bluuebunny/crossref_metadata_2025 huggingface dataset:
A peptide that binds and stabilizes p53 core domain: Chaperone strategy for rescue of oncogenic mutants
Authors: Friedler Assaf , Hansson Lars O. , Veprintsev Dmitry B. , Freund Stefan M. V. , Rippin Thomas M. , Nikolova Penka V. , Proctor Mark R. , Rüdiger Stefan , Fersht Alan R.
Published: July 2002

Conformationally compromised oncogenic mutants of the tumor suppressor protein p53 can, in principle, be rescued by small molecules that bind the native, but not the denatured state. We describe a strategy for the rational search for such molecules. A nine-residue peptide, CDB3, which was derived from a p53 binding protein, binds to p53 core domain and stabilizes it
in vitro
. NMR studies showed that CDB3 bound to p53 at the edge of the DNA binding site, partly overlapping it. The fluorescein-labeled peptide, FL-CDB3, binds wild-type p53 core domain with a dissociation constant of 0.5 μM, and raises the apparent melting temperatures of wild-type and a representative oncogenic mutant, R249S core domain. gadd45 DNA competes with CDB3 and displaces it from its binding site. But this competition does not preclude CDB3 from being a lead compound. CDB3 may act as a “chaperone” that maintains existing or newly synthesized destabilized p53 mutants in a native conformation and then allows transfer to specific DNA, which binds more tightly. Indeed, CDB3 restored specific DNA binding activity to a highly destabilized mutant I195T to close to that of wild-type level.
Predicting durability of a disease resistance gene based on an assessment of the fitness loss and epidemiological consequences of avirulence gene mutation
Authors: Vera Cruz Casiana M. , Bai Jianfa , Oña Isabelita , Leung Hei , Nelson Rebecca J. , Mew Twng-Wah , Leach Jan E.
Published: July 2002

Durability of plant disease resistance (
R
) genes may
be predicted if the cost of pathogen adaptation to overcome resistance
is understood. Adaptation of the bacterial blight pathogen,
Xanthomonas oryzae
pv.
oryzae
(
Xoo
), to virulence in rice is the result of the loss of
pathogen avirulence gene function, but little is known about its effect
on aggressiveness under field conditions. We evaluated the cost in
pathogenic fitness (aggressiveness and persistence) associated with
adaptation of
Xoo
to virulence on near-isogenic rice
lines with single
R
genes (
Xa7
,
Xa10
, and
Xa4
) at two field sites endemic
for bacterial blight. Disease severity was high in all 3 years on all
lines except the line with
Xa7
. Of two
Xoo
lineages (groups of strains inferred to be clonally
related based on DNA fingerprinting) detected, one, lineage C,
dominated the pathogen population at both sites. All
Xoo
strains were virulent to
Xa4
, whereas only lineage C
strains were virulent to
Xa10.
Only a few strains of
lineage C were virulent to
Xa7
. Adaptation to virulence
on
Xa7
occurred through at least four different pathways
and was associated with a reduction in aggressiveness. Loss of
avirulence and reduced aggressiveness were associated with mutations at
the 3′ terminus of the
avrXa7
allele. Strains most
aggressive to
Xa7
were not detected after the second
year, suggesting they were less persistent than less aggressive
strains. These experiments support the prediction that
Xa7
would be a durable
R
gene because of
a fitness penalty in
Xoo
associated with adaptation to
Xa7
.
Aβ amyloid fibrils possess a core structure highly resistant to hydrogen exchange
Authors: Kheterpal Indu , Zhou Shaolian , Cook Kelsey D. , Wetzel Ronald
Published: July 2002

We describe here experiments designed to characterize the secondary
structure of amyloid fibrils of the Alzheimer's amyloid plaque peptide
Aβ, using hydrogen-deuterium exchange measurements evaluated by mass
spectrometry. The results show that ≈50% of the amide protons of the
polypeptide backbone of Aβ(1–40) resist exchange in aqueous, neutral
pH buffer even after more than 1,000 h of incubation at room
temperature. We attribute this extensive, strong protection to
H-bonding by residues in core regions of β-sheet structure within the
fibril. The backbone amide hydrogens exchange at variable rates,
suggesting different degrees of protection within the fibril. These
data suggest that it is unlikely that the entire Aβ sequence is
involved in H-bonded secondary structure within the amyloid fibril.
Future studies using the methods described here should reveal further
details of Aβ fibril structure and assembly. These methods also
should be amenable to studies of other amyloid fibrils and protein
aggregates.
Genome-wide study of aging and oxidative stress response inDrosophilamelanogaster
Authors: Zou Sige , Meadows Sarah , Sharp Linda , Jan Lily Y. , Jan Yuh Nung
Published: July 2002

Aging is a universal but poorly understood biological process. Free radicals accumulate with age and have been proposed to be a major cause of aging. We measured genome-wide changes in transcript levels as a function of age inDrosophila melanogasterand compared these changes with those caused by paraquat, a free-radical generator. A number of genes exhibited changes in transcript levels with both age and paraquat treatment. We also found genes whose transcript levels changed with age but not with paraquat treatment. This study suggests that free radicals play an important role in regulating transcript levels in aging but that they are not the only factors. This genome-wide survey also identifies candidates for molecular markers of aging.
Memory loss in old rats is associated with brain mitochondrial decay and RNA/DNA oxidation: Partial reversal by feeding acetyl-
l
-carnitine and/or
R
-α-lipoic acid
Authors: Liu Jiankang , Head Elizabeth , Gharib Afshin M. , Yuan Wenjun , Ingersoll Russell T. , Hagen Tory M. , Cotman Carl W. , Ames Bruce N.
Published: July 2002

