bluuebunny/crossref_metadata_2025 huggingface dataset:
Pleiotropy in microdeletion syndromes: neurologic and spermatogenic abnormalities in mice homozygous for the p6H deletion are likely due to dysfunction of a single gene.
Authors: Rinchik E M , Carpenter D A , Handel M A
Published: May 2006

Variability and complexity of phenotypes observed in microdeletion syndromes can be due to deletion of a single gene whose product participates in several aspects of development or can be due to the deletion of a number of tightly linked genes, each adding its own effect to the syndrome. The p6H deletion in mouse chromosome 7 presents a good model with which to address this question of multigene vs. single-gene pleiotropy. Mice homozygous for the p6H deletion are diluted in pigmentation, are smaller than their littermates, and manifest a nervous jerky-gait phenotype. Male homozygotes are sterile and exhibit profound abnormalities in spermiogenesis. By using N-ethyl-N-nitrosourea (EtNU) mutagenesis and a breeding protocol designed to recover recessive mutations expressed hemizygously opposite a large p-locus deletion, we have generated three noncomplementing mutations that map to the p6H deletion. Each of these EtNU-induced mutations has adverse effects on the size, nervous behavior, and progression of spermiogenesis that characterize p6H deletion homozygotes. Because EtNU is thought to induce primarily intragenic (point) mutations in mouse stem-cell spermatogonia, we propose that the trio of phenotypes (runtiness, nervous jerky gait, and male sterility) expressed in p6H deletion homozygotes is the result of deletion of a single highly pleiotropic gene. We also predict that a homologous single locus, quite possibly tightly linked and distal to the D15S12 (P) locus in human chromosome 15q11-q13, may be associated with similar developmental abnormalities in humans.
Oligomerization of CD4 is required for stable binding to class II major histocompatibility complex proteins but not for interaction with human immunodeficiency virus gp120.
Authors: Sakihama T , Smolyar A , Reinherz E L
Published: May 2006

Previous studies have failed to detect an interaction between monomeric soluble CD4 (sCD4) and class II major histocompatibility complex (MHC) proteins, suggesting that oligomerization of CD4 on the cell surface may be required to form a stable class II MHC binding site. To test this possibility, we transfected the F43I CD4 mutant, which is incapable of binding to class II MHC or human immunodeficiency virus (HIV) gp120, into COS-7 cells together with wild-type CD4 (wtCD4). Expression of F43I results in a dominant negative effect: no class II MHC binding is observed even though wtCD4 expression is preserved. Apparently, F43I associates with wtCD4 oligomers and interferes with the formation of functional class II MHC binding structures. In contrast, F43I does not affect the binding of gp120 to wtCD4, implying that gp120 binds to a CD4 monomer. By production and characterization of chimeric CD4 molecules, we show that domains 3 and/or 4 appear to be involved in oligomerization. Several models of the CD4-class II MHC interaction are offered, including the possibility that one or two CD4 molecules initially interact with class II MHC dimers and further associate to create larger complexes important for facilitating T-cell receptor crosslinking.
Incorporation of glutamine repeats makes protein oligomerize: implications for neurodegenerative diseases.
Authors: Stott K , Blackburn J M , Butler P J , Perutz M
Published: May 2006

Many transcription factors and some other proteins contain glutamine repeats; their abnormal expansion has been linked to several dominantly inherited neuro-degenerative diseases. Having found that poly(L-glutamine) alone forms beta-strands held together by hydrogen bonds between their amide groups, we surmised that glutamine repeats may form polar zippers, an unusual motif for protein-protein interactions. To test this hypothesis, we have engineered a Gly-Gln10-Gly peptide into the inhibitory loop of truncated chymotrypsin inhibitor 2 (CI2), a small protein from barley seeds, by both insertion and replacement. Gel filtration resolved both mutant inhibitors into at least three fractions, which analytical ultracentrifugation identified as monomers, dimers, and trimers of the recombinant protein; the truncated wild-type CI2 formed only monomers. CD difference spectra of the dimers and trimers versus wild type indicated that their glutamine repeats formed beta-pleated sheets, while those of the monomers versus wild type were more suggestive of type I beta-turns. The CD spectra of all three fractions remained unchanged even after incubation at 70 degrees C; neither the dimers nor the trimers dissociated at this temperature. We argue that the stability of all three fractions is due to the multiplicity of hydrogen bonds between extended strands of glutamine repeats in the oligomers or within a beta-hairpin formed by the single glutamine repeat of each monomer. Pathological effects may arise when expanded glutamine repeats cause proteins to acquire excessively high affinities for each other or for other proteins with glutamine repeats.
Evidence that neuronal G-protein-gated inwardly rectifying K+ channels are activated by G beta gamma subunits and function as heteromultimers.
Authors: Kofuji P , Davidson N , Lester H A
Published: May 2006

