bluuebunny/crossref_metadata_2025 huggingface dataset:
Three small nucleolar RNAs that are involved in ribosomal RNA precursor processing
Authors: Mishra Rakesh K. , Eliceiri George L.
Published: July 2002

Three small nucleolar RNAs (snoRNAs), E1, E2 and E3, have been described that have unique sequences and interact directly with unique segments of pre-rRNA
in vivo
. In this report, injection of antisense oligodeoxynucleotides into
Xenopus laevis
oocytes was used to target the specific degradation of these snoRNAs. Specific disruptions of pre-rRNA processing were then observed, which were reversed by injection of the corresponding
in vitro
-synthesized snoRNA. Degradation of each of these three snoRNAs produced a unique rRNA maturation phenotype. E1 RNA depletion shut down 18 rRNA formation, without overaccumulation of 20S pre-rRNA. After E2 RNA degradation, production of 18S rRNA and 36S pre-rRNA stopped, and 38S pre-rRNA accumulated, without overaccumulation of 20S pre-rRNA. E3 RNA depletion induced the accumulation of 36S pre-rRNA. This suggests that each of these snoRNAs plays a different role in pre-rRNA processing and indicates that E1 and E2 RNAs are essential for 18S rRNA formation. The available data support the proposal that these snoRNAs are at least involved in pre-rRNA processing at the following pre-rRNA cleavage sites: E1 at the 5′ end and E2 at the 3′ end of 18S rRNA, and E3 at or near the 5′ end of 5.8S rRNA.
Neurotrophin regulation of ionic currents and cell size depends on cell context
Authors: Sherwood Nina Tang , Lesser Steven S. , Lo Donald C.
Published: July 2002

Trk receptor activation by neurotrophins is often considered to have a defined set of actions on target neurons, including supporting neuronal survival, inducing morphological differentiation, and regulating a host of target genes that specify neuronal phenotype. It is not known if all such regulatory effects are obligatory, or if some may vary depending on the cell context in which the receptors are expressed. We have examined this issue by comparing neurotrophin effects on the regulation of electrical excitability and morphological differentiation in two strains of PC12 cells. We found that while neurotrophins induced neurite extension and increased calcium currents in both PC12 cell types, sodium current levels were regulated in only one of these strains. Moreover, we found little correlation between calcium current levels and the extent of morphological differentiation when compared in individual cells of a single strain. Thus, the regulatory effects of neurotrophins on cell phenotype are not fully determined by the Trk receptors that they activate; rather, they can vary with differences in cell context that arise not only between different cell lineages, but also between individual cells of clonal relation.
Bipartite substrate discrimination by human nucleotide excision repair
Authors: Hess Martin T. , Schwitter Urs , Petretta Mario , Giese Bernd , Naegeli Hanspeter
Published: July 2002

Mammalian nucleotide excision repair (NER) eliminates carcinogen–DNA adducts by double endonucleolytic cleavage and subsequent release of 24–32 nucleotide-long single-stranded fragments. Here we manipulated the deoxyribose–phosphate backbone of DNA to analyze the mechanism by which damaged strands are discriminated as substrates for dual incision. We found that human NER is completely inactive on DNA duplexes containing single C4′-modified backbone residues. However, the same C4′ backbone variants, which by themselves do not perturb complementary hydrogen bonds, induced strong NER reactions when incorporated into short segments of mispaired bases. No oligonucleotide excision was detected when DNA contained abnormal base pairs without concomitant changes in deoxyribose–phosphate composition. Thus, neither C4′ backbone lesions nor improper base pairing stimulated human NER, but the combination of these two substrate alterations constituted an extremely potent signal for double DNA incision. In summary, we used C4′-modified backbone residues as molecular tools to dissect DNA damage recognition by human NER into separate components and identified a bipartite discrimination mechanism that requires changes in DNA chemistry with concurrent disruption of Watson–Crick base pairing.
Nucleus reticularis neurons mediate diverse inhibitory effects in thalamus
Authors: Cox Charles L. , Huguenard John R. , Prince David A.
Published: July 2002

Detailed information regarding the contribution of individual γ-aminobutyric acid (GABA)-containing inhibitory neurons to the overall synaptic activity of single postsynaptic cells is essential to our understanding of fundamental elements of synaptic integration and operation of neuronal circuits. For example, GABA-containing cells in the thalamic reticular nucleus (nRt) provide major inhibitory innervation of thalamic relay nuclei that is critical to thalamocortical rhythm generation. To investigate the contribution of individual nRt neurons to the strength of this internuclear inhibition, we obtained whole-cell recordings of unitary inhibitory postsynaptic currents (IPSCs) evoked in ventrobasal thalamocortical (VB) neurons by stimulation of single nRt cells in rat thalamic slices, in conjunction with intracellular biocytin labeling. Two types of monosynaptic IPSCs could be distinguished. “Weak” inhibitory connections were characterized by a significant number of postsynaptic failures in response to presynaptic nRt action potentials and relatively small IPSCs. In contrast, “strong” inhibition was characterized by the absence of postsynaptic failures and significantly larger unitary IPSCs. By using miniature IPSC amplitudes to infer quantal size, we estimated that unitary IPSCs associated with weak inhibition resulted from activation of 1–3 release sites, whereas stronger inhibition would require simultaneous activation of 5–70 release sites. The inhibitory strengths were positively correlated with the density of axonal swellings of the presynaptic nRt neurons, an indicator that characterizes different nRt axonal arborization patterns. These results demonstrate that there is a heterogeneity of inhibitory interactions between nRt and VB neurons, and that variations in gross morphological features of axonal arbors in the central nervous system can be associated with significant differences in postsynaptic response characteristics.
Cell death induction by the acute promyelocytic leukemia-specific PML/RARα fusion protein
Authors: Ferrucci Pier Francesco , Grignani Francesco , Pearson Mark , Fagioli Marta , Nicoletti Ildo , Pelicci Pier Giuseppe
Published: July 2002

