bluuebunny/crossref_metadata_2025 huggingface dataset:
Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype
Authors: Devling Tim W. P. , Lindsay Christopher D. , McLellan Lesley I. , McMahon Michael , Hayes John D.
Published: May 2005

A duplex 21 nucleotide small interfering RNA (siRNA) against human Keap1 is described that represents a unique class of cancer chemopreventive agent. This siRNA can knockdown Keap1 mRNA and thereby relieve negative regulation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2)-mediated gene expression. The siRNA lowered endogenous Keap1 mRNA to <30% of control levels between 24 and 72 h after transfection in human HaCaT keratinocyte cells and was capable of blocking ectopic expression of FLAG-tagged human Keap1 protein but not that of ectopic V5-tagged mouse Keap1 protein. Transfection of human HaCaT cells with Keap1 siRNA markedly enhanced endogenous levels of nuclear factor erythroid 2 p45-related factor 2 (Nrf2) protein and increased transcription of an antioxidant response element-driven reporter gene by 2.3-fold. Furthermore, 48 h after transfection of these cells with Keap1 siRNA, expression of aldo-keto reductase 1C1/2 and the glutamate cysteine ligase catalytic and modifier subunits was elevated between 5- and 14-fold. A modest increase of 3-fold in NAD(P)H:quinone oxidoreductase 1 was also observed. The Keap1 siRNA produced a 1.75-fold increase in intracellular glutathione 48 h after transfection. Thus, antagonism of Keap1 by siRNA can be used to preadapt human cells to oxidative stress without the need to expose them to redox stressors.
Clathrin heavy and light chain isoforms originated by independent mechanisms of gene duplication during chordate evolution
Authors: Wakeham Diane E. , Abi-Rached Laurent , Towler Mhairi C. , Wilbur Jeremy D. , Parham Peter , Brodsky Frances M.
Published: May 2005

In humans, there are two isoforms each of clathrin heavy chain (CHC17 and CHC22) and light chain (LCa and LCb) subunits, all encoded by separate genes. CHC17 forms the ubiquitous clathrin-coated vesicles that mediate membrane traffic. CHC22 is implicated in specialized membrane organization in skeletal muscle. CHC17 is bound and regulated by LCa and LCb, whereas CHC22 does not functionally interact with either light chain. The imbalanced interactions between clathrin subunit isoforms suggest a distinct evolutionary history for each isoform pair. Phylogenetic and sequence analysis placed both heavy and light chain gene duplications during chordate evolution, 510-600 million years ago. Genes encoding CHC22 orthologues were found in several vertebrate species, with only a pseudogene present in mice. Multiple paralogons surrounding the CHC genes (
CLTC
and
CLTD
) were identified, evidence that genomic or large-scale gene duplication produced the two CHC isoforms. In contrast, clathrin light chain genes (
CLTA
and
CLTB
) apparently arose by localized duplication, within 1-11 million years of CHC gene duplication. Analysis of sequence divergence patterns suggested that structural features of the CHCs were maintained after gene duplication, but new interactions with regulatory proteins evolved for the CHC22 isoform. Thus, independent mechanisms of gene duplication expanded clathrin functions, concomitant with development of neuromuscular sophistication in chordates.
Genomic and proteomic comparisons between bacterial and archaeal genomes and related comparisons with the yeast and fly genomes
Authors: Karlin Samuel , Brocchieri Luciano , Campbell Allan , Cyert Martha , Mrázek Jan
Published: May 2005