Guanine nucleotide-binding proteins (G proteins) activate K+ conductances in cardiac atrial cells to slow heart rate and in neurons to decrease excitability. cDNAs encoding three isoforms of a G-protein-coupled, inwardly rectifying K+ channel (GIRK) have recently been cloned from cardiac (GIRK1/Kir 3.1) and brain cDNA libraries (GIRK2/Kir 3.2 and GIRK3/Kir 3.3). Here we report that GIRK2 but not GIRK3 can be activated by G protein subunits G beta 1 and G gamma 2 in Xenopus oocytes. Furthermore, when either GIRK3 or GIRK2 was coexpressed with GIRK1 and activated either by muscarinic receptors or by G beta gamma subunits, G-protein-mediated inward currents were increased by 5- to 40-fold. The single-channel conductance for GIRK1 plus GIRK2 coexpression was intermediate between those for GIRK1 alone and for GIRK2 alone, and voltage-jump kinetics for the coexpressed channels displayed new kinetic properties. On the other hand, coexpression of GIRK3 with GIRK2 suppressed the GIRK2 alone response. These studies suggest that formation of heteromultimers involving the several GIRKs is an important mechanism for generating diversity in expression level and function of neurotransmitter-coupled, inward rectifier K+ channels.
The GA5 locus of Arabidopsis thaliana encodes a multifunctional gibberellin 20-oxidase: molecular cloning and functional expression.
Authors: Xu Y L , Li L , Wu K , Peeters A J , Gage D A , Zeevaart J A
Published: May 2006

The biosynthesis of gibberellins (GAs) after GA12-aldehyde involves a series of oxidative steps that lead to the formation of bioactive GAs. Previously, a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-] was isolated by immunoscreening a cDNA library from liquid endosperm of pumpkin (Cucurbita maxima L.) with antibodies against partially purified GA 20-oxidase. Here, we report isolation of a genomic clone for GA 20-oxidase from a genomic library of the long-day species Arabidopsis thaliana Heynh., strain Columbia, by using the pumpkin cDNA clone as a heterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. The three exons are 1131-bp long and encode 377 amino acid residues. A cDNA clone corresponding to the putative GA 20-oxidase genomic sequence was constructed with the reverse transcription-PCR method, and the identity of the cDNA clone was confirmed by analyzing the capability of the fusion protein expressed in Escherichia coli to convert GA53 to GA44 and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55% identity and > 80% similarity with the pumpkin GA 20-oxidase at the derived amino acid level. Both GA 20-oxidases share high homology with other 2-oxoglutarate-dependent dioxygenases (2-ODDs), but the highest homology was found between the two GA 20-oxidases. Mapping results indicated tight linkage between the cloned GA 20-oxidase and the GA5 locus of Arabidopsis. The ga5 semidwarf mutant contains a G-->A point mutation that inserts a translational stop codon in the protein-coding sequence, thus confirming that the GA5 locus encodes GA 20-oxidase. Expression of the GA5 gene in Ara-bidopsis leaves was enhanced after plants were transferred from short to long days; it was reduced by GA4 treatment, suggesting end-product repression in the GA biosynthetic pathway.
Complexity, contingency, and criticality.
Authors: Bak P , Paczuski M
Published: May 2006

Complexity originates from the tendency of large dynamical systems to organize themselves into a critical state, with avalanches or "punctuations" of all sizes. In the critical state, events which would otherwise be uncoupled become correlated. The apparent, historical contingency in many sciences, including geology, biology, and economics, finds a natural interpretation as a self-organized critical phenomenon. These ideas are discussed in the context of simple mathematical models of sandpiles and biological evolution. Insights are gained not only from numerical simulations but also from rigorous mathematical analysis.
Identification of NAB1, a repressor of NGFI-A- and Krox20-mediated transcription.
Authors: Russo M W , Sevetson B R , Milbrandt J
Published: May 2006

NGFI-A (also called Egr1, Zif268, or Krox24) and the closely related proteins Krox20, NGFI-C, and Egr3 are zinc-finger transcription factors encoded by immediate-early genes which are induced by a wide variety of extracellular stimuli. NGFI-A has been implicated in cell proliferation, macrophage differentiation, synaptic activation, and long-term potentiation, whereas Krox20 is critical for proper hindbrain segmentation and peripheral nerve myelination. In previous work, a structure/function analysis of NGFI-A revealed a 34-aa inhibitory domain that was hypothesized to be the target of a cellular factor that represses NGFI-A transcriptional activity. Using the yeast two-hybrid system, we have isolated a cDNA clone which encodes a protein that interacts with this inhibitory domain and inhibits the ability of NGFI-A to activate transcription. This NGFI-A-binding protein, NAB1, is a 570-aa nuclear protein that bears no obvious sequence homology to known proteins. NAB1 also represses Krox20 activity, but it does not influence Egr3 or NGFI-G, thus providing a mechanism for the differential regulation of this family of immediate-early transcription factors.
Homologous recombination promoted by reverse transcriptase during copying of two distinct RNA templates.
Authors: Negroni M , Ricchetti M , Nouvel P , Buc H
Published: May 2006

Retroviruses are known to mutate at high rates. An important source of genetic variability is recombination taking place during reverse transcription of internal regions of the two genomic RNAs. We have designed an in vitro model system, involving genetic markers carried on two RNA templates, to allow a search for individual recombination events and to score their frequency of occurrence. We show that Moloney murine leukemia virus reverse transcriptase alone promotes homologous recombination efficiently. While RNA concentration has little effect on recombination frequency, there is a clear correlation between the amount of reverse transcriptase used in the assay and the extent of recombination observed. Under conditions mimicking the in vivo situation, a rate compatible with ex vivo estimates has been obtained.
Mapping sites of interaction of p47-phox and flavocytochrome b with random-sequence peptide phage display libraries.
Authors: DeLeo F R , Yu L , Burritt J B , Loetterle L R , Bond C W , Jesaitis A J , Quinn M T
Published: May 2006

