model of Parkinson disease
Authors: Bilang-Bleuel Alicia , Revah Frédéric , Colin Phillippe , Locquet Isabelle , Robert Jean-Jacques , Mallet Jacques , Horellou Philippe
Published: July 2002

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for adult nigral dopamine neurons
in vivo
. GDNF has both protective and restorative effects on the nigro-striatal dopaminergic (DA) system in animal models of Parkinson disease. Appropriate administration of this factor is essential for the success of its clinical application. Since it cannot cross the blood–brain barrier, a gene transfer method may be appropriate for delivery of the trophic factor to DA cells. We have constructed a recombinant adenovirus (Ad) encoding GDNF and injected it into rat striatum to make use of its ability to infect neurons and to be retrogradely transported by DA neurons. Ad-GDNF was found to drive production of large amounts of GDNF, as quantified by ELISA. The GDNF produced after gene transfer was biologically active: it increased the survival and differentiation of DA neurons
in vitro
. To test the efficacy of the Ad-mediated GDNF gene transfer
in vivo
, we used a progressive lesion model of Parkinson disease. Rats received injections unilaterally into their striatum first of Ad and then 6 days later of 6-hydroxydopamine. We found that mesencephalic nigral dopamine neurons of animals treated with the Ad-GDNF were protected, whereas those of animals treated with the Ad-β-galactosidase were not. This protection was associated with a difference in motor function: amphetamine-induced turning was much lower in animals that received the Ad-GDNF than in the animals that received Ad-β-galactosidase. This finding may have implications for the development of a treatment for Parkinson disease based on the use of neurotrophic factors.
Identification and classification of 16 new kinesin superfamily (KIF) proteins in mouse genome
Authors: Nakagawa Terunaga , Tanaka Yosuke , Matsuoka Eiji , Kondo Satoru , Okada Yasushi , Noda Yasuko , Kanai Yoshimitsu , Hirokawa Nobutaka
Published: July 2002

KIF (kinesin superfamily) proteins are microtubule-dependent molecular motors that play important roles in intracellular transport and cell division. The extent to which KIFs are involved in various transporting phenomena, as well as their regulation mechanism, are unknown. The identification of 16 new KIFs in this report doubles the existing number of KIFs known in the mouse. Conserved nucleotide sequences in the motor domain were amplified by PCR using cDNAs of mouse nervous tissue, kidney, and small intestine as templates. The new KIFs were studied with respect to their expression patterns in different tissues, chromosomal location, and molecular evolution. Our results suggest that (
i
) there is no apparent tendency among related subclasses of KIFs of cosegregation in chromosomal mapping, and (
ii
) according to their tissue distribution patterns, KIFs can be divided into two classes–i.e., ubiquitous and specific tissue-dominant. Further characterization of KIFs may elucidate unknown fundamental phenomena underlying intracellular transport. Finally, we propose a straightforward nomenclature system for the members of the mouse kinesin superfamily.
Targeted ablation of the vitamin D receptor: An animal model of vitamin D-dependent rickets type II with alopecia
Authors: Li Yan Chun , Pirro Alison E. , Amling Michael , Delling Gunter , Baron Roland , Bronson Roderick , Demay Marie B.
Published: July 2002

Vitamin D, the major steroid hormone that controls mineral ion homeostasis, exerts its actions through the vitamin D receptor (VDR). The VDR is expressed in many tissues, including several tissues not thought to play a role in mineral metabolism. Studies in kindreds with VDR mutations (vitamin D-dependent rickets type II, VDDR II) have demonstrated hypocalcemia, hyperparathyroidism, rickets, and osteomalacia. Alopecia, which is not a feature of vitamin D deficiency, is seen in some kindreds. We have generated a mouse model of VDDR II by targeted ablation of the second zinc finger of the VDR DNA-binding domain. Despite known expression of the VDR in fetal life, homozygous mice are phenotypically normal at birth and demonstrate normal survival at least until 6 months. They become hypocalcemic at 21 days of age, at which time their parathyroid hormone (PTH) levels begin to rise. Hyperparathyroidism is accompanied by an increase in the size of the parathyroid gland as well as an increase in PTH mRNA levels. Rickets and osteomalacia are seen by day 35; however, as early as day 15, there is an expansion in the zone of hypertrophic chondrocytes in the growth plate. In contrast to animals made vitamin D deficient by dietary means, and like some patients with VDDR II, these mice develop progressive alopecia from the age of 4 weeks.
Effects of
p
53 mutations on apoptosis in mouse intestinal and human colonic adenomas
Authors: Fazeli Amin , Steen Robert G. , Dickinson Stephanie L. , Bautista Dolores , Dietrich William F. , Bronson Roderick T. , Bresalier Robert S. , Lander Eric S. , Costa Jose , Weinberg Robert A.
Published: July 2002

We have examined the effects of inactivation of the
p53
tumor suppressor gene on the incidence of apoptotic cell death in two stages of the adenoma-to-carcinoma progression in the intestine: in early adenomas where
p53
mutations are rare and in highly dysplastic adenomas where loss of
p53
occurs frequently. Homozygosity for an inactivating germ-line mutation of
p53
had no effect on the incidence or the rate of progression of