PML/RARα is the abnormal protein product generated by the acute promyelocytic leukemia-specific t(15;17). Expression of PML/RARα in hematopoietic precursor cell lines induces block of differentiation and promotes survival. We report here that PML/RARα has a potent growth inhibitory effect on all nonhematopoietic cell lines and on the majority of the hematopoietic cell lines tested. Inducible expression of PML/RARα in fibroblasts demonstrated that the basis for the growth suppression is induction of cell death. Deletion of relevant promyelocytic leukemia (PML) and retinoic acid receptor (RARα) domains within the fusion protein revealed that its growth inhibitory effect depends on the integrity of the PML aminoterminal region (RING, B1, B2, and coiled coil regions) and the RARα DNA binding region. Analysis of the nuclear localization of the same PML/RARα deletion mutants by immunofluorescence and cell fractionation revealed that the biological activity of the fusion protein correlates with its microspeckled localization and its association to the nuclear matrix. The PML aminoterminal region, but not the RARα zinc fingers, is required for the proper nuclear localization of PML/RARα. We propose that the matrix-associated microspeckles are the active sites of PML/RARα and that targeting of RARα sequences to this specific nuclear subdomain through PML sequences is crucial to the activity of the fusion protein on survival regulation.
Incomplete penetrance of familial retinoblastoma linked to germ-line mutations that result in partial loss of RB function
Authors: Otterson Gregory A. , Chen Wei-dong , Coxon Amy B. , Khleif Samir N. , Kaye Frederic J.
Published: July 2002

To study the molecular basis for the clinical phenotype of incomplete penetrance of familial retinoblastoma, we have examined the functional properties of three
RB
mutations identified in the germ line of five different families with low penetrance.
RB
mutants isolated from common adult cancers and from classic familial retinoblastoma (designated as classic
RB
mutations) are unstable and generally do not localize to the nucleus, do not undergo cyclin-dependent kinase (cdk)-mediated hyperphosphorylation, show absent protein “pocket” binding activity, and do not suppress colony growth of RB(−) cells. In contrast, two low-penetrant alleles (661W and “deletion of codon 480”) retained the ability to localize to the nucleus, showed normal cdk-mediated hyperphosphorylation
in vivo
, exhibited a binding pattern to simian virus 40 large T antigen using a quantitative yeast two-hybrid assay that was intermediate between classic mutants (null) and wild-type RB, and had absent E2F1 binding
in vitro
. A third, low-penetrant allele, “deletion of
RB
exon 4,” showed minimal hyperphosphorylation
in vivo
but demonstrated detectable E2F1 binding
in vitro
. In addition, each low-penetrant RB mutant retained the ability to suppress colony growth of RB(−) tumor cells. These findings suggest two categories of mutant, low-penetrant RB alleles. Class 1 alleles correspond to promoter mutations, which are believed to result in reduced or deregulated levels of wild-type RB protein, whereas class 2 alleles result in mutant proteins that retain partial activity. Characterization of the different subtypes of class 2 low-penetrant genes may help to define more precisely functional domains within the RB product required for tumor suppression.
Mammalian capping enzyme complements mutant
Saccharomyces cerevisiae
lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II
Authors: Yue Zhenyu , Maldonado Edio , Pillutla Renuka , Cho Helen , Reinberg Danny , Shatkin Aaron J.
Published: July 2002

5′-Capping is an early mRNA modification that has important consequences for downstream events in gene expression. We have isolated mammalian cDNAs encoding capping enzyme. They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily. The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical. Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5′-triphosphatase activity and catalyzed formation of RNA 5′-terminal GpppG. A haploid strain of
Saccharomyces cerevisiae
lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA. Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site. The K294A mutant retained RNA 5′-triphosphatase activity, which was eliminated by N-terminal truncation. Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase. The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation. These results also explain the selective capping of RNA polymerase II transcripts.
Smad8 mediates the signaling of the receptor serine kinase
Authors: Chen Yan , Bhushan Anil , Vale Wylie
Published: July 2002

Smad proteins are critical intracellular mediators of signaling by growth and differentiation factors of the transforming growth factor β superfamily. We have isolated a member of the Smad family, Smad8, from a rat brain cDNA library and biochemically and functionally characterized its ability to transduce signals from serine kinase receptors. In
Xenopus
embryo, Smad8 is able to transcriptionally activate a subset of mesoderm target genes similar to those induced by the receptor serine kinase, activin receptor-like kinase (ALK)-2. Smad8 can be specifically phosphorylated by a constitutively active ALK-2 but not the related receptor serine kinase, ALK-4. In response to signaling from ALK-2, Smad8 associates with a common regulatory molecule, Smad4, and this association leads to a synergistic effect on gene transcription. Furthermore, Smad8 is able to rescue the expression of mesoderm genes blocked by truncated ALK-2 in the embryo. These results indicate that Smad8 can function as a downstream signaling mediator of ALK-2.
A yeast genetic system for selecting small molecule inhibitors of protein–protein interactions in nanodroplets
Authors: Huang Jing , Schreiber Stuart L.
Published: July 2002