During assembly of the phagocyte NADPH oxidase, cytosolic p47-phox translocates to the plasma membrane and binds to flavocytochrome b, and binding domains for p47-phox have been identified on the C-terminal tails of both flavocytochrome b subunits. In the present report, we further examine the interaction of these two oxidase components by using random-sequence peptide phage display library analysis. Screening p47-phox with the peptide libraries identified five potential sites of interaction with flavocytochrome b, including three previously reported regions of interaction and two additional regions of interaction of p47-phox with gp91-phox and p22-phox. The additional sites were mapped to a domain on the first predicted cytosolic loop of gp91-phox encompassing residues S86TRVRRQL93 and to a domain near the cytosolic C-terminal tail of gp91-phox encompassing residues F450EWFADLL457. The mapping also confirmed a previously reported binding domain on gp91-phox (E554SGPRGVHFIF564) and putative Src homology 3 domain binding sites on p22-phox (P156PRPP160 and G177GPPGGP183). To demonstrate that the additional regions identified were biologically significant, peptides mimicking the gp91-phox sequences F77LRGSSACCSTRVRRQL93 and E451WFADLLQLLESQ463 were synthesized and assayed for their ability to inhibit NADPH oxidase activity. These peptides had EC50 values of 1 microM and 230 microM, respectively, and inhibited activation when added prior to assembly but did not affect activity of the preassembled oxidase. Our data demonstrate the usefulness of phage display library analysis for the identification of biologically relevant sites of protein-protein interaction and show that the binding of p47-phox to flavocytochrome b involves multiple binding sites along the C-terminal tails of both gp91- and p22-phox and other regions of gp91-phox nearer to the N terminus.
Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in human lymphocytes.
Authors: Bacon C M , Petricoin E F , Ortaldo J R , Rees R C , Larner A C , Johnston J A , O'Shea J J
Published: May 2006

Interleukin 12 (IL-12) is an important immunoregulatory cytokine whose receptor is a member of the hematopoietin receptor superfamily. We have recently demonstrated that stimulation of human T and natural killer cells with IL-12 induces tyrosine phosphorylation of the Janus family tyrosine kinase JAK2 and Tyk2, implicating these kinases in the immediate biochemical response to IL-12. Recently, transcription factors known as STATs (signal transducers and activators of transcription) have been shown to be tyrosine phosphorylated and activated in response to a number of cytokines that bind hematopoietin receptors and activate JAK kinases. In this report we demonstrate that IL-12 induces tyrosine phosphorylation of a recently identified STAT family member, STAT4, and show that STAT4 expression is regulated by T-cell activation. Furthermore, we show that IL-12 stimulates formation of a DNA-binding complex that recognizes a DNA sequence previously shown to bind STAT proteins and that this complex contains STAT4. These data, and the recent demonstration of JAK phosphorylation by IL-12, identify a rapid signal-transduction pathway likely to mediate IL-12-induced gene expression.
WW6: an embryonic stem cell line with an inert genetic marker that can be traced in chimeras.
Authors: Ioffe E , Liu Y , Bhaumik M , Poirier F , Factor S M , Stanley P
Published: May 2006

Mutant mice produced by gene targeting in embryonic stem (ES) cells often have a complex or embryonic lethal phenotype. In these cases, it would be helpful to identify tissues and cell types first affected in mutant embryos by following the contribution to chimeras of ES cells homozygous for the mutant allele. Although a number of strategies for following ES cell development in vivo have been reported, each has limitations that preclude its general application. In this paper, we describe ES cell lines that can be tracked to every nucleated cell type in chimeras at all developmental stages. These lines were derived from blastocysts of mice that carry an 11-Mb beta-globin transgene on chromosome 3. The transgene is readily detected by DNA in situ hybridization, providing an inert, nuclear-localized marker whose presence is not affected by transcriptional or translational controls. The "WW" series of ES lines possess the essential features of previously described ES lines, including giving rise to a preponderance of male chimeras, all of which have to date exhibited germ-line transmission. In addition, clones selected for single or double targeting events form strong chimeras, demonstrating the feasibility of using WW6 cells to identify phenotypes associated with the creation of a null mutant.
Two distinct oscillators in the rat suprachiasmatic nucleus in vitro.
Authors: Shinohara K , Honma S , Katsuno Y , Abe H , Honma K
Published: May 2006

In the rat suprachiasmatic nucleus slice culture, circadian rhythms in the release of arginine vasopressin and vasoactive intestinal polypeptide were measured simultaneously and longitudinally. The phase relationship between the two peptide rhythms was relatively constant in the culture without a treatment of antimitotic drugs but became diverse by an introduction of antimitotics, which is generally used to reduce the number of glial cells. By monitoring the two rhythms continuously for 6 days, different periods were detected in culture with the antimitotic treatment. Furthermore, N-methyl-D-aspartate shifted the phase of the two peptide rhythms in the same culture differently. These results indicate that the arginine vasopressin and vasoactive intestinal polypeptide release are under control of different circadian oscillators.
Plaque-associated expression of human herpesvirus 6 in multiple sclerosis.
Authors: Challoner P B , Smith K T , Parker J D , MacLeod D L , Coulter S N , Rose T M , Schultz E R , Bennett J L , Garber R L , Chang M
Published: May 2006