Apc
Min/+

-induced adenomas in mice and also did not affect the frequency of apoptosis in the cells of these adenomas. To examine the effect of
p53
loss on apoptosis in late-stage adenomas, we compared the incidence of apoptotic cell death before and after the appearance of highly dysplastic cells in human colonic adenomas. The appearance of highly dysplastic cells, which usually coincides during colon tumor progression with loss of heterozygosity at the
p53
locus, did not correlate with a reduction in the incidence of apoptosis. These studies suggest that
p53
is only one of the genes that determine the incidence of apoptotic in colon carcinomas and that wild-type
p53
retards the progression of many benign colonic adenoma to malignant carcinomas by mechanism(s) other than the promotion of apoptosis.
Structure and function in rhodopsin: Packing of the helices in the transmembrane domain and folding to a tertiary structure in the intradiscal domain are coupled
Authors: Hwa John , Garriga Pere , Liu Xun , Khorana H. Gobind
Published: July 2002

A previous study of the retinitis pigmentosa mutation L125R and two designed mutations at this site, L125A and L125F, showed that these mutations cause partial or total misfolding of the opsins expressed in COS cells from the corresponding mutant opsin genes. We now report on expression and characterization of the opsins from the following retinitis pigmentosa mutants in the transmembrane domain of rhodopsin that correspond to six of the seven helices: G51A and G51V (helix A), G89D (helix B), A164V (helix D), H211P (helix E), P267L and P267R (helix F), and T297R (helix G). All the mutations caused partial misfolding of the opsins as observed by the UV/visible absorption characteristics and by separation of the expressed opsins into fractions that bound 11-
cis
-retinal to form the corresponding mutant rhodopsins and those that did not bind 11-
cis
-retinal. Further, all the mutant rhodopsins prepared from the above mutants, except for G51A, showed strikingly abnormal bleaching behavior with abnormal metarhodopsin II photointermediates. The results show that retinitis pigmentosa mutations in every one of the transmembrane helices can cause misfolding of the opsin. Therefore, on the basis of these and previous results, we conclude that defects in the packing of the transmembrane helices resulting from these mutations are relayed to the intradiscal domain, where they cause misfolding of the opsin by inducing the formation of a disulfide bond other than the native Cys-110—Cys-187 disulfide bond. Thus, there is coupling between packing of the helices in the transmembrane domain and folding to a tertiary structure in the intradiscal domain.
RB and hbrm cooperate to repress the activation functions of E2F1
Authors: Trouche Didier , Le Chalony Catherine , Muchardt Christian , Yaniv Moshe , Kouzarides Tony
Published: July 2002

Forced expression of the retinoblastoma (RB) gene product inhibits the proliferation of cells in culture. A major target of the RB protein is the S-phase-inducing transcription factor E2F1. RB binds directly to the activation domain of E2F1 and silences it, thereby preventing cells from entering S phase. To induce complete G
1
arrest, RB requires the presence of the hbrm/BRG-1 proteins, which are components of the coactivator SWI/SNF complex. This cooperation is mediated through a physical interaction between RB and hbrm/BRG-1. We show here that in transfected cells RB can contact both E2F1 and hbrm at the same time, thereby targeting hbrm to E2F1. E2F1 and hbrm are indeed found within the same complex
in vivo
. Furthermore, RB and hbrm cooperate to repress E2F1 activity in transient transfection assays. The ability of hbrm to cooperate with RB to repress E2F1 is dependent upon several distinct domains of hbrm, including the RB binding domain and the NTP binding site. However, the bromodomain seems dispensable for this activity. Taken together, our results point out an unexpected role of corepressor for the hbrm protein. The ability of hbrm and RB to cooperate in repressing E2F1 activity could be an underlying mechanism for the observed cooperation between hbrm and RB to induce G
1
arrest. Finally, we demonstrate that the domain of hbrm that binds RB has transcriptional activation potential which RB can repress. This suggest that RB not only targets hbrm but also regulates its activity.
Evolution of gilled mushrooms and puffballs inferred from ribosomal DNA sequences
Authors: Hibbett David S. , Pine Elizabeth M. , Langer Ewald , Langer Gitta , Donoghue Michael J.
Published: July 2002

Homobasidiomycete fungi display many complex fruiting body morphologies, including mushrooms and puffballs, but their anatomical simplicity has confounded efforts to understand the evolution of these forms. We performed a comprehensive phylogenetic analysis of homobasidiomycetes, using sequences from nuclear and mitochondrial ribosomal DNA, with an emphasis on understanding evolutionary relationships of gilled mushrooms and puffballs. Parsimony-based optimization of character states on our phylogenetic trees suggested that strikingly similar gilled mushrooms evolved at least six times, from morphologically diverse precursors. Approximately 87% of gilled mushrooms are in a single lineage, which we call the “euagarics.” Recently discovered 90 million-year-old fossil mushrooms are probably euagarics, suggesting that (
i
) the origin of this clade must have occurred no later than the mid-Cretaceous and (
ii
) the gilled mushroom morphology has been maintained in certain lineages for tens of millions of years. Puffballs and other forms with enclosed spore-bearing structures (Gasteromycetes) evolved at least four times. Derivation of Gasteromycetes from forms with exposed spore-bearing structures (Hymenomycetes) is correlated with repeated loss of forcible spore discharge (ballistospory). Diverse fruiting body forms and spore dispersal mechanisms have evolved among Gasteromycetes. Nevertheless, it appears that Hymenomycetes have never been secondarily derived from Gasteromycetes, which suggests that the loss of ballistospory has constrained evolution in these lineages.
Dynamic regulation of c-Jun N-terminal kinase activity in mouse brain by environmental stimuli
Authors: Xu Xiao , Raber Jacob , Yang Daseng , Su Bing , Mucke Lennart
Published: July 2002