Cellular processes are mediated by complex networks of molecular interactions. Dissection of their role most commonly is achieved by using genetic mutations that alter, for example, protein–protein interactions. Small molecules that accomplish the same result would provide a powerful complement to the genetic approach, but it generally is believed that such molecules are rare. There are several natural products, however, that illustrate the feasibility of this approach. Split-pool synthesis now provides a simple mechanical means to prepare vast numbers of complex, even natural product-like, molecules individually attached to cell-sized polymer beads. Here, we describe a genetic system compatible with split-pool synthesis that allows the detection of cell-permeable, small molecule inhibitors of protein–protein interactions in 100- to 200-nl cell culture droplets, prepared by a recently described technique that arrays large numbers of such droplets. These “nanodroplets” contain defined media, cells, and one or more beads containing ≈100 pmol of a photoreleasable small molecule and a controlled number of cells. The engineered
Saccharomyces cerevisiae
cells used in this study express two interacting proteins after induction with galactose whose interaction results in cell death in the presence of 5-fluoroorotic acid (inducible reverse two-hybrid assay). Disruption of the interaction by a small molecule allows growth, and the small molecule can be introduced into the system hours before induction of the toxic interaction. We demonstrate that the interaction between the activin receptor R1 and the immunophilin protein FKBP12 can be disrupted by the small molecule FK506 at nanomolar concentrations in nanodroplets. This system should provide a general method for selecting cell-permeable ligands that can be used to study the relevance of protein–protein interactions in living cells or organisms.
Inherited somatic mosaicism caused by an intracisternal A particle insertion in the mouse tyrosinase gene
Authors: Wu Min , Rinchik Eugene M. , Wilkinson Erby , Johnson Dabney K.
Published: July 2002

A recessive, fully penetrant mutation (
c

m1OR

) at the mouse
albino
locus that results in coat-color mottling has been characterized at the molecular level. Restriction mapping and DNA sequencing analyses provide evidence that mutants carry a 5.4-kb intracisternal A particle (IAP) element insertion upstream of the tyrosinase (
Tyr
) promoter. Northern blot analysis and reverse transcription–PCR results show that the tyrosinase gene is expressed at much lower levels in mutant than in wild-type mice. The mutant
Tyr
gene still retains the tissue-specific expression pattern, and the
Tyr
transcript is not initiated from the IAP long terminal repeat promoter. We propose that the IAP insertion isolates the promoter of the tyrosinase gene from upstream cis-acting regulatory elements, leading to a substantially decreased level of
Tyr
gene expression in mutants.
dissatisfaction
, a gene involved in sex-specific behavior and neural development of
Drosophila melanogaster
Authors: Finley Kim D. , Taylor Barbara J. , Milstein Marc , McKeown Michael
Published: July 2002

Few mutations link well defined behaviors with individual neurons and the activity of specific genes. In
Drosophila
, recent evidence indicates the presence of a
doublesex
-independent pathway controlling sexual behavior and neuronal differentiation. We have identified a gene,
dissatisfaction
(
dsf
), that affects sex-specific courtship behaviors and neural differentiation in both sexes without an associated general behavioral debilitation. Male and female mutant animals exhibit abnormalities in courtship behaviors, suggesting a requirement for
dsf
in the brain. Virgin
dsf
females resist males during courtship and copulation and fail to lay mature eggs.
dsf
males actively court and attempt copulation with both mature males and females but are slow to copulate because of maladroit abdominal curling. Structural abnormalities in specific neurons indicate a role for
dsf
in the differentiation of sex-specific abdominal neurons. The egg-laying defect in females correlates with the absence of motor neuronal innervation on uterine muscles, and the reduced abdominal curling in males correlates with alteration in motor neuronal innervation of male ventral abdominal muscles. Epistasis experiments show that
dsf
acts in a
tra
-dependent and
dsx
-independent manner, placing
dsf
in the
dsx
-independent portion of the sex determination cascade.
Requirement for an initial signal from the membrane-proximal region of the interleukin 2 receptor γ
c
chain for Janus kinase activation leading to T cell proliferation
Authors: Nelson Brad H. , McIntosh Bryan C. , Rosencrans Lori L. , Greenberg Philip D.
Published: July 2002

The interleukin 2 receptor (IL-2R) generates proliferative signals in T lymphocytes by ligand-induced heterodimerization of two chains, IL-2Rβ and γ
c
, which associate with the tyrosine kinases Jak1 and Jak3, respectively. Genetic and molecular studies have demonstrated that Jak3 is essential for mitogenic signaling by the γ
c
chain; because it is also the only molecule known to associate with γ
c
, we speculated that Jak3 might be sufficient for signaling by this chain. Therefore, fusion proteins were constructed in which all or part of the cytoplasmic domain of γ
c
was replaced by Jak3. Signaling was evaluated in the IL-2-dependent T cell line CTLL-2 using chimeric IL-2Rβ and γ
c
chains that bind and are activated by the cytokine granulocyte–macrophage colony-stimulating factor. Chimeric γ
c
chains containing only Jak3 in the cytoplasmic domain failed to mediate proliferation of CTLL-2 cells, but addition of a conserved membrane-proximal (PROX) domain of γ
c
in tandem with Jak3 fully reconstituted γ
c
function. The requirement for the PROX domain reflected an essential role in the activation of Jak3
in vivo
. Despite lacking defined catalytic motifs, PROX induced an early Jak-independent signal, including tyrosine phosphorylation of IL-2Rβ and the tyrosine phosphatase SHP-2. The results define the minimal signaling components of γ
c
and suggest a new mechanism by which the IL-2R initiates signaling in response to ligand.
Pivotal role for the NFIL3/E4BP4 transcription factor in interleukin 3-mediated survival of pro-B lymphocytes
Authors: Ikushima Satoshi , Inukai Takeshi , Inaba Toshiya , Nimer Stephen D. , Cleveland John L. , Look A. Thomas
Published: July 2002