Accumulation of oxidative damage to mitochondria, protein, and nucleic acid in the brain may lead to neuronal and cognitive dysfunction. The effects on cognitive function, brain mitochondrial structure, and biomarkers of oxidative damage were studied after feeding old rats two mitochondrial metabolites, acetyl-
l
-carnitine (ALCAR) [0.5% or 0.2% (wt/vol) in drinking water], and/or
R
-α-lipoic acid (LA) [0.2% or 0.1% (wt/wt) in diet]. Spatial memory was assessed by using the Morris water maze; temporal memory was tested by using the peak procedure (a time-discrimination procedure). Dietary supplementation with ALCAR and/or LA improved memory, the combination being the most effective for two different tests of spatial memory (
P
< 0.05;
P
< 0.01) and for temporal memory (
P
< 0.05). Immunohistochemical analysis showed that oxidative damage to nucleic acids (8-hydroxyguanosine and 8-hydroxy-2′-deoxyguanosine) increased with age in the hippocampus, a region important for memory. Oxidative damage to nucleic acids occurred predominantly in RNA. Dietary administration of ALCAR and/or LA significantly reduced the extent of oxidized RNA, the combination being the most effective. Electron microscopic studies in the hippocampus showed that ALCAR and/or LA reversed age-associated mitochondrial structural decay. These results suggest that feeding ALCAR and LA to old rats improves performance on memory tasks by lowering oxidative damage and improving mitochondrial function.
DEC1 is a downstream target of TGF-β with sequence-specific transcriptional repressor activities
Authors: Zawel Leigh , Yu Jian , Torrance Christopher J. , Markowitz Sanford , Kinzler Kenneth W. , Vogelstein Bert , Zhou Shibin
Published: July 2002

To identify genes that mediate transforming growth factor-β (TGF-β) signaling, a colorectal cancer cell line that was sensitive to the growth inhibitory effects of this cytokine was created. We then determined the global gene expression profiles of these cells, and those of HaCaT human keratinocytes, in the presence and absence of TGF-β. Of the several genes identified in this screen,
DEC1
was of particular note in light of the rapidity and consistency of its induction and its potential biochemical activities. We identified a consensus DNA-binding site for DEC1 and showed that DEC1 could repress the transcription of a reporter containing this binding site in its promoter. Finally, both alleles of the
DEC1
locus in HaCaT cells were inactivated through targeted homologous recombination. This approach revealed that DEC1 induction was not required for the growth inhibition mediated by TGF-β in this line. However, DEC1 may function in concert with other signaling components to mediate certain biologic effects of TGF-β.
Codon repeats in genes associated with human diseases: fewer repeats in the genes of nonhuman primates and nucleotide substitutions concentrated at the sites of reiteration.
Authors: Djian P , Hancock J M , Chana H S
Published: July 2002

Five human diseases are due to an excessive number of CAG repeats in the coding regions of five different genes. We have analyzed the repeat regions in four of these genes from nonhuman primates, which are not known to suffer from the diseases. These primates have CAG repeats at the same sites as in human alleles, and there is similar polymorphism of repeat number, but this number is smaller than in the human genes. In some of the genes, the segment of poly(CAG) has expanded in nonhuman primates, but the process has advanced further in the human lineage than in other primate lineages, thereby predisposing to diseases of CAG reiteration. Adjacent to stretches of homogeneous present-day codon repeats, previously existing codons of the same kind have undergone nucleotide substitutions with high frequency. Where these lead to amino acid substitutions, the effect will be to reduce the length of the original homopolymeric stretch in the protein.
Dimerization specificity of Arabidopsis MADS domain homeotic proteins APETALA1, APETALA3, PISTILLATA, and AGAMOUS.
Authors: Riechmann J L , Krizek B A , Meyerowitz E M
Published: July 2002

The MADS domain homeotic proteins APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) act in a combinatorial manner to specify the identity of Arabidopsis floral organs. The molecular basis for this combinatorial mode of action was investigated. Immunoprecipitation experiments indicate that all four proteins are capable of interacting with each other. However, these proteins exhibit "partner-specificity" for the formation of DNA-binding dimers; only AP1 homodimers, AG homodimers, and AP3/PI heterodimers are capable of binding to CArG-box sequences. Both the AP3/PI heterodimer and the AP1 or AG homodimers are formed when the three corresponding proteins are present together. The use of chimeric proteins formed by domain swapping indicates that the L region (which follows the MADS box) constitutes a key molecular determinant for the selective formation of DNA-binding dimers. The implications of these results for the ABC genetic model of flower development are discussed.
Dopamine transporters are markedly reduced in Lesch-Nyhan disease in vivo.
Authors: Wong D F , Harris J C , Naidu S , Yokoi F , Marenco S , Dannals R F , Ravert H T , Yaster M , Evans A , Rousset O , Bryan R N , Gjedde A , Kuhar M J , Breese G R
Published: July 2002