Activation of the recently identified c-Jun N-terminal kinases (JNKs) typically results in programmed cell death (apoptosis) in neurons and other cell types grown in culture. However, the effects of JNK activation in the central nervous systemin vivoare unknown. At baseline, JNK activity in mice was on average 17-fold higher in brain than in peripheral organs, whereas JNK protein levels were similar. In brain, JNK was expressed primarily in neurons. Restraining mice or allowing them to explore a novel environment rapidly increased JNK activity 3- to 15-fold in various brain regions, but these manipulations did not increase brain activity of the extracellular signal-regulated kinase. Because noninvasive environmental stimuli that do not induce neurodegeneration elicited prominent increases in JNK activity in the brain, we conclude that acute activation of the JNK cascade in central nervous system neurons does not induce neuronal apoptosisin vivo. In contrast, the high baseline activity of JNK in the brain and the activation of the JNK cascade by environmental stimuli suggest that this kinase may play an important physiological role in neuronal function.
In vivotelomere dynamics of human hematopoietic stem cells
Authors: Notaro Rosario , Cimmino Amelia , Tabarini Diane , Rotoli Bruno , Luzzatto Lucio
Published: July 2002

Agingin vivoand cell divisionin vitroare associated with telomere shortening. Several lines of evidence suggest that telomere length may be a good predictor of the long term replicative capacity of cells. To investigate the natural fate of chromosome telomeres of hematopoietic stem cellsin vivo, we measured the telomere length of peripheral blood granulocytes from 11 fully engrafted bone marrow transplant recipients and from their respective donors. In 10 of 11 donor–recipient pairs, the telomere length was significantly reduced in the recipient and the extent of reduction correlated inversely with the number of nucleated cells infused. These data provide internally controlledin vivoevidence that, concomitantly with their proliferation, hematopoietic stem cells lose telomere length; it is possible that, as a result, their proliferative potential is reduced. These findings must be taken into account when developing new protocols in which few stem cells are used for bone marrow transplantation or for gene therapy.
The Dr1/DRAP1 heterodimer is a global repressor of transcription
in vivo
Authors: Kim SungJoon , Na Jong G. , Hampsey Michael , Reinberg Danny
Published: July 2002

A general repressor extensively studied
in vitro
is the human Dr1/DRAP1 heterodimeric complex. To elucidate the function of Dr1 and DRAP1
in vivo
, the yeast
Saccharomyces cerevisiae
Dr1/DRAP1 repressor complex was identified. The repressor complex is encoded by two essential genes, designated
YDR1
and
BUR6
. The inviability associated with deletion of the yeast genes can be overcome by expressing the human genes. However, the human corepressor DRAP1 functions in yeast only when human Dr1 is coexpressed. The yDr1/Bur6 complex represses transcription
in vitro
in a reconstituted RNA polymerase II transcription system. Repression of transcription could be overcome by increasing the concentration of TATA-element binding protein (TBP). Consistent with the
in vitro
results, overexpression of
YDR1 in vivo
resulted in decreased mRNA accumulation. Furthermore,
YDR1
overexpression impaired cell growth, an effect that could be rescued by overexpression of TBP. In agreement with our previous studies
in vitro
, we found that overexpression of Dr1
in vivo
also affected the accumulation of RNA polymerase III transcripts, but not of RNA polymerase I transcripts. Our results demonstrate that Dr1 functions as a repressor of transcription
in vivo
and, moreover, directly targets TBP, a global regulator of transcription.
Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin
Authors: Wilson Carole L. , Heppner Kathleen J. , Labosky Patricia A. , Hogan Brigid L. M. , Matrisian Lynn M.
Published: July 2002

Matrix metalloproteinases (MMPs) classically have been implicated in basement membrane destruction associated with late-stage tumor cell invasion and metastasis. However, recent studies have demonstrated that one MMP family member, matrilysin, is expressed in a high percentage of early-stage human colorectal tumors. We analyzed matrilysin expression in benign intestinal tumors from mice heterozygous for the
Apc
Min
allele (
Min
/+) and found that the mRNA was induced in the majority (88%) of these adenomas. Protein was detected in the tumor cells, where, surprisingly, it was predominantly immunolocalized to the lumenal surface of dysplastic glands rather than the basement membrane or extracellular matrix. To address the role of matrilysin in
Min
intestinal tumorigenesis, we generated
Min
/+ mice deficient in this MMP by gene targeting and homologous recombination. The absence of matrilysin resulted in a reduction in mean tumor multiplicity in
Min
/+ animals of approximately 60% and a significant decrease in the average tumor diameter. Based on these findings, we conclude that matrilysin is a suppressor of the
Min
phenotype, possibly by functioning in a capacity independent of matrix degradation. These results argue for the use of MMP inhibitors in the treatment and prevention of early-stage colon cancer.
Chirality of DNA trefoils: Implications in intramolecular synapsis of distant DNA segments
Authors: Shaw Stanley Y. , Wang James C.
Published: July 2002