Representational difference analysis was used to search for pathogens in multiple sclerosis brains. We detected a 341-nucleotide fragment that was 99.4% identical to the major DNA binding protein gene of human herpesvirus 6 (HHV-6). Examination of 86 brain specimens by PCR demonstrated that HHV-6 was present in > 70% of MS cases and controls and is thus a commensal virus of the human brain. By DNA sequencing, 36/37 viruses from MS cases and controls were typed as HHV-6 variant B group 2. Other herpesviruses, retroviruses, and measles virus were detected infrequently or not at all. HHV-6 expression was examined by immunocytochemistry with monoclonal antibodies against HHV-6 virion protein 101K and DNA binding protein p41. Nuclear staining of oligodendrocytes was observed in MS cases but not in controls, and in MS cases it was observed around plaques more frequently than in uninvolved white matter. MS cases showed prominent cytoplasmic staining of neurons in gray matter adjacent to plaques, although neurons expressing HHV-6 were also found in certain controls. Since destruction of oligodendrocytes is a hallmark of MS, these studies suggest an association of HHV-6 with the etiology or pathogenesis of MS.
Identification of four acidic amino acids that constitute the catalytic center of the RuvC Holliday junction resolvase.
Authors: Saito A , Iwasaki H , Ariyoshi M , Morikawa K , Shinagawa H
Published: May 2006

Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. Since the endonucleolytic activity of RuvC requires a divalent cation and since 3 or 4 acidic residues constitute the catalytic centers of several nucleases that require a divalent cation for the catalytic activity, we examined whether any of the acidic residues of RuvC were required for the nucleolytic activity. By site-directed mutagenesis, we constructed a series of ruvC mutant genes with similar amino acid replacements in 1 of the 13 acidic residues. Among them, the mutant genes with an alteration at Asp-7, Glu-66, Asp-138, or Asp-141 could not complement UV sensitivity of a ruvC deletion strain, and the multicopy mutant genes showed a dominant negative phenotype when introduced into a wild-type strain. The products of these mutant genes were purified and their biochemical properties were studied. All of them retained the ability to form a dimer and to bind specifically to a synthetic Holliday junction. However, they showed no, or extremely reduced, endonuclease activity specific for the junction. These 4 acidic residues, which are dispersed in the primary sequence, are located in close proximity at the bottom of the putative DNA binding cleft in the three-dimensional structure. From these results, we propose that these 4 acidic residues constitute the catalytic center for the Holliday junction resolvase and that some of them play a role in coordinating a divalent metal ion in the active center.
Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice.
Authors: Douglas G R , Jiao J , Gingerich J D , Gossen J A , Soper L M
Published: May 2006

The lacZ transgenic mouse (Muta mouse) model was used to examine the timing of ethylnitrosourea (ENU)-induced mutations in germ cells. The spectrum of mutations was also determined. Animals received five daily treatments with ENU at 50 mg/kg and were sampled at times up to 55 days after treatment. In mixed germ-cell populations isolated from seminiferous tubules, there was little increase in the mutant frequency 5 days after treatment; subsequently, there was a continuous increase until the maximum (17.5-fold above background) was reached by approximately 35 days. In the spermatozoa, an increase in mutant frequency was not seen until 20 days after treatment, with the maximum (4.3-fold above background) being achieved no sooner than approximately 35 days. Based on the timing of sampling, these data demonstrate the detection of both spermatogonial and postspermatogonial, mutations. The most prominent feature of the ENU-induced base-pair mutations in testicular germ cells sampled 55 days after treatment is that 70% are induced in A.T base pairs, compared to only 16% in spontaneous mutations. These findings are consistent with comparable data from ENU studies using assays for inherited germ-cell mutations in mice. This study has demonstrated the utility and potential of the transgenic mouse lacZ model (Muta mouse) for the detection and study of germ-cell mutations and provides guidance in the selection of simplified treatment and sampling protocols.
Gene for the catalytic subunit of the human DNA-activated protein kinase maps to the site of the XRCC7 gene on chromosome 8.
Authors: Sipley J D , Menninger J C , Hartley K O , Ward D C , Jackson S P , Anderson C W
Published: May 2006

The DNA-activated serine/threonine protein kinase (DNA-PK) is composed of a large (approximately 460 kDa) catalytic polypeptide (DNA-PKcs) and Ku, a heterodimeric DNA-binding component (p70/p80) that targets DNA-PKcs to DNA. A 41-kbp segment of the DNA-PKcs gene was isolated, and a 7902-bp segment was sequenced. The sequence contains a polymorphic Pvu II restriction enzyme site, and comparing the sequence with that of the cDNA revealed the positions of nine exons. The DNA-PKcs gene was mapped to band q11 of chromosome 8 by in situ hybridization. This location is coincident with that of XRCC7, the gene that complements the DNA double-strand break repair and V(D)J recombination defects (where V is variable, D is diversity, and J is joining) of hamster V3 and murine severe combined immunodeficient (scid) cells.
Similarities and differences in signal transduction by interleukin 4 and interleukin 13: analysis of Janus kinase activation.
Authors: Keegan A D , Johnston J A , Tortolani P J , McReynolds L J , Kinzer C , O'Shea J J , Paul W E
Published: May 2006