The E2A-HLF (hepatic leukemia factor) oncoprotein, generated in pro-B lymphocytes by fusion of the trans-activation domain ofE2Ato the basic region/leucine zipper (bZIP) domain ofHLF,functions as an anti-apoptotic transcription factor in leukemic cell transformation. When introduced into interleukin 3 (IL-3)-dependent mouse pro-B lymphocytes, E2A-HLF prevents apoptosis induced by growth factor deprivation, suggesting that IL-3 mediates cell survival through activation of a transcription factor whose activity can be constitutively replaced by the chimeric oncoprotein. We considered four bZIP transcription factors as candidates for this putative IL-3-regulated factor, each of which binds avidly to the DNA consensus sequence recognized by E2A-HLF and is related to theCaenorhabditis elegansCES-2 (cell death specification protein) neuron-specific mediator of cell death. The expression and binding activity of the Nfil3 protein (also called E4bp4), but not of Hlf, Dbp, or Tef, was found to be regulated by IL-3 in mouse pro-B cell lines (Baf-3 and FL5.12). Northern blot analysis showed thatNfil3/E4bp4is regulated as a “delayed-early” IL-3-responsive gene, requiringde novoprotein synthesis. In the absence of IL-3, enforced expression of the humanNFIL3/E4BP4cDNA promoted the survival but not the growth of IL-3-dependent pro-B cells. Our results implicate NFIL3/E4BP4 (nuclear factor regulated by IL-3/adenovirus E4 promoter binding protein) in a distinct growth factor-regulated signaling pathway that is responsible for the survival of early B-cell progenitors, and whose alteration by E2A-HLF leads to childhood B lineage leukemia.
Is polarization important in cation-π interactions?
Authors: Cubero Elena , Luque F. Javier , Orozco Modesto
Published: July 2002

The importance of cation->aromatic polarization effects on cation-π interactions has been explored. Theoretical calculations demonstrate that polarization is a large contribution to cation-aromatic interactions, and particularly to cation-π interactions. For a series of compounds with a similar aromatic core, polarization is constant and makes small influence in the relative cation-binding energies. However, when the aromatic core changes polarization contributions might be very different. We found that the generalized molecular interaction potential with polarization is a very fast and powerful tool for the prediction of cation binding of aromatic compounds.
Endothelin-induced conversion of embryonic heart muscle cells into impulse-conducting Purkinje fibers
Authors: Gourdie Robert G. , Wei Yan , Kim Darlene , Klatt Sandra C. , Mikawa Takashi
Published: July 2002

A regular heart beat is dependent on a specialized network of pacemaking and conductive cells. There has been a longstanding controversy regarding the developmental origin of these cardiac tissues which also manifest neural-like properties. Recently, we have shown conclusively that during chicken embryogenesis, impulse-conducting Purkinje cells are recruited from myocytes in spatial association with developing coronary arteries. Here, we report that cultured embryonic myocytes convert to a Purkinje cell phenotype after exposure to the vascular cytokine, endothelin. This inductive response declined gradually during development. These results yield further evidence for a role of arteriogenesis in the induction of impulse-conducting Purkinje cells within the heart muscle lineage and also may provide a basis for tissue engineering of cardiac pacemaking and conductive cells.
Fas-independent death of activated CD4+T lymphocytes induced by CTLA-4 crosslinking
Authors: Scheipers Peter , Reiser Hans
Published: July 2002

The CTLA-4 receptor is a critical inhibitory regulator of T cell proliferation and effector function. However, the mechanisms through which CTLA-4 modulates the activation of T cells remain uncertain. Initial studies, using activated human T cells, have suggested that CTLA-4 crosslinking may induce apoptosis. However, more recent experiments have demonstrated that crosslinking of the CTLA-4 receptor on the surface of resting murine T cells blocks cell cycle progression without inducing apoptosis. Here we provide evidence that CTLA-4 crosslinking on the surface of activated murine CD4+T lymphocytes leads to death of a substantial fraction of the cells whereas in resting CD4+T cells the same stimulation conditions induce cell cycle arrest without apoptosis. Cell death induced by CTLA-4 stimulation occurs independently of Fas and therefore may involve a novel pathway. CTLA-4-mediated apoptosis may be a means of terminating the function of previously stimulated T cells. Exploitation of this mechanism also may provide a therapeutic strategy to eliminate alloreactive or autoreactive T cells.
Biliary cholesterol excretion: A novel mechanism that regulates dietary cholesterol absorption
Authors: Sehayek Ephraim , Ono Jennie G. , Shefer Sarah , Nguyen Lien B. , Wang Nan , Batta Ashok K. , Salen Gerald , Smith Jonathan D. , Tall Alan R. , Breslow Jan L.
Published: July 2002