We show that supercoiling of a DNA trefoil, the simplest knotted ring, perturbs differently the spatial writhe of its two chiral forms. As a consequence, the negative-noded and positive-noded DNA trefoils can be resolved by gel electrophoresis. Analysis of the chirality of trefoils produced by cyclization of two linear DNAs demonstrates that the two chiral trefoils are produced in equal amounts, suggesting that these DNAs do not prefer intrinsic writhe of one chirality or the other. In contrast, knotting of nicked DNA rings by a molar excess of
Saccharomyces cerevisiae
DNA topoisomerase II produces more negative-noded than positive-noded trefoils, indicating an asymmetry in the interaction between the enzyme and DNA crossovers of different signs. These results suggest that asymmetry in DNA crossovers and intrinsic or ligand-induced writhe in a DNA might be detectable from an analysis of trefoil chirality.
Single amino acid residue as a functional determinant of rod and cone visual pigments
Authors: Imai Hiroo , Kojima Daisuke , Oura Tomonori , Tachibanaki Shuji , Terakita Akihisa , Shichida Yoshinori
Published: July 2002

The visual transduction processes in rod and cone photoreceptor cells begin with photon absorption by the different types of visual pigments. Cone visual pigments exhibit faster regeneration from 11-
cis
-retinal and opsin and faster decay of physiologically active intermediate (meta II) than does the rod visual pigment, rhodopsin, as expected, due to the functional difference between rod and cone photoreceptor cells. To identify the amino acid residue(s) responsible for the difference in molecular properties between rod and cone visual pigments, we selected three amino acid positions (64, 122, and 150), where cone visual pigments have amino acid residues electrically different from those of rhodopsin, and prepared mutants of rhodopsin and chicken green-sensitive cone visual pigment. The results showed that the replacement of Glu-122 of rhodopsin by the residue containing green- or red-sensitive cone pigment converted rhodopsin’s rates of regeneration and meta II decay into those of the respective cone pigments, whereas the introduction of Glu-122 into green-sensitive cone visual pigment changed the rates of these processes into rates similar to those of rhodopsin. Furthermore, exchange of the residue at position 122 between rhodopsin and chicken green-sensitive cone pigment interchanges their efficiencies in activating retinal G protein transducin. Thus, the amino acid residue at position 122 is a functional determinant of rod and cone visual pigments.
T cells cooperate with passive antibody to modifyCryptococcus neoformansinfection in mice
Authors: Yuan RuiRong , Casadevall Arturo , Oh Jin , Scharff Matthew D.
Published: July 2002

Cryptococcus neoformansis an encapsulated fungus that is a major cause of meningitis in patients with AIDS. In immunocompetent mice, administration of IgG1 mAb protects against cryptococcal infection, whereas administration of IgG3 is not protective and can accelerate the infection. In beige mice with impaired natural killer cell function, the effects of IgG1 and IgG3 are similar to those observed in immunocompetent mice, suggesting that natural killer cells are not crucial for antibody-mediated modulation of cryptococcal infection. In mice lacking CD4+T cells, IgG1 is not protective and IgG3 accelerates infection, indicating that CD4+T cells are required for antibody-mediated protection. In mice lacking CD8+T cells, both IgG1 and IgG3 antibodies prolong survival, indicating that acceleration of the disease process by IgG3 involves CD8+T cells. Both IgG1-mediated protection and IgG3-mediated acceleration of infection require interferon γ. These results reveal a functional dependence of passively administered antibody on cellular immunity in cryptococcal infection in mice and have implications for antibody-based therapies in humans in the setting of CD4+lymphopenia.
mcm5/cdc46-bob1
bypasses the requirement for the S phase activator Cdc7p
Authors: Hardy Christopher F. J. , Dryga Olga , Seematter Sara , Pahl Paula M. B. , Sclafani Robert A.
Published: July 2002

Cdc7p is a protein kinase that is required for G
1
/S transition and initiation of DNA replication in
Saccharomyces cerevisiae
. The mechanisms whereby Cdc7p and its substrates exerts their effects are unknown. We report here the characterization in
S. cerevisiae
of a recessive mutation in a member of the MCM family, MCM5/
CDC46
, which bypasses the requirement for Cdc7p and its interacting factor Dbf4p. Because the MCM family of evolutionarily conserved proteins have been implicated in restricting DNA replication to once per cell cycle, our studies suggest that Cdc7p is required late in G
1
because in its absence the Mcm5p/Cdc46p blocks the initiation of DNA replication. Moreover, Mcm5p/Cdc46p may have both positive and negative effects on the ability of cell to initiate replication.
Transduction of interleukin-2 antiapoptotic and proliferative signals via Akt protein kinase
Authors: Ahmed Naheed N. , Grimes H. Leighton , Bellacosa Alfonso , Chan Tung O. , Tsichlis Philip N.
Published: July 2002