The cytokines interleukin (IL) 4 and IL-13 induce many of the same biological responses, including class switching to IgE and induction of major histocompatibility complex class II antigens and CD23 on human B cells. It has recently been shown that IL-4 induces the tyrosine phosphorylation of a 170-kDa protein, a substrate called 4PS, and of the Janus kinase (JAK) family members JAK1 and JAK3. Because IL-13 has many functional effects similar to those of IL-4, we compared the ability of IL-4 and IL-13 to activate these signaling molecules in the human multifactor-dependent cell line TF-1. In this report we demonstrate that both IL-4 and IL-13 induced the tyrosine phosphorylation of 4PS and JAK1. Interestingly, although IL-4 induced the tyrosine phosphorylation of JAK3, we did not detect JAK3 phosphorylation in response to IL-13. These data suggest that IL-4 and IL-13 signal in similar ways via the activation of JAK1 and 4PS. However, our data further indicate that there are significant differences because IL-13 does not activate JAK3.
Proenzyme of Manduca sexta phenol oxidase: purification, activation, substrate specificity of the active enzyme, and molecular cloning.
Authors: Hall M , Scott T , Sugumaran M , Söderhäll K , Law J H
Published: May 2006

Phenol oxidase (PO) was isolated as a proenzyme (pro-phenol oxidase, pro-PO) from the hemolymph of Manduca sexta larvae and purified to homogeneity. Pro-PO exhibits a M(r) of 130,000 on gel filtration and two bands with an apparent M(r) of approximately 100,000 on SDS/PAGE, as well as size-exclusion HPLC. Activation of pro-PO was achieved either by specific proteolysis by a cuticular protease or by the detergent cetylpyridinium chloride at a concentration below the critical micellar concentration. A cDNA clone for M. sexta pro-PO was obtained from a larval hemocyte cDNA library. The clone encodes a polypeptide of approximately 80,000 Da that contains two copper-binding sites and shows high sequence similarity to POs, hemocyanins, and storage proteins of arthropods. The M. Sexta pro-PO, together with other arthropod pro-POs, contains a short stretch of amino acids with sequence similarity to the thiol ester region of alpha-macroglobulins and complement proteins C3 and C4.
Role of pili and the phase-variable PilC protein in natural competence for transformation of Neisseria gonorrhoeae.
Authors: Rudel T , Facius D , Barten R , Scheuerpflug I , Nonnenmacher E , Meyer T F
Published: May 2006

The Gram-negative bacterial pathogen Neisseria gonorrhoeae is naturally competent for transformation with species-related DNA. We show here that two phase-variable pilus-associated proteins, the major pilus subunit (pilin, or PilE) and PilC, a factor known to function in the assembly and adherence of gonococcal pili, are essential for transformation competence. The PilE and PilC proteins are necessary for the conversion of linearized plasmid DNA carrying the Neisseria-specific DNA uptake signal into a DNase-resistant form. The biogenesis of typical pilus fibers is neither essential nor sufficient for this process. DNA uptake deficiency of defined piliated pilC1,2 double mutants can be complemented by expression of a cloned pilC2 gene in trans. The PilC defect can also be restored by the addition of purified PilC protein, or better, pili containing PilC protein, to the mutant gonococci. Our data suggest that the two phase-variable Pil proteins act on the bacterial cell surface and cooperate in DNA recognition and/or outer membrane translocation.
Molecular design of the N-methyl-D-aspartate receptor binding site for phencyclidine and dizolcipine.
Authors: Ferrer-Montiel A V , Sun W , Montal M
Published: May 2006

The N-methyl-D-aspartate receptor (NMDAR), a pivotal entity for synaptic plasticity and excitotoxicity in the brain, is a target of psychotomimetic drugs such as phencyclidine (PCP) and dizolcipine (MK-801). In contrast, a related glutamate receptor, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor GluR1, is weakly sensitive to these drugs. Three point mutations on GluR1, mimicking homologous residues on the NMDAR, confer the PCP and MK-801 blockade properties that are characteristic of the NMDAR--namely, high potency, voltage dependence, and use dependence. The molecular determinants that specify the PCP block appear confined to the putative M2 transmembrane segment, whereas the sensitivity to MK-801 requires an interplay between residues from M2 and M3. Given the plausible involvement of the NMDAR in the etiology of several neurodegenerative diseases and in excitotoxic neuronal cell death, tailored glutamate receptors with specific properties may be models for designing and screening new drugs targeted to prevent glutamate-mediated neural damage.
Rapid acquisition of dendritic spines by visual thalamic neurons after blockade of N-methyl-D-aspartate receptors.
Authors: Rocha M , Sur M
Published: May 2006

N-Methyl-D-aspartate (NMDA) receptors play an important role in the development of retinal axon arbors in the mammalian lateral geniculate nucleus (LGN). We investigated whether blockade of NMDA receptors in vivo or in vitro affects the dendritic development of LGN neurons during the period that retinogeniculate axons segregate into on-center and off-center sublaminae. Osmotic minipumps containing either the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV) or saline were implanted in ferret kits at postnatal day 14. After 1 week, LGN neurons were intracellularly injected with Lucifer yellow. Infusion of D-APV in vivo led to an increase in the number of branch points and in the density of dendritic spines compared with age-matched normal or saline-treated animals. To examine the time course of spine formation, crystals of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate were placed in the LGN in brain slices from 14- to 18-day-old ferrets. Labeled LGN cell dendrites were imaged on-line in living slices by confocal microscopy, with slices maintained either in normal perfusion medium or with the addition of D-APV or NMDA to the medium. Addition of D-APV in vitro at doses specific for blocking NMDA receptors led to a > 6-fold net increase in spine density compared with control or NMDA-treated slices. Spines appeared within a few hours of NMDA receptor blockade, indicating a rapid local response by LGN cells in the absence of NMDA receptor activation. Thus, activity-dependent structural changes in postsynaptic cells act together with changes in presynaptic arbors to shape projection patterns and specific retinogeniculate connections.
Brain-derived neurotrophic factor rapidly enhances synaptic transmission in hippocampal neurons via postsynaptic tyrosine kinase receptors.
Authors: Levine E S , Dreyfus C F , Black I B , Plummer M R
Published: May 2006