Dopamine (DA) deficiency has been implicated in Lesch-Nyhan disease (LND), a genetic disorder that is characterized by hyperuricemia, choreoathetosis, dystonia, and compulsive self-injury. To establish that DA deficiency is present in LND, the ligand WIN-35,428, which binds to DA transporters, was used to estimate the density of DA-containing neurons in the caudate and putamen of six patients with classic LND. Comparisons were made with 10 control subjects and 3 patients with Rett syndrome. Three methods were used to quantify the binding of the DA transporter so that its density could be estimated by a single dynamic positron emission tomography study. These approaches included the caudate- or putamen-to-cerebellum ratio of ligand at 80-90 min postinjection, kinetic analysis of the binding potential [Bmax/(Kd x Vd)] using the assumption of equal partition coefficients in the striatum and the cerebellum, and graphical analysis of the binding potential. Depending on the method of analysis, a 50-63% reduction of the binding to DA transporters in the caudate, and a 64-75% reduction in the putamen of the LND patients was observed compared to the normal control group. When LND patients were compared to Rett syndrome patients, similar reductions were found in the caudate (53-61%) and putamen (67-72%) in LND patients. Transporter binding in Rett syndrome patients was not significantly different from the normal controls. Finally, volumetric magnetic resonance imaging studies detected a 30% reduction in the caudate volume of LND patients. To ensure that a reduction in the caudate volume would not confound the results, a rigorous partial volume correction of the caudate time activity curve was performed. This correction resulted in an even greater decrease in the caudate-cerebellar ratio in LND patients when contrasted to controls. To our knowledge, these findings provide the first in vivo documentation of a dopaminergic reduction in LND and illustrate the role of positron emission tomography imaging in investigating neurodevelopmental disorders.
The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes.
Authors: Solomon K R , Rudd C E , Finberg R W
Published: July 2002

Glycosylphosphatidylinositol (GPI)-anchored proteins are nonmembrane spanning cell surface proteins that have been demonstrated to be signal transduction molecules. Because these proteins do not extend into the cytoplasm, the mechanism by which cross-linking of these molecules leads to intracellular signal transduction events is obscure. Previous analysis has indicated that these proteins are associated with src family member tyrosine kinases; however, the role this interaction plays in the generation of intracellular signals is not clear. Here we show that GPI-anchored proteins are associated with alpha subunits of heterotrimeric GTP binding proteins (G proteins) in both human and murine lymphocytes. When the GPI-anchored proteins CD59, CD48, and Thy-1 were immunoprecipitated from various cell lines or freshly isolated lymphocytes, all were found to be associated with a 41-kDa phosphoprotein that we have identified, by using specific antisera, as a mixture of tyrosine phosphorylated G protein alpha subunits: a small amount of Gialpha1, and substantial amounts of Gialpha2 and Gialpha3. GTP binding assays performed with immunoprecipitations of CD59 indicated that there was GTP-binding activity associated with this molecule. Thus, we have shown by both immunochemical and functional criteria that GPI-anchored proteins are physically associated with G proteins. These experiments suggest a potential role of G proteins in the transduction of signals generated by GPI-anchored molecules expressed on lymphocytes of both mouse and human.
Mitochondria are selective targets for the protective effects of heat shock against oxidative injury.
Authors: Polla B S , Kantengwa S , François D , Salvioli S , Franceschi C , Marsac C , Cossarizza A
Published: July 2002

Heat shock (HS) proteins (HSPs) induce protection against a number of stresses distinct from HS, including reactive oxygen species. In the human premonocytic line U937, we investigated in whole cells the effects of preexposure to HS and exposure to hydrogen peroxide (H2O2) on mitochondrial membrane potential, mass, and ultrastructure. HS prevented H2O2-induced alterations in mitochondrial membrane potential and cristae formation while increasing expression of HSPs and the protein product of bcl-2. Protection correlated best with the expression of the 70-kDa HSP, hsp70. We propose that mitochondria represent a selective target for HS-mediated protection against oxidative injury.
The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins.
Authors: Yuryev A , Patturajan M , Litingtung Y , Joshi R V , Gentile C , Gebara M , Corden J L
Published: July 2002

Although transcription and pre-mRNA processing are colocalized in eukaryotic nuclei, molecules linking these processes have not previously been described. We have identified four novel rat proteins by their ability to interact with the repetitive C-terminal domain (CTD) of RNA polymerase II in a yeast two-hybrid assay. A yeast homolog of one of the rat proteins has also been shown to interact with the CTD. These CTD-binding proteins are all similar to the SR (serine/arginine-rich) family of proteins that have been shown to be involved in constitutive and regulated splicing. In addition to alternating Ser-Arg domains, these proteins each contain discrete N-terminal or C-terminal CTD-binding domains. We have identified SR-related proteins in a complex that can be immunoprecipitated from nuclear extracts with antibodies directed against RNA polymerase II. In addition, in vitro splicing is inhibited either by an antibody directed against the CTD or by wild-type but not mutant CTD peptides. Thus, these results suggest that the CTD and a set of CTD-binding proteins may act to physically and functionally link transcription and pre-mRNA processing.
Fused polycationic peptide mediates delivery of diphtheria toxin A chain to the cytosol in the presence of anthrax protective antigen.
Authors: Blanke S R , Milne J C , Benson E L , Collier R J
Published: July 2002