The interleukin-2 (IL-2) receptor (IL-2R) is composed of three subunits. Of these, IL-2Rα is required for high-affinity IL-2 binding, while IL-2Rβ and IL-2Rγ
c
are required for the transduction of IL-2-generated signals. Signals transduced via the S region of the IL-2Rβ (amino acids 267–322) in BAF/3 cells activate the phosphatidylinositol 3-kinase (PI3-kinase) and induce the expression of Bcl-2 and c-myc. Through the induction of Bcl-2, IL-2 inhibits apoptosis and through the combination of Bcl-2 and c-myc it stimulates progression through the cell cycle. Here we show that the protein kinase encoded by the
Akt
proto-oncogene is activated by IL-2. Akt activation by IL-2 depends on PI3-kinase signals transduced via the S region of the IL-2Rβ and is linked to the translocation of Akt to the cell membrane. Expression of catalytically active Akt mutants in BAF/3 cells expressing IL-2Rβ[A0]ΔS promotes the expression of Bcl-2 and c-myc, inhibits apoptosis induced by IL-3 deprivation or staurosporine, and stimulates cell cycle progression. The same mutants also stimulate cell cycle progression in 2780a, an IL-2-dependent T cell line that undergoes G
1
arrest rather than apoptosis after IL-2 deprivation. The activation of Akt by IL-2 via the PI3-kinase and the rescue of the PI3-kinase-mediated antiapoptotic and proliferative IL-2 signals by catalytically active Akt indicate that these signals are transduced by Akt.
DNA ligands that bind tightly and selectively to cellobiose
Authors: Yang Qing , Goldstein Irwin J. , Mei Houng-Yau , Engelke David R.
Published: July 2002

Cell surface oligosaccharides have been shown to play essential biological roles in such diverse biological phenomena as cellular adhesion, molecular recognition, and inflammatory response. The development of high-affinity ligands capable of selectively recognizing a variety of small motifs in different oligosaccharides would be of significant interest as experimental and diagnostic tools. As a step toward this goal we have developed DNA ligands that recognize the disaccharide cellobiose, whether in soluble form or as the repeating unit of the polymer, cellulose. These DNA “aptamers” bind with high selectivity to cellobiose with little or no affinity for the related disaccharides lactose, maltose, and gentiobiose. Thus, the DNA ligands can discriminate sugar epimers, anomers, and disaccharide linkages.
A unified statistical framework for sequence comparison and structure comparison
Authors: Levitt Michael , Gerstein Mark
Published: July 2002

We present an approach for assessing the significance of
sequence and structure comparisons by using nearly identical
statistical formalisms for both sequence and structure. Doing so
involves an all-vs.-all comparison of protein domains [taken here from
the Structural Classification of Proteins (scop) database] and then
fitting a simple distribution function to the observed scores. By using
this distribution, we can attach a statistical significance to each
comparison score in the form of a
P
value, the
probability that a better score would occur by chance. As expected, we
find that the scores for sequence matching follow an extreme-value
distribution. The agreement, moreover, between the
P
values that we derive from this distribution and those reported by
standard programs (e.g.,
blast
and
fasta
validates our approach. Structure comparison scores also follow an
extreme-value distribution when the statistics are expressed in terms
of a structural alignment score (essentially the sum of reciprocated
distances between aligned atoms minus gap penalties). We find that the
traditional metric of structural similarity, the rms deviation in atom
positions after fitting aligned atoms, follows a different distribution
of scores and does not perform as well as the structural alignment
score. Comparison of the sequence and structure statistics for pairs of
proteins known to be related distantly shows that structural comparison
is able to detect approximately twice as many distant relationships as
sequence comparison at the same error rate. The comparison also
indicates that there are very few pairs with significant similarity in
terms of sequence but not structure whereas many pairs have significant
similarity in terms of structure but not sequence.
Resistance gene
N
-mediated
de novo
synthesis and activation of a tobacco mitogen-activated protein kinase by tobacco mosaic virus infection
Authors: Zhang Shuqun , Klessig Daniel F.
Published: July 2002

Salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two distinct members of the mitogen-activated protein (MAP) kinase family, are activated in tobacco resisting infection by tobacco mosaic virus (TMV). WIPK activation by TMV depends on the disease-resistance gene
N
because infection of susceptible tobacco not carrying the
N
gene failed to activate WIPK. Activation of WIPK required not only posttranslational phosphorylation but also a preceding rise in its mRNA and
de novo
synthesis of WIPK protein. The induction by TMV of WIPK mRNA and protein also occurred systemically. Its activation at the mRNA, protein, and enzyme levels was independent of salicylic acid. The regulation of WIPK at multiple levels by an
N
gene-mediated signal(s) suggests that this MAP kinase may be an important component upstream of salicylic acid in the signal-transduction pathway(s) leading to local and systemic resistance to TMV.
Low-dose expression of a human apolipoprotein E transgene in macrophages restores cholesterol efflux capacity of apolipoprotein E-deficient mouse plasma
Authors: Zhu Yenhong , Bellosta Stefano , Langer Claus , Bernini Franco , Pitas Robert E. , Mahley Robert W. , Assmann Gerd , von Eckardstein Arnold
Published: July 2002