The regulation of dietary cholesterol absorption was examined in C57BL/6 and transgenic mice with liver overexpression of the scavenger receptor BI (SR-BI Tg). In C57BL/6 animals, feeding 0.02 to 1% (wt/wt) dietary cholesterol resulted in a dose-dependent decrease in the percentage of dietary cholesterol absorbed. A plot of total daily mass of dietary cholesterol absorbed versus the percentage by weight of cholesterol in the diet yielded a curve suggesting a saturable process with a
K
m
of 0.4% (wt/wt) and a
V
max
of 0.65 mg cholesterol/g body weight per day. Dietary cholesterol suppressed hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity, stimulated cholesterol 7α-hydroxylase activity, and enhanced fecal excretion of bile acids, but none of these changes correlated with the percentage of dietary cholesterol absorption. Dietary cholesterol also caused an increase in biliary cholesterol concentration, and in this case the concentration of biliary cholesterol was strongly and inversely correlated with the percentage dietary cholesterol absorption (
r
= −0.63,
P
< 0.0001). Biliary cholesterol concentration was also directly correlated with daily cholesterol intake, dietary cholesterol mass absorption, and liver cholesterol ester content. Transgene-induced overexpression of SR-BI resulted in a stimulation of excretion of cholesterol into the bile and suppressed percentage dietary cholesterol absorption. Furthermore, biliary cholesterol levels in SR-BI Tg mice were strongly and inversely correlated with the percentage of dietary cholesterol absorbed (
r
= −0.99,
P
< 0.0008). In summary, these results suggest that the excretion of cholesterol into the bile plays an important role in regulating the percentage absorption of dietary cholesterol.
Quantitative analysis of the immunopotency of genetically transfected dendritic cells
Authors: Timares Laura , Takashima Akira , Johnston Stephen Albert
Published: July 2002

Dendritic cells (DCs) instruct and activate a naive immune system to mount a response toward foreign proteins. Therefore, it has been hypothesized that an ideal vaccine strategy would be to directly introduce genes encoding antigens into DCs. To test this strategy quantitatively, we have compared the immune response elicited by a genetically transfected DC line to that induced by a fibroblast line, or standard genetic immunization. We observe that a single injection of 500–1,000 transfected DCs can produce a response comparable to that of standard genetic immunization, whereas fibroblasts, with up to 50-fold greater transfection efficiency, were less potent. We conclude that transfection of a small number of DCs is sufficient to initiate a wide variety of immune responses. These results indicate that targeting genes to DCs will be important for controlling and augmenting the immunological outcome in genetic immunization.
Rapid elimination of mature autoreactive B cells demonstrated by Cre-induced change in B cell antigen receptor specificity
in vivo
Authors: Lam Kong-Peng , Rajewsky Klaus
Published: July 2002

Developing autoreactive B cells edit their B cell antigen receptor (BCR) in the bone marrow and are clonally deleted when they fail to reexpress an innocent BCR. Here, inducible Cre-
lox
P-mediated gene inversion is used to change the specificity of the BCR on mature IgM
+
IgD
+
B cells
in vivo
to address the fate of lymphocytes encountering self-antigens at this developmental stage. Expression of an autoreactive BCR on mature B cells leads to their rapid elimination from the periphery, a process that is inhibited by constitutive
bcl-2
transgene expression in an antigen dose-dependent manner. Thus, selection of mature B cells into the long-lived peripheral pool does not prevent their deletion upon encounter of self-antigens.
Protein phosphatase 2A is required for the initiation of chromosomal DNA replication
Authors: Lin Xiao-Hong , Walter Johannes , Scheidtmann Karl , Ohst Kim , Newport John , Walter Gernot
Published: July 2002

Protein phosphatase 2A (PP2A) is an abundant, multifunctional serine/threonine-specific phosphatase that stimulates simian virus 40 DNA replication. The question as to whether chromosomal DNA replication also depends on PP2A was addressed by using a cell-free replication system derived from
Xenopus laevis
eggs. Immunodepletion of PP2A from
Xenopus
egg extract resulted in strong inhibition of DNA replication. PP2A was required for the initiation of replication but not for the elongation of previously engaged replication forks. Therefore, the initiation of chromosomal DNA replication depends not only on phosphorylation by protein kinases but also on dephosphorylation by PP2A.
p21
WAF-1
reorganizes the nucleus in tumor suppression
Authors: Linares-Cruz Gustavo , Bruzzoni-Giovanelli Heriberto , Alvaro Véronique , Roperch Jean-Pierre , Tuynder Marcel , Schoevaert Damien , Nemani Mona , Prieur Sylvie , Lethrosne Florence , Piouffre Laurence , Reclar Valérie , Faille Annick , Chassoux Danièle , Dausset Jean , Amson Robert B. , Calvo Fabien , Telerman Adam
Published: July 2002