Bacterial, archaeal, yeast, and fly genomes are compared with respect to predicted highly expressed (PHX) genes and several genomic properties. There is a striking difference in the status of PHX ribosomal protein (RP) genes where the archaeal genome generally encodes more RP genes and fewer PHX RPs compared with bacterial genomes. The increase in RPs in archaea and eukaryotes compared with that in bacteria may reflect a more complex set of interactions in archaea and eukaryotes in regulating translation, e.g., differences in structure requiring scaffolding of longer rRNA molecules, expanded interactions with the chaperone machinery, and, in eukaryotic interactions with endoplasmic reticulum components. The yeast genome is similar to fast-growing bacteria in PHX genes but also features several cytoskeletal genes, including actin and tropomyosin, and several signal transduction regulatory proteins from the 14.3.3 family. The most PHX genes of
Drosophila
encode cytoskeletal and exoskeletal proteins. We found that the preference of a microorganism for an anaerobic metabolism correlates with the number of PHX enzymes of the glycolysis pathway that well exceeds the number of PHX enzymes acting in the tricarboxylic acid cycle. Conversely, if the number of PHX enzymes of the tricarboxylic acid cycle well exceeds the PHX enzymes of glycolysis, an aerobic metabolism is preferred. Where the numbers are approximately commensurate, a facultative growth behavior prevails.
Laser-capture microdissection of plasma cells from subacute sclerosing panencephalitis brain reveals intrathecal disease-relevant antibodies
Authors: Burgoon Mark P. , Keays Kathryne M. , Owens Gregory P. , Ritchie Alanna M. , Rai Pradeep R. , Cool Carlyne D. , Gilden Donald H.
Published: May 2005

Increased IgG and oligoclonal bands are found in cerebrospinal fluid of humans with chronic infectious CNS disease. Studies have shown that these oligoclonal bands are antibodies directed against the agent that causes disease. Laser-capture microdissection was used to isolate individual CD38+ plasma cells from the brain of a patient with subacute sclerosing panencephalitis, and single-cell RT-PCR was used to analyze individual IgG heavy and light chains expressed by each cell. Based on overrepresented IgG sequences, we constructed functional recombinant antibodies (recombinant IgGs) and determined their specificities. Five of eight recombinant IgGs recognized measles virus, the cause of subacute sclerosing panencephalitis. These results demonstrate that overrepresented IgG sequences in postmortem brains can be used to produce functional recombinant antibodies that recognize their target antigens. This strategy can be used to identify disease-relevant antigens in CNS inflammatory diseases of unknown etiology.
Investigating local conformations of double-stranded DNA by low-energy circular dichroism of pyrrolo-cytosine
Authors: Johnson Neil P. , Baase Walter A. , von Hippel Peter H.
Published: May 2005

Local base stacking and conformational mobility play a major role in the structure and function of nucleic acids. We have recently shown that the low-energy CD spectrum of 2-aminopurine (2-AP), i.e., the CD spectral region above 300 nm, can be used to monitor conformational changes in polynucleotides at or near mono- and dinucleotide 2-AP residues that replace adenine residues in DNA and RNA. Here, we extend this technique to pyrrolo-cytosine (PC), a fluorescent analogue of cytosine. The low-energy CD spectrum of a PC dinucleotide in dsDNA exhibits an exciton couplet with two bands of opposite sign centered at 350 nm. This signal is characteristic of base stacking between adjacent PC residues in a right helical conformation. Isolated PC nucleotide residues inserted into polynucleotide chains also display chirality that reflects the asymmetric environment of their sequence context. Thus, we show that the low-energy CD spectra of C(PC)A and A(PC)C sequences in dsDNA have opposite signs. It appears that the measurement of the low-energy CD spectra of PC residues will usefully complement 2-AP measurements by serving to characterize the local conformations and dynamics of nucleic acids near C residues and G-C base pairs.
Prefrontal cortex and flexible cognitive control: Rules without symbols
Authors: Rougier Nicolas P. , Noelle David C. , Braver Todd S. , Cohen Jonathan D. , O'Reilly Randall C.
Published: May 2005

Human cognitive control is uniquely flexible and has been shown to depend on prefrontal cortex (PFC). But exactly how the biological mechanisms of the PFC support flexible cognitive control remains a profound mystery. Existing theoretical models have posited powerful task-specific PFC representations, but not how these develop. We show how this can occur when a set of PFC-specific neural mechanisms interact with breadth of experience to self organize abstract rule-like PFC representations that support flexible generalization in novel tasks. The same model is shown to apply to benchmark PFC tasks (Stroop and Wisconsin card sorting), accurately simulating the behavior of neurologically intact and frontally damaged people.
The neurotrophin receptor p75
NTR
modulates long-term depression and regulates the expression of AMPA receptor subunits in the hippocampus
Authors: Rösch Harald , Schweigreiter Rüdiger , Bonhoeffer Tobias , Barde Yves-Alain , Korte Martin
Published: May 2005