Although neurotrophins are primarily associated with long-term effects on neuronal survival and differentiation, recent studies have shown that acute changes in synaptic transmission can also be produced. In the hippocampus, an area critically involved in learning and memory, we have found that brain-derived neurotrophic factor (BDNF) rapidly enhanced synaptic efficacy through a previously unreported mechanism--increased postsynaptic responsiveness via a phosphorylation-dependent pathway. Within minutes of BDNF application to cultured hippocampal neurons, spontaneous firing rate was dramatically increased, as were the frequency and amplitude of excitatory postsynaptic currents. The increased frequency of postsynaptic currents resulted from the change in presynaptic firing. However, the increased amplitude was postsynaptic in origin because it was selectively blocked by intracellular injection of the tyrosine kinase receptor (Ntrk2/TrkB) inhibitor K-252a and potentiated by injection of the phosphatase inhibitor okadaic acid. These results suggest a role for BDNF in the modulation of synaptic transmission in the hippocampus.
Amyloid precursor protein processing is stimulated by metabotropic glutamate receptors.
Authors: Lee R K , Wurtman R J , Cox A J , Nitsch R M
Published: May 2006

Stimulation of muscarinic m1 or m3 receptors can, by generating diacylglycerol and activating protein kinase C, accelerate the breakdown of the amyloid precursor protein (APP) to form soluble, nonamyloidogenic derivatives (APPs), as previously shown. This relationship has been demonstrated in human glioma and neuroblastoma cells, as well as in transfected human embryonic kidney 293 cells and PC-12 cells. We now provide evidence that stimulation of metabotropic glutamate receptors (mGluRs), which also are coupled to phosphatidylinositol 4,5-bisphosphate hydrolysis, similarly accelerates processing of APP into nonamyloidogenic APPs. This process is demonstrated both in hippocampal neurons derived from fetal rats and in human embryonic kidney 293 cells transfected with cDNA expression constructs encoding the mGluR 1 alpha subtype. In hippocampal neurons, both an mGluR antagonist, L-(+)-2-amino-3-phosphonopropionic acid, and an inhibitor of protein kinase C, GF 109203X, blocked the APPs release evoked by glutamate receptor stimulation. Ionotropic glutamate agonists, N-methyl-D-aspartate or S(-)-5-fluorowillardiine, failed to affect APPs release. These data show that selective mGluR agonists that initiate signal-transduction events can regulate APP processing in bona fide primary neurons and transfected cells. As glutamatergic neurons in the cortex and hippocampus are damaged in Alzheimer disease, amyloid production in these regions may be enhanced by deficits in glutamatergic neurotransmission.
Interaction of GroEL with a highly structured folding intermediate: iterative binding cycles do not involve unfolding.
Authors: Lilie H , Buchner J
Published: May 2006

The GroE proteins are molecular chaperones involved in protein folding. The general mechanism by which they facilitate folding is still enigmatic. One of the central open questions is the conformation of the GroEL-bound nonnative protein. Several suggestions have been made concerning the folding stage at which a protein can interact with GroEL. Furthermore, the possibility exists that binding of the nonnative protein to GroEL results in its unfolding. We have addressed these issues that are basic for understanding the GroE-mediated folding cycle by using folding intermediates of an Fab antibody fragment as molecular probes to define the binding properties of GroEL. We show that, in addition to binding to an early folding intermediate, GroEL is able to recognize and interact with a late quaternary-structured folding intermediate (Dc) without measurably unfolding it. Thus, the prerequisite for binding is not a certain folding stage of a nonnative protein. In contrast, general surface properties of nonnative proteins seem to be crucial for binding. Furthermore, unfolding of a highly structured intermediate does not necessarily occur upon binding to GroEL. Folding of Dc in the presence of GroEL and ATP involves cycles of binding and release. Because in this system no off-pathway reactions or kinetic traps are involved, a quantitative analysis of the reactivation kinetics observed is possible. Our results indicate that the association reaction of Dc and GroEL in the presence of ATP is rather slow, whereas in the absence of ATP association is several orders of magnitude more efficient. Therefore, it seems that ATP functions by inhibiting reassociation rather than promoting release of the bound substrate.
Modeling and optimization of populations subject to time-dependent mutation.
Authors: Kepler T B , Perelson A S
Published: May 2006