Interphasic nuclear organization has a key function in genome biology. We demonstrate that p21
WAF-1
, by influencing gene expression and inducing chromosomal repositioning in tumor suppression, plays a major role as a nuclear organizer. Transfection of U937 tumor cells with p21
WAF-1
resulted in expression of the
HUMSIAH
(
human seven in absentia homologue
),
Rb
, and
Rbr-2
genes and strong suppression of the malignant phenotype. p21
WAF-1
drastically modified the compartmentalization of the nuclear genome. DNase I genome exposure and fluorescence
in situ
hybridization show, respectively, a displacement of the sensitive sites to the periphery of the nucleus and repositioning of chromosomes 13, 16, 17, and 21. These findings, addressing nuclear architecture modulations, provide potentially significant perspectives for the understanding of tumor suppression.
A dynamic model for PC4 coactivator function in RNA polymerase II transcription
Authors: Malik Sohail , Guermah Mohamed , Roeder Robert G.
Published: July 2002

Human positive cofactor (PC4) acts as a general coactivator for activator-dependent transcription by RNA polymerase II. Here we show that PC4 coactivator function, in contrast to basal (activator-independent) transcription, is dependent both on TATA binding protein (TBP)-associated factors (TAFs) in TFIID and on TFIIH. Surprisingly, PC4 strongly represses transcription initiation by minimal preinitiation complexes in the absence of TAFs and TFIIH, while simultaneously promoting the formation of these complexes. Furthermore, TFIIH and TAF
II
250, the largest subunit of TFIID, can both phosphorylate PC4. These results provide evidence for an inactive, PC4-induced intermediate in preinitiation complex assembly and point to TFIIH and TAF requirements for its progression into a functional preinitiation complex. Thus PC4 coactivator activity is realized in a stepwise series of events reminiscent of prokaryotic activation pathways involving conversion of inactive RNA polymerase-promoter complexes to an initiation-competent state.
Regions on adenylyl cyclase that are necessary for inhibition of activity by βγ and G
iα
subunits of heterotrimeric G proteins
Authors: Wittpoth Claus , Scholich Klaus , Yigzaw Yinges , Stringfield Teresa M. , Patel Tarun B.
Published: July 2002

The two large cytoplasmic domains (C1 and C2) of adenylyl cyclases
(AC), when expressed separately and mixed together, reconstitute enzyme
activity that can be regulated by various modulators. Therefore, we
have used the C1 or its C1a subdomain and C2 regions from type I AC
(ACI) and type V AC (ACV) to identify the region on ACI that interacts
with βγ subunits of heterotrimeric G proteins. In addition, we also
used a chimeric C1 domain (VC1aIC1b) in which the C1a region was
derived from ACV and the C1b region was from ACI. By mixing the C1 or
C1a or VC1aIC1b domains with C2 regions of ACI or ACV, we have shown
that the C1a region (amino acids 236–471) of ACI is sufficient to
observe βγ-mediated inhibition of enzyme activity, which is
stimulated by either constitutively active G
sα
(G
sα
*) or Ca
2+
/calmodulin
(CaM). Although the C1b region and C2 domain of ACI were by themselves
not sufficient for inhibition of activity by βγ subunits, the
presence of both of these regions formed another βγ interaction
site that was sufficient to observe G
sα
*- or
Ca
2+
/CaM-stimulated activity. Inhibition of AC activity
attributable to interaction of βγ subunits at either of the two
sites was blocked by a peptide (QEHA) that has previously been shown to
inhibit the effects of βγ on various effectors. Moreover, the C1
region of ACI was sufficient to observe G
iα1
-elicited
inhibition of Ca
2+
/CaM-stimulated activity. Although the
C1a region of ACV was sufficient for inhibition of activity by
G
iα1
, the presence of C1b region from either ACI or ACV
increased sensitivity to inhibition by the inhibitory G protein. Thus,
the inhibitory influences of G
iα1
are mediated on the C1
regions of both ACI and ACV. The effects of βγ on ACI can be
mediated by interactions with the C1a region and a βγ interacting
site formed by the C1b and C2 domains of this enzyme.
Single-copy transgenic wheat generated through the resolution of complex integration patterns
Authors: Srivastava Vibha , Anderson Olin D. , Ow David W.
Published: July 2002

Genetic transformation of plants often results in multiple copies of the introduced DNA at a single locus. To ensure that only a single copy of a foreign gene resides in the plant genome, we used a strategy based on site-specific recombination. The transformation vector consists of a transgene flanked by recombination sites in an inverted orientation. Regardless of the number of copies integrated between the outermost transgenes, recombination between the outermost sites resolves the integrated molecules into a single copy. An example of this strategy has been demonstrated with wheat transformation, where four of four multiple-copy loci were resolved successfully into single-copy transgenes.
Structurally similar oxidized phospholipids differentially regulate endothelial binding of monocytes and neutrophils
Authors: Leitinger Norbert , Tyner Timothy R. , Oslund Laura , Rizza Cristina , Subbanagounder Ganesamoorthy , Lee Hans , Shih Peggy T. , Mackman Nigel , Tigyi Gabor , Territo Mary C. , Berliner Judith A. , Vora Devendra K.
Published: July 2002