Neurotrophins are involved in the modulation of synaptic transmission, including the induction of long-term potentiation (LTP) through the receptor TrkB. Because previous studies have revealed a bidirectional mode of neurotrophin action by virtue of signaling through either the neurotrophin receptor p75
NTR
or the Trk receptors, we tested the hypothesis that p75
NTR
is important for longterm depression (LTD) to occur. Although LTP was found to be unaffected in hippocampal slices of two different strains of mice carrying mutations of the p75
NTR
gene, hippocampal LTD was impaired in both p75
NTR
-deficient mouse strains. Furthermore, the expression levels of two (
RS
)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits, GluR2 and GluR3, but not GluR1 or GluR4, were found to be significantly altered in the hippocampus of p75
NTR
-deficient mice. These results implicate p75
NTR
in activity-dependent synaptic plasticity and extend the concept of functional antagonism of the neurotrophin signaling system.
Late-emigrating neural crest cells in the roof plate are restricted to a sensory fate by GDF7
Authors: Lo Liching , Dormand Emma L. , Anderson David J.
Published: May 2005

Lineage-tracing experiments have shown that some premigratory neural crest cells generate both sensory (S) and autonomic (A) derivatives, whereas others generate only S derivatives. Whether this lineage heterogeneity reflects random variation in a homogeneous population or an early sensory specification of some premigratory crest cells has not been clear. Using Cre recombinase-based fate mapping, we show that GDF7, which is exclusively expressed in the roof plate, marks neural crest cells with a 10-fold higher bias to the sensory lineage than those marked (at the same stage of development) by an inducible Wnt1-Cre, which is expressed more broadly in the dorsal neural tube.
In vitro
, GDF7 has potent sensory neuron-inducing activity. These data suggest that some premigratory crest cells are deterministically restricted to the S lineage and implicate GDF7 itself in this restriction process.
Perinatal ω-3 polyunsaturated fatty acid supply modifies brain zinc homeostasis during adulthood
Authors: Jayasooriya Anura P. , Ackland M. Leigh , Mathai Michael L. , Sinclair Andrew J. , Weisinger Harrison S. , Weisinger Richard S. , Halver John E. , Kitajka Klára , Puskás László G.
Published: May 2005

Dietary ω-3 polyunsaturated fatty acid (PUFA) influences the expression of a number of genes in the brain. Zinc transporter (ZnT) 3 has been identified as a putative transporter of zinc into synaptic vesicles of neurons and is found in brain areas such as hippocampus and cortex. Neuronal zinc is involved in the formation of amyloid plaques, a major characteristic of Alzheimer's disease. The present study evaluated the influence of dietary ω-3 PUFA on the expression of the
ZnT3
gene in the brains of adult male Sprague-Dawley rats. The rats were raised and/or maintained on a control (CON) diet that contained ω-3 PUFA or a diet deficient (DEF) in ω-3 PUFA.
ZnT3
gene expression was analyzed by using real-time PCR, free zinc in brain tissue was determined by zinquin staining, and total zinc concentrations in plasma and cerebrospinal fluid were determined by atomic absorption spectrophotometry. Compared with CON-raised animals, DEF-raised animals had increased expression of
ZnT3
in the brain that was associated with an increased level of free zinc in the hippocampus. In addition, compared with CON-raised animals, DEF-raised animals had decreased plasma zinc level. No difference in cerebrospinal fluid zinc level was observed. The results suggest that overexpression of
ZnT3
due to a perinatal ω-3 PUFA deficiency caused abnormal zinc metabolism in the brain. Conceivably, the influence of dietary ω-3 PUFA on brain zinc metabolism could explain the observation made in population studies that the consumption of fish is associated with a reduced risk of dementia and Alzheimer's disease.
Yeast genome-wide screen reveals dissimilar sets of host genes affecting replication of RNA viruses
Authors: Panavas Tadas , Serviene Elena , Brasher Jeremy , Nagy Peter D.
Published: May 2005