Apolipoprotein E- (apoE) deficient (
E
−/−
) mice develop severe hyperlipidemia and diffuse atherosclerosis. Low-dose expression of a human apoE3 transgene in macrophages of apoE-deficient mice (
E
−/−
hTgE
+/0
), which results in about 5% of wild-type apoE plasma levels, did not correct hyperlipidemia but significantly reduced the extent of atherosclerotic lesions. To investigate the contribution of apoE to reverse cholesterol transport, we compared plasmas of wild-type (
E
+/+
),
E
−/−
, and
E
−/−
hTgE
+/0
mice for the appearance of apoE-containing lipoproteins by electrophoresis and their capacity to take up and esterify
3
H-labeled cholesterol from radiolabeled fibroblasts or J774 macrophages. Wild-type plasma displayed lipoproteins containing apoE that were the size of high density lipoprotein and that had either electrophoretic α or γ mobilities. Similar particles were also present in
E
−/−
hTgE
+/0
plasma. Depending on incubation time,
E
−/−
plasma released 48–74% less
3
H-labeled cholesterol from fibroblasts than
E
+/+
plasma, whereas cholesterol efflux into
E
−/−
hTgE
+/0
plasma was only 11–25% lower than into
E
+/+
plasma.
E
−/−
hTgE
+/0
plasma also released 10% more
3
H-labeled cholesterol from radiolabeled J774 macrophages than
E
−/−
plasma.
E
+/+
and
E
−/−
hTgE
+/0
plasma each esterified significantly more cell-derived
3
H-labeled cholesterol than
E
−/−
plasma. Moreover,
E
−/−
plasma accumulated much smaller proportions of fibroblast-derived
3
H-labeled cholesterol in fractions with electrophoretic γ and α mobility than
E
+/+
and
E
−/−
hTgE
+/0
plasma. Thus, low-dose expression of apoE in macrophages nearly restored the cholesterol efflux capacity of apoE-deficient plasma through the formation of apoE-containing particles, which efficiently take up cell-derived cholesterol, and through the increase of cholesterol esterification activity. Thus, macrophage-derived apoE may protect against atherosclerosis by increasing cholesterol efflux from arterial wall cells.
The specific features of methionine biosynthesis and metabolism in plants
Authors: Ravanel Stéphane , Gakière Bertrand , Job Dominique , Douce Roland
Published: July 2002

Plants, unlike other higher eukaryotes, possess all the necessary enzymatic equipment for
de novo
synthesis of methionine, an amino acid that supports additional roles than simply serving as a building block for protein synthesis. This is because methionine is the immediate precursor of
S
-adenosylmethionine (AdoMet), which plays numerous roles of being the major methyl-group donor in transmethylation reactions and an intermediate in the biosynthesis of polyamines and of the phytohormone ethylene. In addition, AdoMet has regulatory function in plants behaving as an allosteric activator of threonine synthase. Among the AdoMet-dependent reactions occurring in plants, methylation of cytosine residues in DNA has raised recent interest because impediment of this function alters plant morphology and induces homeotic alterations in flower organs. Also, AdoMet metabolism seems somehow implicated in plant growth via an as yet fully understood link with plant-growth hormones such as cytokinins and auxin and in plant pathogen interactions. Because of this central role in cellular metabolism, a precise knowledge of the biosynthetic pathways that are responsible for homeostatic regulation of methionine and AdoMet in plants has practical implications, particularly in herbicide design.
Positional cloning of
ZNF
217
and
NABC
1
: Genes amplified at 20q13.2 and overexpressed in breast carcinoma
Authors: Collins Colin , Rommens Johanna M. , Kowbel David , Godfrey Tony , Tanner Minna , Hwang Soo-in , Polikoff Daniel , Nonet Genevieve , Cochran Joanne , Myambo Ken , Jay Karen E. , Froula Jeff , Cloutier Thomas , Kuo Wen-Lin , Yaswen Paul , Dairkee Shanaz , Giovanola Jennifer , Hutchinson Gordon B. , Isola Jorma , Kallioniemi Olli-P , Palazzolo Mike , Martin Chris , Ericsson Cheryl , Pinkel Dan , Albertson Donna , Li Wu-Bo , Gray Joe W.
Published: July 2002

We report here the molecular cloning of an ≈1-Mb region of recurrent amplification at 20q13.2 in breast cancer and other tumors and the delineation of a 260-kb common region of amplification. Analysis of the 1-Mb region produced evidence for five genes,
ZNF217
,
ZNF218
, and
NABC1
,
PIC1L
(
PIC1-l
ike),
CYP24
, and a pseudogene
CRP
(
C
yclophillin
R
elated
P
seudogene).
ZNF217
and
NABC1
emerged as strong candidate oncogenes and were characterized in detail.
NABC1
is predicted to encode a 585-aa protein of unknown function and is overexpressed in most but not all breast cancer cell lines in which it was amplified.
ZNF217
is centrally located in the 260-kb common region of amplification, transcribed in multiple normal tissues, and overexpressed in all cell lines and tumors in which it is amplified and in two in which it is not.
ZNF217
is predicted to encode alternately spliced, Kruppel-like transcription factors of 1,062 and 1,108 aa, each having a DNA-binding domain (eight C2H2 zinc fingers) and a proline-rich transcription activation domain.
Small heat shock protein of
Methanococcus jannaschii
, a hyperthermophile
Authors: Kim Rosalind , Kim Kyeong Kyu , Yokota Hisao , Kim Sung-Hou
Published: July 2002