We previously have demonstrated that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a component of minimally modified low density lipoprotein (MM-LDL), activates endothelial cells to bind monocytes. 1-Palmitoyl-2- (5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC) and 1- palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), which are present in OxPAPC, MM-LDL, and atherosclerotic lesions, were shown to have a major role in the activation of endothelial cells. We now demonstrate that these two highly similar molecules have dramatically different effects on leukocyte endothelial interactions. POVPC is a potent regulator of monocyte-specific endothelial interactions. Treatment of endothelial cells with POVPC increased monocyte binding by inducing the surface expression of the connecting segment 1 domain of fibronectin; no increase in neutrophil binding was observed. In addition, POVPC strongly inhibited lipopolysaccharide-mediated induction of neutrophil binding and expression of E-selectin protein and mRNA. This inhibition was mediated by a protein kinase A-dependent pathway, resulting in down-regulation of NF-κB-dependent transcription. In contrast, PGPC induced both monocyte and neutrophil binding and expression of E-selectin and vascular cell adhesion molecule 1. We present evidence to suggest that the two phospholipids act by different novel receptors present inXenopuslaevis oocytes and that POVPC, but not PGPC, stimulates a cAMP-mediated pathway. At concentrations equal to that present in MM-LDL, the effect of POVPC dominates and inhibits PGPC-induced neutrophil binding and E-selectin expression in endothelial cells. In summary, our data provide evidence that both POVPC and PGPC are important regulators of leukocyte-endothelial interactions and that POVPC may play a dominant role in a number of chronic inflammatory processes where oxidized phospholipids are known to be present.
Atom density in protein structures
Authors: Karlin Samuel , Zhu Zhan-Yang , Baud Franck
Published: July 2002

The residue environment in protein structures is studied with respect to the density of carbon (C), oxygen (O), and nitrogen (N) atoms within a certain distance (say 5 Å) of each residue. Two types of environments are evaluated: one based on side-chain atom contacts (abbreviated S-S) and the other based on all atom (side-chain + backbone) contacts (abbreviated A-A). Different atom counts are observed about nine-residue structural categories defined by three solvent accessibility levels and three secondary structure states. Among the structural categories, the S-S atom count ratios generally vary more than the A-A atom count ratios because of the fact that the backbone (O) and (N) atoms contribute equal counts. Secondary structure affects the (C) density for the A-A contacts whereas secondary structure has little influence on the (C) density for the S-S contacts. For S-S contacts, a greater density of (O) over (N) atom neighbors stands out in the environment of most amino acid types. By contrast, for A-A contacts, independent of the solvent accessibility levels, the ratio (O)/(N) is ≈1 in helical states, consistent with the geometry of α-helical residues whose side-chains tilt oppositely to the amino to carboxy α-helical axis. The highest ratio of neighbor (O)/(N) is achieved under solvent exposed conditions. This (O) vs. (N) prevalence is advantageous at the protein surface that generally exhibits an acid excess that helps to enhance protein solubility in the cell and to avoid nonspecific interactions with phosphate groups of DNA, RNA, and other plasma constituents.
A novel bacterial tyrosine kinase essential for cell division and differentiation
Authors: Wu Jianguo , Ohta Noriko , Zhao Ji-Liang , Newton Austin
Published: July 2002

Protein kinases play central roles in the regulation of eukaryotic and prokaryotic cell growth, division, and differentiation. The
Caulobacter crescentus divL
gene encodes a novel bacterial tyrosine kinase essential for cell viability and division. Although the DivL protein is homologous to the ubiquitous bacterial histidine protein kinases (HPKs), it differs from previously studied members of this protein kinase family in that it contains a tyrosine residue (Tyr-550) in the conserved H-box instead of a histidine residue, which is the expected site of autophosphorylation. DivL is autophosphorylated on Tyr-550
in vitro
, and this tyrosine residue is essential for cell viability and regulation of the cell division cycle. Purified DivL also catalyzes phosphorylation of CtrA and activates transcription
in vitro
of the cell cycle-regulated
fliF
promoter. Suppressor mutations in
ctrA
bypass the conditional cell division phenotype of cold-sensitive
divL
mutants, providing genetic evidence that DivL function in cell cycle and developmental regulation is mediated, at least in part, by the global response regulator CtrA. DivL is the only reported HPK homologue whose function has been shown to require autophosphorylation on a tyrosine, and, thus, it represents a new class of kinases within this superfamily of protein kinases.
Orientation of the transcription preinitiation complex in Archaea
Authors: Bell Stephen D. , Kosa Peter L. , Sigler Paul B. , Jackson Stephen P.
Published: July 2002

The basal transcription machinery of Archaea corresponds to the minimal subset of factors required for RNA polymerase II transcription in eukaryotes. Using just two factors, Archaea recruit the RNA polymerase to promoters and define the direction of transcription. Notably, the principal determinant for the orientation of transcription is not the recognition of the TATA box by the TATA-box-binding protein. Instead, transcriptional polarity is governed by the interaction of the archaeal TFIIB homologue with a conserved motif immediately upstream of the TATA box. This interaction yields an archaeal preinitiation complex with the same orientation as the analogous eukaryal complex.
A novel multigene family encodes diversified variable regions
Authors: Strong Scott J. , Mueller M. Gail , Litman Ronda T. , Hawke Noel A. , Haire Robert N. , Miracle Ann L. , Rast Jonathan P. , Amemiya Chris T. , Litman Gary W.
Published: July 2002