The lethal factor (LF) and edema factor (EF) of anthrax toxin bind by means of their amino-terminal domains to protective antigen (PA) on the surface of toxin-sensitive cells and are translocated to the cytosol, where they act on intracellular targets. Genetically fusing the amino-terminal domain of LF (LFN; residues 1-255) to certain heterologous proteins has been shown to potentiate these proteins for PA-dependent delivery to the cytosol. We report here that short tracts of lysine, arginine, or histidine residues can also potentiate a protein for such PA-dependent delivery. Fusion of these polycationic tracts to the amino terminus of the enzymic A chain of diphtheria toxin (DTA; residues 1-193) enabled it to be translocated to the cytosol by PA and inhibit protein synthesis. The efficiency of translocation was dependent on tract length: (LFN > Lys8 > Lys6 > Lys3). Lys6 was approximately 100-fold more active than Arg6 or His6, whereas Glu6 and (SerSerGly)2 were inactive. Arg6DTA was partially degraded in cell culture, which may explain its low activity relative to that of Lys6DTA. The polycationic tracts may bind to anionic sites at the cell surface (possibly on PA), allowing the fusion proteins to be coendocytosed with PA and delivered to the endosome, where translocation to the cytosol occurs. Excess free LFN blocked the action of LFNDTA, but not of Lys6DTA. This implies that binding to the LF/EF site is not an obligatory step in translocation and suggests that the polycationic tag binds to a different site. Besides elucidating the process of translocation in anthrax toxin, these findings may aid in developing systems to deliver heterologous proteins and peptides to the cytoplasm of mammalian cells.
Auxin as a positional signal in pattern formation in plants.
Authors: Uggla C , Moritz T , Sandberg G , Sundberg B
Published: July 2002

By using a novel, extremely sensitive and specific gas chromatography-mass spectrometry technique we demonstrate in Pinus sylvestris (L.) trees the existence of a steep radial concentration gradient of the endogenous auxin, indole-3-acetic acid, over the lateral meristem responsible for the bulk of plant secondary growth, the vascular cambium. This is the first evidence that plant morphogens, such as indole-3-acetic acid, occur in concentration gradients over developing tissues. This finding gives evidence for a regulatory system in plants based on positional signaling, similar to animal systems.
Guanidinium-cholesterol cationic lipids: efficient vectors for the transfection of eukaryotic cells.
Authors: Vigneron J P , Oudrhiri N , Fauquet M , Vergely L , Bradley J C , Basseville M , Lehn P , Lehn J M
Published: July 2002

Two cationic lipids, bis-guanidinium-spermidine-cholesterol (BGSC) and bis-guanidinium-trencholesterol (BGTC)-cholesterol derivatives bearing two guanidinium groups-have been synthesized and tested as artificial vectors for gene transfer. They combine the membrane compatible features of the cholesterol subunit and the favorable structural and high pKa features of the guanidinium functions for binding DNA via its phosphate groups. Reagent BGTC is very efficient for transfection into a variety of mammalian cell lines when used as a micellar solution. In addition, both BGTC and BGSC present also a high transfection activity when formulated as liposomes with the neutral phospholipid dioleoylphosphatidyl ethanolamine. These results reveal the usefulness of cholesterol derivatives bearing guanidinium groups for gene transfer.
Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.
Authors: Herman J G , Graff J R , Myöhänen S , Nelkin B D , Baylin S B
Published: July 2002

Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.
Female mating preference for bold males in the guppy, Poecilia reticulata.
Authors: Godin J G , Dugatkin L A
Published: July 2002

Although females prefer to mate with brightly colored males in numerous species, the benefits accruing to such females are virtually unknown. According to one hypothesis of sexual selection theory, if the expression of costly preferred traits in males (such as conspicuous colors) is proportional to the male's overall quality or reveals his quality, a well-developed trait should indicate good condition and/or viability for example. A female choosing such a male would therefore stand to gain direct or indirect fitness benefits, or both. Among potential phenotypic indicators of an individual's quality are the amount and brightness of its carotenoid-based colors and its boldness, as measured by its willingness to risk approaching predators without being killed. Here, we show experimentally that in the Trinidadian guppy (Poecilia reticulata) the visual conspicuousness of the color pattern of males correlates positively with boldness toward, and with escape distance from, a cichlid fish predator. Bold individuals are thus more informed about nearby predators and more likely to survive encounters with them. Mate-choice experiments showed that females prefer colorful males as mates, but prefer bolder males irrespective of their coloration when given the opportunity to observe their behavior toward a potential fish predator. By preferentially mating with colorful males, female guppies are thus choosing on average, relatively bold, and perhaps more viable, individuals. In doing so, and to the extent that viability is heritable, they potentially gain indirect fitness benefits by producing more viable offspring than otherwise.
Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification.
Authors: Gottlieb R A , Nordberg J , Skowronski E , Babior B M
Published: July 2002