Small heat shock proteins (sHSPs) belong to a family of 12- to 43-kDa proteins that are ubiquitous and are conserved in amino acid sequence among all organisms. A sHSP homologue of
Methanococcus jannaschii
, a hyperthermophilic Archaeon, forms a homogeneous multimer comprised of 24 monomers with a molecular mass of 400 kDa in contrast to other sHSPs that show heterogeneous oligomeric complexes. Electron microscopy analysis revealed a spherically shaped oligomeric structure ≈15–20 nm in diameter. The protein confers thermal protection of other proteins
in vitro
as found in other sHSPs.
Escherichia coli
cell extracts containing the protein were protected from heat-denatured precipitation when heated up to 100°C, whereas extracts from cells not expressing the protein were heat-sensitive at 60°C. Similar results were obtained when purified sHSP protein was added to an
E. coli
cell lysate. The protein also prevented the aggregation of two purified proteins: single-chain monellin (SCM) at 80°C and citrate synthase at 40°C.
Brain-derived neurotrophic factor mediates estradiol-induced dendritic spine formation in hippocampal neurons
Authors: Murphy Diane D. , Cole Nelson B. , Segal Menahem
Published: July 2002

Dendritic spines are of major importance in information processing and memory formation in central neurons. Estradiol has been shown to induce an increase of dendritic spine density on hippocampal neurons
in vivo
and
in vitro
. The neurotrophin brain-derived neurotrophic factor (BDNF) recently has been implicated in neuronal maturation, plasticity, and regulation of GABAergic interneurons. We now demonstrate that estradiol down-regulates BDNF in cultured hippocampal neurons to 40% of control values within 24 hr of exposure. This, in turn, decreases inhibition and increases excitatory tone in pyramidal neurons, leading to a 2-fold increase in dendritic spine density. Exogenous BDNF blocks the effects of estradiol on spine formation, and BDNF depletion with a selective antisense oligonucleotide mimics the effects of estradiol. Addition of BDNF antibodies also increases spine density, and diazepam, which facilitates GABAergic neurotransmission, blocks estradiol-induced spine formation. These observations demonstrate a functional link between estradiol, BDNF as a potent regulator of GABAergic interneurons, and activity-dependent formation of dendritic spines in hippocampal neurons.
Reactivity of the HIV-1 nucleocapsid protein p7 zinc finger domains from the perspective of density-functional theory
Authors: Maynard A. T. , Huang M. , Rice W. G. , Covell D. G.
Published: July 2002

The reaction of the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein p7 (NCp7) with a variety of electrophilic agents was investigated by experimental measurements of Trp
37
fluorescence decay and compared with theoretical measures of reactivity based on density-functional theory in the context of the hard and soft acids and bases principle. Statistically significant correlations were found between rates of reaction and the ability of these agents to function as soft electrophiles. Notably, the molecular property that correlated strongest was the ratio of electronegativity to hardness, χ
2
/η, a quantity related to the capacity of an electrophile to promote a soft (covalent) reaction. Electronic and steric determinants of the reaction were also probed by Fukui function and frontier-orbital overlap analysis in combination with protein–ligand docking methods. This analysis identified selective ligand docking regions within the conserved zinc finger domains that promoted reaction. The Cys
49
thiolate was found overall to be the NCp7 site most susceptible to electrophilic attack.
A common mutation in the methylenetetrahydrofolate reductase gene is associated with an accumulation of formylated tetrahydrofolates in red blood cells
Authors: Bagley Pamela J. , Selhub Jacob
Published: July 2002

A common mutation (C677T) in the gene encoding for methylenetetrahydrofolate reductase (MTHFR) (5-methyltetrahydrofolate:(acceptor) oxidoreductase, EC
1.7.99.5
), a key regulatory enzyme in one-carbon metabolism, results in a thermolabile variant of the MTHFR enzyme with reduced activity
in vitro
. In the present study we used a chromatographic method for folate analysis to test the hypothesis that this mutation would be associated with altered distribution of red blood cell (RBC) folates. An alteration was found as manifested by the presence of formylated tetrahydrofolate polyglutamates in addition to methylated derivatives in the RBCs from homozygous mutant individuals. 5-Methyltetrahydrofolate polyglutamates were the only folate form found in RBCs from individuals with the wild-type genotype. Existence of formylated folates in RBCs only from individuals with the thermolabile MTHFR is consistent with the hypothesis that there is
in vivo
impairment in the activity of the thermolabile variant of MTHFR and that this impairment results in an altered distribution of RBC folates.
Defects in embryonic hindbrain development and fetal resorption resulting from vitamin A deficiency in the rat are prevented by feeding pharmacological levels of all-
trans-
retinoic acid
Authors: White Jeffrey C. , Shankar V. Narayanaswamy , Highland Margaret , Epstein Miles L. , DeLuca Hector F. , Clagett-Dame Margaret
Published: July 2002