Viruses are devastating pathogens of humans, animals, and plants. To further our understanding of how viruses use the resources of infected cells, we systematically tested the yeast single-gene-knockout library for the effect of each host gene on the replication of tomato bushy stunt virus (TBSV), a positive-strand RNA virus of plants. The genome-wide screen identified 96 host genes whose absence either reduced or increased the accumulation of the TBSV replicon. The identified genes are involved in the metabolism of nucleic acids, lipids, proteins, and other compounds and in protein targeting/transport. Comparison with published genome-wide screens reveals that the replication of TBSV and brome mosaic virus (BMV), which belongs to a different supergroup among plus-strand RNA viruses, is affected by vastly different yeast genes. Moreover, a set of yeast genes involved in vacuolar targeting of proteins and vesicle-mediated transport both affected replication of the TBSV replicon and enhanced the cytotoxicity of the Parkinson's disease-related α-synuclein when this protein was expressed in yeast. In addition, a set of host genes involved in ubiquitin-dependent protein catabolism affected both TBSV replication and the cytotoxicity of a mutant huntingtin protein, a candidate agent in Huntington's disease. This finding suggests that virus infection and disease-causing proteins might use or alter similar host pathways and may suggest connections between chronic diseases and prior virus infection.
Chromosomal clustering of genes controlled by the aire transcription factor
Authors: Johnnidis Jonathan B. , Venanzi Emily S. , Taxman Debra J. , Ting Jenny P.-Y. , Benoist Christophe O. , Mathis Diane J.
Published: May 2005

Autoimmune regulator (aire) is a transcription factor that controls the self-reactivity of the T cell repertoire. Although previous results indicate that it exerts this function in part by promoting ectopic expression of a battery of peripheral-tissue antigens in epithelial cells of the thymic medulla, recent data argue for additional roles in negative selection of thymocytes by medullary cells. As one approach to exploring such roles, we performed computational analyses of microarray data on medullary RNA transcripts from aire-deficient versus wild-type mice, focusing on the genomic localization of aire-controlled genes. Our results highlight this molecule's transcriptional activating and silencing roles and reveal a significant degree of clustering of its target genes. On a local scale, aire-regulated clusters appeared punctate, with aire-controlled and aire-independent genes often being interspersed. This pattern suggests that aire's action may not be a simple reflection of the wide action of a chromatin remodeling enzyme. Analysis of the identity of certain of the clustered genes was evocative of aire's potential roles in antigen presentation and the coordination of intrathymic cell migration: for example, major histocompatibility complex class I and class II gene products and certain chemokine genes are targets of aire-regulated transcription.
Noncholinergic excitatory actions of motoneurons in the neonatal mammalian spinal cord
Authors: Mentis George Z. , Alvarez Francisco J. , Bonnot Agnes , Richards Dannette S. , Gonzalez-Forero David , Zerda Ricardo , O'Donovan Michael J.
Published: May 2005

Mammalian spinal motoneurons are considered to be output elements of the spinal cord that generate exclusively cholinergic actions on Renshaw cells, their intraspinal synaptic targets. Here, we show that antidromic stimulation of motor axons evokes depolarizing monosynaptic potentials in Renshaw cells that are depressed, but not abolished, by cholinergic antagonists. This residual potential was abolished by 2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione. In the presence of cholinergic antagonists, motor axon stimulation triggered locomotor-like activity that was blocked by 2-amino-5-phosphonovaleric acid. Some cholinergic motoneuronal terminals on both Renshaw cells and motoneurons were enriched in glutamate, but none expressed vesicular glutamate transporters. Our results raise the possibility that motoneurons release an excitatory amino acid in addition to acetylcholine and that they may be more directly involved in the genesis of mammalian locomotion than previously believed.
Glucose and urea kinetics in cows in early lactation
Authors: Bruckental I. , Oldham J. D. , Sutton J. D.
Published: May 2005

1. Four mature Friesian cows were used. For 2 weeks after calving they were fed 3 kg hay (147 g crude protein (nitrogen × 6·25; CP)/kg dry matter (DM)) plus 9 kg concentrates (177 g CP/kg DM) per d and thereafter 4 kg hay plus 12 kg concentrates/d.