We have previously shown that in neutrophils deprived of granulocyte colony-stimulating factor, apoptosis is preceded by acidification and that the protection against apoptosis conferred on neutrophils by granulocyte colony-stimulating factor is dependent upon delay of this acidification. To test the hypothesis that acidification could be a general feature of apoptosis, we examined intracellular pH changes in another cell line. Jurkat cells, a T-lymphoblastoid line, were induced to undergo apoptosis with anti-Fas IgM, cycloheximide, or exposure to short-wavelength UV light. We found that acidification occurred in response to treatment with these agents and that acidification preceded DNA fragmentation. Jurkat cells were also found to possess an acid endonuclease that is active below pH 6.8, compatible with a possible role for this enzyme in chromatin digestion during apoptosis. Incubation of the cells with the bases imidazole or chloroquine during treatment with anti-Fas antibody or cycloheximide or after UV exposure decreased apoptosis as assessed by nuclear morphology and DNA content. The alkalinizing effect of imidazole and chloroquine was shown by the demonstration that the percentage of cells with an intracellular pH below 6.8 after treatment with anti-Fas antibody, cycloheximide, or UV was diminished in the presence of base as compared with similarly treated cells incubated in the absence of base. We conclude that acidification is an early event in programmed cell death and may be essential for genome destruction.
Analysis of the genetic differences between Neisseria meningitidis and Neisseria gonorrhoeae: two closely related bacteria expressing two different pathogenicities.
Authors: Tinsley C R , Nassif X
Published: July 2002

We have investigated genetic differences between the closely related pathogenic Neisseria species, Neisseria meningitidis and Neisseria gonorrhoeae, as a novel approach to the elucidation of the genetic basis for their different pathogenicities. N. meningitidis is a major cause of cerebrospinal meningitis, whereas N. gonorrhoeae is the agent of gonorrhoea. The technique of representational difference analysis was adapted to the search for genes present in the meningococcus but absent from the gonococcus. The libraries achieved are comprehensive and specific in that they contain sequences corresponding to the presently identified meningococcus-specific genes (capsule, frp, rotamase, and opc) but lack genes more or less homologous between the two species, e.g., ppk and pilC1. Of 35 randomly chosen clones specific to N. meningitidis, DNA sequence analysis has confirmed that the large majority have no homology with published neisserial sequences. Mapping of the cloned DNA fragments onto the chromosome of N. meningitidis strain Z2491 has revealed a nonrandom distribution of meningococcus-specific sequences. Most of the genetic differences between the meningococcus and gonococcus appear to be clustered in three distinct regions, one of which (region 1) contains the capsule-related genes. Region 3 was found only in strains of serogroup A, whereas region 2 is present in a variety of meningococci belonging to different serogroups. At a time when bacterial genomes are being sequenced, we believe that this technique is a powerful tool for a rapid and directed analysis of the genetic basis of inter- or intraspecific phenotypic variations.
msg1, a novel melanocyte-specific gene, encodes a nuclear protein and is associated with pigmentation.
Authors: Shioda T , Fenner M H , Isselbacher K J
Published: July 2002

Messenger RNA transcripts of the highly pigmented murine melanoma B16-F1 cells were compared with those from their weakly pigmented derivative B16-F10 cells by differential display. A novel gene called msg1 (melanocyte-specific gene) was found to be expressed at high levels in B16-F1 cells but at low levels in B16-F10 cells. Expression of msg1 was undetectable in the amelanotic K1735 murine melanoma cells. The pigmented murine melanocyte cell line melan-a expressed msg1, as did pigmented primary cultures of murine and human melanocytes; however, seven amelanotic or very weakly pigmented human melanoma cell lines were negative. Transformation of murine melanocytes by transfection with v-Ha-ras or Ela was accompanied by depigmentation and led to complete loss of msg1 expression. The normal tissue distribution of msg1 mRNA transcripts in adult mice was confined to melanocytes and testis. Murine msg1 and human MSG1 genes encode a predicted protein of 27 kDa with 75% overall amino acid identity and 96% identity within the C-terminal acidic domain of 54 amino acids. This C-terminal domain was conserved with 76% amino acid identity in another protein product of a novel human gene, MRG1 (msg1-related gene), isolated from normal human melanocyte cDNA by 5'-rapid amplification of cDNA ends based on the homology to msg1. The msg1 protein was localized to the melanocyte nucleus by immunofluorescence cytochemistry. We conclude that msg1 encodes a nuclear protein, is melanocyte-specific, and appears to be lost in depigmented melanoma cells.
Identification of a novel p53 functional domain that is necessary for efficient growth suppression
Authors: Walker Kristen K. , Levine Arnold J.
Published: July 2002