It has become clear that many organisms possess the ability to regulate their mutation rate in response to environmental conditions. So the question of finding an optimal mutation rate must be replaced by that of finding an optimal mutation schedule. We show that this task cannot be accomplished with standard population-dynamic models. We then develop a "hybrid" model for populations experiencing time-dependent mutation that treats population growth as deterministic but the time of first appearance of new variants as stochastic. We show that the hybrid model agrees well with a Monte Carlo simulation. From this model, we derive a deterministic approximation, a "threshold" model, that is similar to standard population dynamic models but differs in the initial rate of generation of new mutants. We use these techniques to model antibody affinity maturation by somatic hypermutation. We had previously shown that the optimal mutation schedule for the deterministic threshold model is phasic, with periods of mutation between intervals of mutation-free growth. To establish the validity of this schedule, we now show that the phasic schedule that optimizes the deterministic threshold model significantly improves upon the best constant-rate schedule for the hybrid and Monte Carlo models.
Decreased atherosclerosis in mice deficient in both macrophage colony-stimulating factor (op) and apolipoprotein E.
Authors: Smith J D , Trogan E , Ginsberg M , Grigaux C , Tian J , Miyata M
Published: May 2006

To develop a murine model system to test the role of monocyte-derived macrophage in atherosclerosis, the osteopetrotic (op) mutation in the macrophage colony-stimulating factor gene was bred onto the apolipoprotein E (apoE)-deficient background. The doubly mutant (op/apoE-deficient) mice fed a low-fat chow diet had significantly smaller proximal aortic lesions at an earlier stage of progression than their apoE-deficient control littermates. These lesions in the doubly mutant mice were composed of macrophage foam cells. The op/apoE-deficient mice also had decreased body weights, decreased blood monocyte differentials, and increased mean cholesterol levels of approximately 1300 mg/dl. Statistical analysis determined that atherosclerosis lesion area was significantly affected by the op genotype and gender. The confounding variables of body weight, plasma cholesterol, and monocyte differential, which were all affected by op genotype, had no significant additional effect on lesion area once they were adjusted for the effects of op genotype and gender. Unexpectedly, there was a significant inverse correlation between plasma cholesterol and lesion area, implying that each may be the result of a common effect of macrophage colony-stimulating factor levels. The data support the hypothesis that macrophage colony-stimulating factor and its effects on macrophage development and function play a key role in atherogenesis.
A general model of invariant chain association with class II major histocompatibility complex proteins.
Authors: Lee C , McConnell H M
Published: May 2006

The binding of invariant chain to major histocompatibility complex (MHC) proteins is an important step in processing of MHC class II proteins and in antigen presentation. The question of how invariant chain can bind to all MHC class II proteins is central to understanding these processes. We have employed molecular modeling to predict the structure of class II-associated invariant chain peptide (CLIP)-MHC protein complexes and to ask whether the predicted mode of association could be general across all MHC class II proteins. CLIP fits identically into the MHC class II alleles HLA-DR3, I-Ak, I-Au, and I-Ad, with a consistent pattern of hydrogen bonds, contacts, and hydrophobic burial and without bad contacts. Our model predicts the burial of CLIP residues Met-91 and Met-99 in the deep P1 and P9 anchor pockets and other detailed interactions, which we have compared with available data. The predicted pattern of I-A allele-specific effects on CLIP binding is very similar to that observed experimentally by alanine-scanning mutations of CLIP. Together, these results indicate that CLIP may bind in a single, general way across products of MHC class II alleles.
Four p53 DNA-binding domain peptides bind natural p53-response elements and bend the DNA.
Authors: Balagurumoorthy P , Sakamoto H , Lewis M S , Zambrano N , Clore G M , Gronenborn A M , Appella E , Harrington R E
Published: May 2006

Recent structural studies of the minimal core DNA-binding domain of p53 (p53DBD) complexed to a single consensus pentamer sequence and of the isolated p53 tetramerization domain have provided valuable insights into their functions, but many questions about their interacting roles and synergism remain unanswered. To better understand these relationships, we have examined the binding of the p53DBD to two biologically important full-response elements (the WAF1 and ribosomal gene cluster sites) by using DNA circularization and analytical ultracentrifugation. We show that the p53DBD binds DNA strongly and cooperatively with p53DBD to DNA binding stoichiometries of 4:1. For the WAF1 element, the mean apparent Kd is (8.3 +/- 1.4) x 10(-8) M, and no intermediate species of lower stoichiometries can be detected. We show further that complex formation induces an axial bend of at least 60 degrees in both response elements. These results, taken collectively, demonstrate that p53DBD possesses the ability to direct the formation of a tight nucleoprotein complex having the same 4:1 DNA-binding stoichiometry as wild-type p53 which is accompanied by a substantial conformational change in the response-element DNA. This suggests that the p53DBD may play a role in the tetramerization function of p53. A possible role in this regard is proposed.
Cytochrome c oxidase in Neurospora crassa contains myristic acid covalently linked to subunit 1.
Authors: Vassilev A O , Plesofsky-Vig N , Brambl R
Published: May 2006

Radiolabel from [3H]myristic acid was incorporated by Neurospora crassa into the core catalytic subunit 1 of cytochrome c oxidase (EC 1.9.3.1), as indicated by immunoprecipitation. This modification of the subunit, which was specific for myristic acid, represents an uncommon type of myristoylation through an amide linkage at an internal lysine, rather than an N-terminal glycine. The [3H]myristate, which was chemically recovered from the radiolabeled subunit peptide, modified an invariant Lys-324, based upon analyses of proteolysis products. This myristoylated lysine is found within one of the predicted transmembrane helices of subunit 1 and could contribute to the environment of the active site of the enzyme. The myristate was identified by mass spectrometry as a component of mature subunit 1 of a catalytically active, purified enzyme. To our knowledge, fatty acylation of a mitochondrially synthesized inner-membrane protein has not been reported previously.
On constructing folding heteropolymers.
Authors: Ebeling M , Nadler W
Published: May 2006