2. At approximately the 2nd, 4th and 9th weeks post-partum each cow was given a single intravenous dose of [14C]urea (1 mCi) and [6-3H]glucose (1·5 mCi). Following this, jugular venous blood samples were withdrawn up to 26 h post-injection.

3. The log (specific activity)vtime curves were not linear for either metabolite. Glucose irreversible loss rates (IRL) were calculated by a stochastic procedure. The linear part of the urea log (specific activity)v.time curve gave the same estimate for urea IRL rate as a stochastic method of calculation, but urea pool sizes were overestimated so that stochastic analysis of results was preferred.

4. Mean milk yields at the time of the three measurements were 27·1, 30·8 and 27·9 kg milk/d.

5. Urea IRL was significantly lower (P< 0·05) in the first 4 weeks of lactation than in the 9th, and blood urea concentration was lower (P< 0·05) in the 4th than in the 2nd and 9th week, but there was no close correlation between urea IRL and concentration.

6. Glucose IRL rose (P< 0·05) between the 2nd and 9th weeks post-partum, perhaps due to the increase in feed intake. The results were generally consistent with the relationship between milk yield and glucose IRL established by Paterson & Linzell (1974).

7. Urea IRL (y) and milk protein-N (x) production values were used to calculate the efficiency of protein utilization (EPU) asx÷ (x+ 0·35y). This assumes that 0·35 of urea IRL was due to protein catabolism. EPU was found to be 0·59–0·80 (mean 0·69).

8. The possible contribution of catabolized amino acids to glucose IRL was calculated by assuming that 0·35 of urea IRL was due to protein catabolism and 0·2 of catabolized protein-C passed through the glucose pool. On this basis the contribution of protein-C to glucose-C was < 25 g/kg glucose IRL.
Growth and the development of dietary obesity in adulthood of rats which have been undernourished during development
Authors: Stephens D. N.
Published: May 2005

1. The effect of feeding a highly-palatable and varied diet on growth and body composition was assessed in male rats, some of which had undergone a period of undernutrition early in their development.

2. Undernutrition during gestation had no effects on weight, length or fat content of offspring in adulthood. Rats underfed during the first 3 weeks of postnatal life were shorter, lighter and more lean as adults than controls which had been well nourished through life.

3. Feeding the palatable diet from weaning led to increases in length and fat-free mass, and to comparable extents of obesity in all groups, irrespective of whether they had suffered either period of undernutrition. Access to palatable food for 30 d in adulthood also led to obesity, but to increases in length and fat-free mass in only the groups undernourished during suckling.

4. Withdrawal of the palatable diet led to some initial weight loss in all groups, irrespective of whether they had been undernourished during development; and 100 d following the replacement of the palatable with stock diet, there were no longer differences in weight between groups which had received palatable food, and those given stock diet throughout.

5. Nevertheless, those rats which had been undernourished during the suckling period and subsequently fed on the palatable diet still showed increased length and fat-free mass relative to their controls fed on the stock diet throughout.

6. All groups which had received the palatable diet, whether from weaning or as adults, and irrespective of early nutrition, were significantly less fat 100 d after its withdrawal than were those rats fed on the stock diet throughout life.
A cytomegaloviral protein reveals a dual role for STAT2 in IFN-γ signaling and antiviral responses
Authors: Zimmermann Albert , Trilling Mirko , Wagner Markus , Wilborn Manuel , Bubic Ivan , Jonjic Stipan , Koszinowski Ulrich , Hengel Hartmut
Published: May 2005

A mouse cytomegalovirus (MCMV) gene conferring interferon (IFN) resistance was identified. This gene, M27, encodes a 79-kD protein that selectively binds and down-regulates for signal transducer and activator of transcription (STAT)-2, but it has no effect on STAT1 activation and signaling. The absence of pM27 conferred MCMV susceptibility to type I IFNs (α/β), but it had a much more dramatic effect on type II IFNs (γ) in vitro and in vivo. A comparative analysis of M27+ and M27− MCMV revealed that the antiviral efficiency of IFN-γ was partially dependent on the synergistic action of type I IFNs that required STAT2. Moreover, STAT2 was directly activated by IFN-γ. This effect required IFN receptor expression and was independent of type I IFNs. IFN-γ induced increasing levels of tyrosine-phosphorylated STAT2 in M27− MCMV-infected cells that were essential for the antiviral potency of IFN-γ. pM27 represents a new strategy for simultaneous evasions from types I and II IFNs, and it documents an unknown biological significance for STAT2 in antiviral IFN-γ responses.
Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopicHOXB4expression
Authors: Wang Lisheng , Menendez Pablo , Shojaei Farbod , Li Li , Mazurier Frederick , Dick John E. , Cerdan Chantal , Levac Krysta , Bhatia Mickie
Published: May 2005