Antigen recognition in the adaptive immune response by Ig and T-cell antigen receptors (TCRs) is effected through patterned differences in the peptide sequence in the V regions. V-region specificity forms through genetically programmed rearrangement of individual, diversified segmental elements in single somatic cells. Other Ig superfamily members, including natural killer receptors that mediate cell-surface recognition, do not undergo segmental reorganization, and contain type-2 C (C2) domains, which are structurally distinct from the C1 domains found in Ig and TCR. Immunoreceptor tyrosine-based inhibitory motifs that transduce negative regulatory signals through the cell membrane are found in certain natural killer and other cell surface inhibitory receptors, but not in Ig and TCR. In this study, we employ a genomic approach by using the pufferfish (Spheroides nephelus) to characterize a nonrearranging novel immune-type receptor gene family. Twenty-six different nonrearranging genes, which each encode highly diversified V as well as a V-like C2 extracellular domain, a transmembrane region, and in most instances, an immunoreceptor tyrosine-based inhibitory motif-containing cytoplasmic tail, are identified in an ≈113 kb P1 artificial chromosome insert. The presence in novel immune-type receptor genes of V regions that are related closely to those found in Ig and TCR as well as regulatory motifs that are characteristic of inhibitory receptors implies a heretofore unrecognized link between known receptors that mediate adaptive and innate immune functions.
Mycobacterial infection of macrophages results in membrane-permeable phagosomes
Authors: Teitelbaum R. , Cammer M. , Maitland M. L. , Freitag N. E. , Condeelis J. , Bloom B. R.
Published: July 2002

Cell-mediated immunity is critical for host resistance to tuberculosis. T lymphocytes recognizing antigens presented by the major histocompatibility complex (MHC) class I and class II molecules have been found to be necessary for control of mycobacterial infection. Mice genetically deficient in the generation of MHC class I and class Ia responses are susceptible to mycobacterial infection. Although soluble protein antigens are generally presented by macrophages to T cells through MHC class II molecules, macrophages infected with
Mycobacterium tuberculosis
or bacille Calmette-Guerin have been shown to facilitate presentation of ovalbumin through the MHC class I presentation pathway via a TAP-dependent mechanism. How mycobacteria, thought to reside within membrane-bound vacuoles, facilitate communication with the cytoplasm and enable MHC class I presentation presents a paradox. By using confocal microscopy to study the localization of fluorescent-tagged dextrans of varying size microinjected intracytoplasmically into macrophages infected with bacille Calmette-Guerin expressing the green fluorescent protein, molecules as large as 70 kilodaltons were shown to gain access to the mycobacterial phagosome. Possible biological consequences of the permeabilization of vacuolar membranes by mycobacteria would be pathogen access to host cell nutrients within the cytoplasm, perhaps contributing to bacterial pathogenesis, and access of microbial antigens to the MHC class I presentation pathway, contributing to host protective immune responses.
Odortypes: Their origin and composition
Authors: Yamazaki Kunio , Beauchamp Gary K. , Singer Alan , Bard Judith , Boyse Edward A.
Published: July 2002

Odors that distinguish one individual from another member of the
species and are determined by polymorphic genes are called odortypes.
Odortypes and their considerable societal significance have been
studied intimately only in mice and mainly with respect to the genes of
the major histocompatibility complex. Further understanding and the
matter of human relevance have been hampered by the apparent
restriction of odortype expression to urine. The present finding that
odorants comprising prerenal odortypes are already present in blood,
albeit in masked form, affords the basis of a comprehensive view of
odortypes. Accordingly, major histocompatibility complex and other
polymorphic genes of antiquity are seen
inter alia
as
agents of normal variation, which entails quantitative variation in
output of odorant metabolites. Relatively few such normal variations
should suffice for a vast range of compound odors whose specificity is
determined by combinative assortment of the same set of individual
volatile compounds.
Human T-cell leukemia retrovirus-Tax protein is a repressor of nuclear receptor signaling
Authors: Doucas Vassilis , Evans Ronald M.
Published: July 2002

The Tax oncoprotein promotes cellular transformation and is associated with the pathogenesis of adult T-cell leukemia. Tax expression activates transcription via the cAMP enhancer binding protein/activating transcription factor (CREB/ATF) and NF-κB pathways. In contrast to its positive action, here we demonstrate that Tax is a potent repressor of steroid and retinoid receptor transcription. The Tax protein becomes localized in the promyelocytic (PML) oncogenic domain, and unexpectedly, expression of the PML protein reverses Tax-induced repression. These results suggest that PML and Tax may act in opposing manners to influence nuclear receptor transcription and human T-cell leukemia retrovirus pathogenesis.
Calcium/calmodulin-dependent protein kinase II is associated with the
N
-methyl-
d
-aspartate receptor
Authors: Leonard A. Soren , Lim Indra A. , Hemsworth Daniel E. , Horne Mary C. , Hell Johannes W.
Published: July 2002

The molecular basis of long-term potentiation (LTP), a long-lasting change in synaptic transmission, is of fundamental interest because of its implication in learning. Usually LTP depends on Ca
2+
influx through postsynaptic
N
-methyl-
d
-aspartate (NMDA)-type glutamate receptors and subsequent activation of Ca
2+
/calmodulin-dependent protein kinase II (CaMKII). For a molecular understanding of LTP it is crucial to know how CaMKII is localized to its postsynaptic targets because protein kinases often are targeted to their substrates by adapter proteins. Here we show that CaMKII directly binds to the NMDA receptor subunits NR1 and NR2B. Moreover, activation of CaMKIIα by stimulation of NMDA receptors in forebrain slices increase this association. This interaction places CaMKII not only proximal to a major source of Ca
2+
influx but also close to α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors, which become phosphorylated upon stimulation of NMDA receptors in these forebrain slices. Identification of the postsynaptic adapter for CaMKII fills a critical gap in the understanding of LTP because CaMKII-mediated phosphorylation of AMPA receptors is an important step during LTP.