Activation of the p53 tumor suppressor protein has been
demonstrated to block cell growth by inducing either a transient cell
cycle arrest or programmed cell death (apoptosis). Although
evidence exists linking p53’s function as an activator of
transcription to its ability to effect cell cycle arrest, the role of
this activity in the induction of apoptosis remains unclear. To
gain insight into the molecular mechanisms underlying p53-mediated
antiproliferative pathways, a study was initiated to explore the
functions of a putative p53 signaling domain. This region of the human
p53 protein is localized between amino acids 61 and 94 (out of 393) and
is noteworthy in that it contains five repeats of the sequence PXXP
(where P represents proline and X any amino acid). This motif has been
shown to play a role in signal transduction via its SH3 domain binding
activity. A p53 cDNA deletion mutant (ΔproAE), which lacks this
entire proline-rich domain (deleted for amino acids 62–91), was
created and characterized for a variety of p53 functions. The entire
domain has been shown to be completely dispensable for transcriptional
activation. On the other hand, this deletion of the p53 proline-rich
domain impairs p53’s ability to suppress tumor cell growth in culture.
Amino acid substitution mutations at residues 22 and 23 of p53
(eliminates transcriptional activity) also impair p53-mediated
inhibition of cell growth in culture. Unlike wild-type p53, the
ΔproAE mutant cDNA can be stably expressed in tumor derived cell
lines with few immediate detrimental effects. These cells express
physiologic levels of p53 protein that are induced normally in response
to DNA damage, indicating that removal of the proline-rich domain does
not disrupt p53’s upstream regulation by DNA damage. These data
indicate that, in addition to the transcriptional activation domain,
the p53 proline-rich domain plays a critical role in the transmission
of antiproliferative signals downstream of the p53 protein and may link
p53 to a direct signal transduction pathway.
Intergenic splicing of MDS1 and EVI1 occurs in normal tissues as well as in myeloid leukemia and produces a new member of the PR domain family.
Authors: Fears S , Mathieu C , Zeleznik-Le N , Huang S , Rowley J D , Nucifora G
Published: July 2002

The EVI1 gene, located at chromosome band 3q26, is overexpressed in some myeloid leukemia patients with breakpoints either 5' of the gene in the t(3;3)(q21;q26) or 3' of the gene in the inv(3)(q21q26). EVI1 is also expressed as part of a fusion transcript with the transcription factor AML1 in the t(3;21)(q26;q22), associated with myeloid leukemia. In cells with t(3;21), additional fusion transcripts are AML1-MDS1 and AML1-MDS1-EVI1. MDS1 is located at 3q26 170-400 kb upstream (telomeric) of EVI1 in the chromosomal region in which some of the breakpoints 5' of EVI1 have been mapped. MDS1 has been identified as a single gene as well as a previously unreported exon(s) of EVI1 We have analyzed the relationship between MDS1 and EVI1 to determine whether they are two separate genes. In this report, we present evidence indicating that MDS1 exists in normal tissues both as a unique transcript and as a normal fusion transcript with EVI1, with an additional 188 codons at the 5' end of the previously reported EVI1 open reading frame. This additional region has about 40% homology at the amino acid level with the PR domain of the retinoblastoma-interacting zinc-finger protein RIZ. These results are important in view of the fact that EVI1 and MDS1 are involved in leukemia associated with chromosomal translocation breakpoints in the region between these genes.
Decreased food intake does not completely account for adiposity reduction after ob protein infusion.
Authors: Levin N , Nelson C , Gurney A , Vandlen R , de Sauvage F
Published: July 2002

The effects of recombinantly produced ob protein were compared to those of food restriction in normal lean and genetically obese mice. Ob protein infusion into ob/ob mice resulted in large decreases in body and fat-depot weight and food intake that persisted throughout the study. Smaller decreases in body and fat-depot weights were observed in vehicle-treated ob/ob mice that were fed the same amount of food as that consumed by ob protein-treated ob/ob mice (pair feeding). In lean mice, ob protein infusion significantly decreased body and fat-depot weights, while decreasing food intake to a much lesser extent than in ob/ob mice. Pair feeding of lean vehicle-treated mice to the intake of ob protein-treated mice did not reduce body fat-depot weights. The potent weight-, adipose-, and appetite-reducing effects exerted by the ob protein in ob protein-deficient mice (ob/ob) confirm hypotheses generated from early parabiotic studies that suggested the existence of a circulating satiety factor of adipose origin. Pair-feeding studies provide compelling evidence that the ob protein exerts adipose-reducing effects in excess of those induced by reductions in food intake.
Measurement of muscle protein synthesis by positron emission tomography with L-[methyl-11C]methionine.
Authors: Hsu H , Yu Y M , Babich J W , Burke J F , Livni E , Tompkins R G , Young V R , Alpert N M , Fischman A J
Published: July 2002

Positron emission tomography (PET) with L-[methyl-11C]methionine was explored as an in vivo, noninvasive, quantitative method for measuring the protein synthesis rate (PSR) in paraspinal and hind limb muscles of anesthetized dogs. Approximately 25 mCi (1 Ci = 37 GBq) of L-[methyl-11C]methionine was injected intravenously, and serial images and arterial blood samples were acquired over 90 min. Data analysis was performed by fitting tissue- and metabolite-corrected arterial blood time-activity curves to a three-compartment model and assuming insignificant transamination and transmethylation in this tissue. PSR was calculated from fitted parameter values and plasma methionine concentrations. PSRs measured by PET were compared with arterio-venous (A-V) difference measurements across the hind limb during primed constant infusion (5-6 h) of L-[1-13C, methyl-2H3]methionine. Results of PET measurements demonstrated similar PSRs for paraspinal and hind limb muscles: 0.172 +/- 0.062 vs. 0.208 +/- 0.048 nmol-1.min-1.(g of muscle)-1 (P = not significant). PSR determined by the stable isotope technique was 0.27 +/- 0.050 nmol-1.min-1.(g of leg tissue)-1 (P < 0.07 from PET) and indicated that the contribution of transmethylation to total hind limb methionine utilization was approximately 10%. High levels of L-[methyl-11C]methionine utilization by bone marrow were observed. We conclude that muscle PSR can be measured in vivo by PET and that this approach offers promise for application in human metabolic studies.
Accelerated evolution and Muller's rachet in endosymbiotic bacteria.
Authors: Moran N A
Published: July 2002