Despite the need for alternative sources of human hematopoietic stem cells (HSCs), the functional capacity of hematopoietic cells generated from human embryonic stem cells (hESCs) has yet to be evaluated and compared with adult sources. Here, we report that somatic and hESC-derived hematopoietic cells have similar phenotype and in vitro clonogenic progenitor activity. However, in contrast with somatic cells, hESC-derived hematopoietic cells failed to reconstitute intravenously transplanted recipient mice because of cellular aggregation causing fatal emboli formation. Direct femoral injection allowed recipient survival and resulted in multilineage hematopoietic repopulation, providing direct evidence of HSC function. However, hESC-derived HSCs had limited proliferative and migratory capacity compared with somatic HSCs that correlated with a distinct gene expression pattern of hESC-derived hematopoietic cells that included homeobox (HOX) A and B gene clusters. Ectopic expression of HOXB4 had no effect on repopulating capacity of hESC-derived cells. We suggest that limitations in the ability of hESC-derived HSCs to activate a molecular program similar to somatic HSCs may contribute to their atypical in vivo behavior. Our study demonstrates that HSCs can be derived from hESCs and provides an in vivo system and molecular foundation to evaluate strategies for the generation of clinically transplantable HSC from hESC lines.
Decay-accelerating factor modulates induction of T cell immunity
Authors: Heeger Peter S. , Lalli Peter N. , Lin Feng , Valujskikh Anna , Liu Jinbo , Muqim Nasima , Xu Yuanyuan , Medof M. Edward
Published: May 2005

Decay-accelerating factor (Daf) dissociates C3/C5 convertases that assemble on host cells and thereby prevents complement activation on their surfaces. We demonstrate that during primary T cell activation, the absence of Daf on antigen-presenting cells (APCs) and on T cells enhances T cell proliferation and augments the induced frequency of effector cells. The effect is factor D- and, at least in part, C5-dependent, indicating that local alternative pathway activation is essential. We show that cognate T cell–APC interactions are accompanied by rapid production of alternative pathway components and down-regulation of Daf expression. The findings argue that local alternative pathway activation and surface Daf protein function respectively as a costimulator and a negative modulator of T cell immunity and explain previously reported observations linking complement to T cell function. The results could have broad therapeutic implications for disorders in which T cell immunity is important.
The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance
Authors: Lin Hsi-Hsien , Faunce Douglas E. , Stacey Martin , Terajewicz Ania , Nakamura Takahiko , Zhang-Hoover Jie , Kerley Marilyn , Mucenski Michael L. , Gordon Siamon , Stein-Streilein Joan
Published: May 2005

We show that the mouse macrophage-restricted F4/80 protein is not required for the development and distribution of tissue macrophages but is involved in the generation of antigen-specific efferent regulatory T (T reg) cells that suppress antigen-specific immunity. In the in vivo anterior chamber (a.c.)–associated immune deviation (ACAID) model of peripheral tolerance, a.c. inoculation of antigen into F4/80−/− mice was unable to induce efferent T reg cells and suppress delayed-type hypersensitivity (DTH) responses. Moreover, the use of anti-F4/80 mAb and F4/80−/− APCs in an in vitro ACAID model showed that all APC cells in the culture must be able to express F4/80 protein if efferent T reg cells were to be generated. In a low-dose oral tolerance model, WT but not F4/80−/− mice generated an efferent CD8+ T reg cell population that suppressed an antigen-specific DTH response. Peripheral tolerance was restored in F4/80−/− mice by adoptive transfer of F4/80+ APCs in both peripheral tolerance models, indicating a central role for the F4/80 molecule in the generation of efferent CD8+ T reg cells.
Complex group algebras of finite groups: Brauer’s Problem 1
Authors: Moretó Alexander
Published: May 2005