Simplified models of the protein-folding process have led to valuable insights into the generic properties of the folding of heteropolymers. On the basis of theoretical arguments, Shakhnovich and Gutin [(1993) Proc. Natl. Acad. Sci. USA 90, 7195-7199] have proposed a specific method to generate folding sequences for one of these. Here we present a model of folding in heteropolymers that is comparable in simplicity but different in spirit to the one studied by Shakhnovich and Gutin. In our model, the proposed recipe for constructing folding sequence fails. We find that, as a rule, the construction of folding sequences is impossible to achieve by looking at the native conformation only. Rather, competing conformations have to be taken into account too. An evolutionary algorithm that generates folding sequences by optimizing both stability of the native state and folding time is described. Remarkably, this algorithm produces, among others, sequences that fold reproducibly to metastable states.
Phorbol ester treatment inhibits phosphatidylinositol 3-kinase activation by, and association with, CD28, a T-lymphocyte surface receptor.
Authors: Hutchcroft J E , Franklin D P , Tsai B , Harrison-Findik D , Varticovski L , Bierer B E
Published: May 2006

CD28 is a costimulatory receptor found on the surface of most T lymphocytes. Engagement of CD28 induces interleukin 2 (IL-2) production and cell proliferation when combined with an additional signal such as treatment with phorbol ester, an activator of protein kinase C. Recent studies have established that after CD28 ligation, the cytoplasmic domain of CD28 can bind to the 85-kDa subunit of phosphatidylinositol 3-kinase (PI3 kinase). There is a concomitant increase in PI3 lipid kinase activity that may be important in CD28 signaling. Despite the requirement of phorbol 12-myristate 13-acetate (PMA) for effector function, we have found, however, that treatment of Jurkat T cells with the phorbol ester PMA dramatically inhibits (i) the association of PI3 kinase with CD28, (ii) the ability of p85 PI3 kinase to be immunoprecipitated by anti-phosphotyrosine antibodies, and (iii) the induction of PI3 kinase activity after stimulation of the cells with the anti-CD28 monoclonal antibody 9.3. These changes occur within minutes of PMA treatment and are persistent. In addition, we have found that wortmannin, a potent inhibitor of PI3 kinase, does not interfere with the induction of IL-2 after stimulation of Jurkat T cells with anti-CD28 monoclonal antibody and PMA. We conclude that PI3 kinase activity may not be required for CD28-dependent IL-2 production from Jurkat T cells in the presence of PMA.
Phosphorylation-dependent conformational changes in OmpR, an osmoregulatory DNA-binding protein of Escherichia coli.
Authors: Kenney L J , Bauer M D , Silhavy T J
Published: May 2006

Osmoregulated porin gene expression in Escherichia coli is controlled by the two-component regulatory system EnvZ and OmpR. EnvZ, the osmosensor, is an inner membrane protein and a histidine kinase. EnvZ phosphorylates OmpR, a cytoplasmic DNA-binding protein, on an aspartyl residue. Phospho-OmpR binds to the promoters of the porin genes to regulate the expression of ompF and ompC. We describe the use of limited proteolysis by trypsin and ion spray mass spectrometry to characterize phospho-OmpR and the conformational changes that occur upon phosphorylation. Our results are consistent with a two-domain structure for OmpR, an N-terminal phosphorylation domain joined to a C-terminal DNA-binding domain by a flexible linker region. In the presence of acetyl phosphate, OmpR is phosphorylated at only one site. Phosphorylation induces a conformational change that is transmitted to the C-terminal domain via the central linker. Previous genetic analysis identified a region in the C-terminal domain that is required for transcriptional activation. Our results indicate that this region is within a surface-exposed loop. We propose that this loop contacts the alpha subunit of RNA polymerase to activate transcription. Mass spectrometry also reveals an unusual dephosphorylated form of OmpR, the potential significance of which is discussed.
Li-Fraumeni syndrome fibroblasts homozygous for p53 mutations are deficient in global DNA repair but exhibit normal transcription-coupled repair and enhanced UV resistance.
Authors: Ford J M , Hanawalt P C
Published: May 2006

We investigated whether mutations in the p53 tumor suppressor gene alter UV sensitivity and/or repair of UV-induced DNA damage in primary human skin fibroblasts from patients with Li-Fraumeni syndrome, heterozygous for mutations in one allele of the p53 gene (p53 wt/mut) and sublines expressing only mutant p53 (p53 mut). The p53 mut cells were more resistant than the p53 wt/mut cells to UV cytotoxicity and exhibited less UV-induced apoptosis. DNA repair analysis revealed reduced removal of cyclobutane pyrimidine dimers from overall genomic DNA in vivo in p53 mut cells compared with p53 wt/mut or normal cells. However, p53 mut cells retained the ability to preferentially repair damage in the transcribed strands of expressed genes (transcription-coupled repair). These results suggest that loss of p53 function may lead to greater genomic instability by reducing the efficiency of DNA repair but that cellular resistance to DNA-damaging agents may be enhanced through elimination of apoptosis.