Many bacteria live only within animal cells and infect hosts through cytoplasmic inheritance. These endosymbiotic lineages show distinctive population structure, with small population size and effectively no recombination. As a result, endosymbionts are expected to accumulate mildly deleterious mutations. If these constitute a substantial proportion of new mutations, endosymbionts will show (i) faster sequence evolution and (ii) a possible shift in base composition reflecting mutational bias. Analyses of 16S rDNA of five independently derived endosymbiont clades show, in every case, faster evolution in endosymbionts than in free-living relatives. For aphid endosymbionts (genus Buchnera), coding genes exhibit accelerated evolution and unusually low ratios of synonymous to nonsynonymous substitutions compared to ratios for the same genes for enterics. This concentration of the rate increase in nonsynonymous substitutions is expected under the hypothesis of increased fixation of deleterious mutations. Polypeptides for all Buchnera genes analyzed have accumulated amino acids with codon families rich in A+T, supporting the hypothesis that substitutions are deleterious in terms of polypeptide function. These observations are best explained as the result of Muller's ratchet within small asexual populations, combined with mutational bias. In light of this explanation, two observations reported earlier for Buchnera, the apparent loss of a repair gene and the overproduction of a chaperonin, may reflect compensatory evolution. An alternative hypothesis, involving selection on genomic base composition, is contradicted by the observation that the speedup is concentrated at nonsynonymous sites.
Developmental abnormalities in cultured mouse embryos deprived of retinoic by inhibition of yolk-sac retinol binding protein synthesis.
Authors: Båvik C , Ward S J , Chambon P
Published: July 2002

Presomitic and 3- to 12-somite pair cultured mouse embryos were deprived of retinoic acid (RA) by yolk-sac injections of antisense oligodeoxynucleotides for retinol binding protein (RBP). Inhibition of yolk-sac RBP synthesis was verified by immunohistochemistry, and the loss of activity of a lacZ-coupled RA-sensitive promoter demonstrated that embryos rapidly became RA-deficient. This deficiency resulted in malformations of the vitelline vessels, cranial neural tube, and eye, depending upon the stage of embryonic development at the time of antisense injection. Addition of RA to the culture medium at the time of antisense injection restored normal development implicating the role of RBP in embryonic RA synthesis. Furthermore, the induced RA deficiency resulted in early down-regulation of developmentally important genes including TGF-beta1 and Shh.
Identification of the promoter of the mouse obese gene.
Authors: de la Brousse F C , Shan B , Chen J L
Published: July 2002

Primer extension and RACE (rapid amplification of cDNA ends) assays were used to identify and sequence the 5' terminus of mouse ob mRNA. This sequence was used to obtain a recombinant bacteriophage containing the first exon of the encoding gene. DNA sequence analysis of the region immediately upstream of the first exon of the mouse ob gene revealed DNA sequences corresponding to presumptive cis-regulatory elements. A canonical TATA box was observed 30-34 base pairs upstream from the start site of transcription and a putative binding site for members of the C/EBP family of transcription factors was identified immediately upstream from the TATA box. Nuclear extracts prepared from primary adipocytes contained a DNA binding activity capable of avid and specific interaction with the putative C/EBP response element; antibodies to C/EBP alpha neutralized the DNA binding activity present in adipocyte nuclear extracts. When linked to a firefly luciferase reporter and transfected into primary adipocytes, the presumptive promoter of the mouse ob gene facilitated luciferase expression. When transfected into HepG2 cells, which lack C/EBP alpha, the mouse ob promoter was only weakly active. Supplementation of C/EBP alpha by cotransfection with a C/EBP alpha expression vector markedly stimulated luciferase expression. Finally, an ob promoter variant mutated at the C/EBP response element was inactive in both primary adipocytes and HepG2 cells. These observations provide evidence for identification of a functional promoter capable of directing expression of the mouse ob gene.
Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase
Authors: Ryazanov Alexey G. , Ward Michael D. , Mendola Charmaine E. , Pavur Karen S. , Dorovkov Maxim V. , Wiedmann Martin , Erdjument-Bromage Hediye , Tempst Paul , Parmer Toni Gestone , Prostko C. Robert , Germino F. Joseph , Hait William N.
Published: July 2002