Vitamin A is required for reproduction and normal embryonic development. We have determined that all-
trans-
retinoic acid (atRA) can support development of the mammalian embryo to parturition in vitamin A-deficient (VAD) rats. At embryonic day (E) 0.5, VAD dams were fed purified diets containing either 12 μg of atRA per g of diet (230 μg per rat per day) or 250 μg of atRA per g of diet (4.5 mg per rat per day) or were fed the purified diet supplemented with a source of retinol (100 units of retinyl palmitate per day). An additional group was fed both 250 μg of atRA per g of diet in combination with retinyl palmitate. Embryonic survival to E12.5 was similar for all groups. However, embryonic development in the group fed 12 μg of atRA per g of diet was grossly abnormal. The most notable defects were in the region of the hindbrain, which included a loss of posterior cranial nerves (IX, X, XI, and XII) and postotic pharyngeal arches as well as the presence of ectopic otic vesicles and a swollen anterior cardinal vein. All embryonic abnormalities at E12.5 were prevented by feeding pharmacological amounts of atRA (250 μg/g diet) or by supplementation with retinyl palmitate. Embryos from VAD dams receiving 12 μg of atRA per g of diet were resorbed by E18.5, whereas those in the group fed 250 μg of atRA per g of diet survived to parturition but died shortly thereafter. Equivalent results were obtained by using commercial grade atRA or atRA that had been purified to eliminate any potential contamination by neutral retinoids, such as retinol. Thus, 250 μg of atRA per g of diet fed to VAD dams (≈4.5 mg per rat per day) can prevent the death of embryos at midgestation and prevents the early embryonic abnormalities that arise when VAD dams are fed insufficient amounts of atRA.
Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases
Authors: Horton Nancy C. , Newberry Kate Juliet , Perona John J.
Published: July 2002

The 2.15-Å resolution cocrystal structure of
Eco
RV endonuclease mutant T93A complexed with DNA and Ca
2+
ions reveals two divalent metals bound in one of the active sites. One of these metals is ligated through an inner-sphere water molecule to the phosphate group located 3′ to the scissile phosphate. A second inner-sphere water on this metal is positioned approximately in-line for attack on the scissile phosphate. This structure corroborates the observation that the pro-S
P
phosphoryl oxygen on the adjacent 3′ phosphate cannot be modified without severe loss of catalytic efficiency. The structural equivalence of key groups, conserved in the active sites of
Eco
RV,
Eco
RI,
Pvu
II, and
Bam
HI endonucleases, suggests that ligation of a catalytic divalent metal ion to this phosphate may occur in many type II restriction enzymes. Together with previous cocrystal structures, these data allow construction of a detailed model for the pretransition state configuration in
Eco
RV. This model features three divalent metal ions per active site and invokes assistance in the bond-making step by a conserved lysine, which stabilizes the attacking hydroxide ion nucleophile.
Insecticide resistance resulting from an absence of target-site gene product
Authors: Wilson Thomas G. , Ashok Mudgapalli
Published: July 2002

Genetic changes in insects that lead to insecticide resistance include point mutations and up-regulation/amplification of detoxification genes. Here, we report a third mechanism, resistance caused by an absence of gene product. Mutations of the
Methoprene-tolerant
(
Met
) gene of
Drosophila melanogaster
result in resistance to both methoprene, a juvenile hormone (JH) agonist insecticide, and JH. Previous results have demonstrated a mechanism of resistance involving an intracellular JH binding protein that has reduced ligand affinity in
Met
flies. We show that a γ-ray induced allele,
Met
27
, completely lacks
Met
transcript during the insecticide-sensitive period in development. Although
Met
27
homozygotes have reduced oogenesis, they are viable, demonstrating that
Met
is not a vital gene. Most target-site resistance genes encode vital proteins and thus have few mutational changes that permit both resistance and viability. In contrast, resistance genes such as
Met
that encode nonvital insecticide target proteins can have a variety of mutational changes that result in an absence of functional gene product and thus should show higher rates of resistance evolution.
Evidence that a prominent cavity in the coiled coil of HIV type 1 gp41 is an attractive drug target
Authors: Chan David C. , Chutkowski Christine T. , Kim Peter S.
Published: July 2002

Synthetic C peptides, corresponding to the C helix of the HIV type 1 (HIV-1) gp41 envelope protein, are potent inhibitors of HIV-1 membrane fusion. One such peptide is in clinical trials. The crystal structure of the gp41 core, in its proposed fusion-active conformation, is a trimer of helical hairpins in which three C helices pack against a central coiled coil. Each C helix shows especially prominent contacts with one of three symmetry-related, hydrophobic cavities on the surface of the coiled coil. We show that the inhibitory activity of the C peptide C34 depends on its ability to bind to this coiled-coil cavity. Moreover, examining a series of C34 peptide variants with modified cavity-binding residues, we find a linear relationship between the logarithm of the inhibitory potency and the stability of the corresponding helical-hairpin complexes. Our results provide strong evidence that this coiled-coil cavity is a good drug target and clarify the mechanism of C peptide inhibition. They also suggest simple, quantitative assays for the identification and evaluation of analogous inhibitors of HIV-1 entry.