The several hundred members of the eukaryotic protein kinase superfamily characterized to date share a similar catalytic domain structure, consisting of 12 conserved subdomains. Here we report the existence and wide occurrence in eukaryotes of a protein kinase with a completely different structure. We cloned and sequenced the human, mouse, rat, and
Caenorhabditis elegans
eukaryotic elongation factor-2 kinase (eEF-2 kinase) and found that with the exception of the ATP-binding site, they do not contain any sequence motifs characteristic of the eukaryotic protein kinase superfamily. Comparison of different eEF-2 kinase sequences reveals a highly conserved region of ≈200 amino acids which was found to be homologous to the catalytic domain of the recently described myosin heavy chain kinase A (MHCK A) from
Dictyostelium.
This suggests that eEF-2 kinase and MHCK A are members of a new class of protein kinases with a novel catalytic domain structure.
The neuron-restrictive silencer element: A dual enhancer/silencer crucial for patterned expression of a nicotinic receptor gene in the brain
Authors: Bessis Alain , Champtiaux Nicolas , Chatelin Laurent , Changeux Jean-Pierre
Published: July 2002

The neuron-restrictive silencer element (NRSE) has been identified in several neuronal genes and confers neuron specificity by silencing transcription in nonneuronal cells. NRSE is present in the promoter of the neuronal nicotinic acetylcholine receptor β2-subunit gene that determines its neuron-specific expression in the nervous system. Using transgenic mice, we show that NRSE may either silence or enhance transcription depending on the cellular context within the nervous system.In vitroin neuronal cells, NRSE activates transcription of synthetic promoters when located downstream in the 5′ untranslated region, or at less than 50 bp upstream from the TATA box, but switches to a silencer when located further upstream. In contrast, in nonneuronal cells NRSE always functions as a silencer. Antisense RNA inhibition shows that the NRSE-binding protein REST contributes to the activation of transcription in neuronal cells.
Requirement of STAT5b for sexual dimorphism of body growth rates and liver gene expression
Authors: Udy Garry B. , Towers Raewyn P. , Snell Russell G. , Wilkins Richard J. , Park Soo-Hee , Ram Prabha A. , Waxman David J. , Davey Helen W.
Published: July 2002

The signal transducer and activator of transcription, STAT5b, has been implicated in signal transduction pathways for a number of cytokines and growth factors, including growth hormone (GH). Pulsatile but not continuous GH exposure activates liver STAT5b by tyrosine phosphorylation, leading to dimerization, nuclear translocation, and transcriptional activation of the STAT, which is proposed to play a key role in regulating the sexual dimorphism of liver gene expression induced by pulsatile plasma GH. We have evaluated the importance of STAT5b for the physiological effects of GH pulses using a mouse gene knockout model.
STAT5b
gene disruption led to a major loss of multiple, sexually differentiated responses associated with the sexually dimorphic pattern of pituitary GH secretion. Male-characteristic body growth rates and male-specific liver gene expression were decreased to wild-type female levels in
STAT5b
−/−
males, while female-predominant liver gene products were increased to a level intermediate between wild-type male and female levels. Although these responses are similar to those observed in GH-deficient
Little
mice,
STAT5b
−/−
mice are not GH-deficient, suggesting that they may be GH pulse-resistant. Indeed, the dwarfism, elevated plasma GH, low plasma insulin-like growth factor I, and development of obesity seen in
STAT5b
−/−
mice are all characteristics of Laron-type dwarfism, a human GH-resistance disease generally associated with a defective GH receptor. The requirement of STAT5b to maintain sexual dimorphism of body growth rates and liver gene expression suggests that STAT5b may be the major, if not the sole, STAT protein that mediates the sexually dimorphic effects of GH pulses in liver and perhaps other target tissues. STAT5b thus has unique physiological functions for which, surprisingly, the highly homologous STAT5a is unable to substitute.
Evolution of the Friedreich’s ataxia trinucleotide repeat expansion: Founder effect and premutations
Authors: Cossée Mireille , Schmitt Michèle , Campuzano Victoria , Reutenauer Laurence , Moutou Céline , Mandel Jean-Louis , Koenig Michel
Published: July 2002

Friedreich’s ataxia, the most frequent inherited ataxia, is caused, in the vast majority of cases, by large GAA repeat expansions in the first intron of the frataxin gene. The normal sequence corresponds to a moderately polymorphic trinucleotide repeat with bimodal size distribution. Small normal alleles have approximately eight to nine repeats whereas a more heterogeneous mode of large normal alleles ranges from 16 to 34 GAA. The latter class accounts for ≈17% of normal alleles. To identify the origin of the expansion mutation, we analyzed linkage disequilibrium between expansion mutations or normal alleles and a haplotype of five polymorphic markers within or close to the frataxin gene; 51% of the expansions were associated with a single haplotype, and the other expansions were associated with haplotypes that could be related to the major one by mutation at a polymorphic marker or by ancient recombination. Of interest, the major haplotype associated with expansion is also the major haplotype associated with the larger alleles in the normal size range and was almost never found associated with the smaller normal alleles. The results indicate that most if not all large normal alleles derive from a single founder chromosome and that they represent a reservoir for larger expansion events, possibly through “premutation” intermediates. Indeed, we found two such alleles (42 and 60 GAA) that underwent cataclysmic expansion to pathological range in a single generation. This stepwise evolution to large trinucleotide expansions already was suggested for myotonic dystrophy and fragile X syndrome and may relate to a common mutational mechanism, despite sequence motif differences.