Brauer’s Problem 1 asks the following: what are the possible complex group algebras of finite groups? It seems that with the present knowledge of representation theory it is not possible to settle this question. The goal of this paper is to announce a partial solution to this problem. We conjecture that if the complex group algebra of a finite group does not have more than a fixed number

m
m

of isomorphic summands, then its dimension is bounded in terms of

m
m

. We prove that this is true for every finite group if it is true for the symmetric groups.
The influence of phospholipid membranes on bovine calcitonin secondary structure and amyloid formation
Authors: Wang Steven S.‐S. , Good Theresa A. , Rymer Dawn L.
Published: May 2005

Calcitonin, a peptide hormone associated with medullary carcinoma of the thyroid, has the potential to form amyloid fibrils and may be a valuable model for investigating the role of peptide–membrane interactions in β‐sheet and amyloid formation. Via a new model peptide system, bovine calcitonin, we found that the exposure of peptide to phospholipid membranes altered its structure relative to the structures formed in aqueous solutions. Of particular relevance to the amyloidoses, incubation of calcitonin with cholesterol‐rich and ganglioside‐containing membranes resulted in significant enrichment in the β‐sheet and amyloid content of the peptide. The formation of amyloid was also accelerated in these systems. A correlation between the phospholipid‐induced structural alterations and calcitonin binding affinities to phospholipid membranes was evident. Bovine calcitonin has considerably higher binding affinity for the phospholipid systems that enhanced its β‐sheet and amyloid structure. Electrostatic forces were not the governing forces behind the observed behavior, as supported by the fact that the ionic strength did not affect the peptide structures or binding affinities. A Van't Hoff analysis of the temperature‐dependent peptide binding affinities indicated that binding led to an increase in enthalpy and possibly an increase in entropy of the peptide–membrane systems. Experiments with other amyloid‐forming peptides such as β‐amyloid of Alzheimer's disease have also shown similar results and may indicate the need to manipulate peptide–membrane interactions in order to control amyloid formation and its associated disease.
p25α is flexible but natively folded and binds tubulin with oligomeric stoichiometry
Authors: Otzen Daniel E. , Lundvig Ditte M.S. , Wimmer Reinhard , Nielsen Lotte H. , Pedersen Jakob R. , Jensen Poul H.
Published: May 2005

p25α is a 219‐residue proteinwhich stimulates aberrant tubulin polymerization and is implicated in a variety of other functions. The protein has unusual secondary structure involving significant amounts of random coil, and binding to microtubules is accompanied by a large structural change, suggesting a high degree of plasticity. p25α has been proposed to be natively unfolded, so that folding is coupled to interaction with its physiological partners. Here we show that recombinant human p25α is folded under physiological conditions, since it has a well structured and solvent‐sequestered aromatic environment and considerable chemical shift dispersion of amide and aliphatic protons. With increasing urea concentrations, p25α undergoes clear spectral changes suggesting significant loss of structure. p25α unfolds cooperatively in urea according to a simple two‐state transition with a stability in water of ∼5 kcal/mol. The protein behaves as a monomer and refolds with a transient on‐pathway folding intermediate. However, high sensitivity to proteolytic attack and abnormal gel filtration migration behavior suggests a relatively extended structure, possibly organized in distinct domains. A deletion mutant of p25α lacking residues 3–43 also unfolds cooperatively and with similar stability, suggesting that the N‐terminal region is largely unstructured. Both proteins undergo significant loss of structure when bound to monomeric tubulin. The stoichiometry of binding is estimated to be 3–4 molecules of tubulin per p25α and is not significantly affected by the deletion of residues 3–43. In conclusion, we dismiss the proposal that p25α is natively unfolded, although the protein is relatively flexible. This flexibility may be linked to its tubulin‐binding properties.
Mass spectrometric analysis of the human 40S ribosomal subunit: Native and HCV IRES‐bound complexes
Authors: Yu Yonghao , Ji Hong , Doudna Jennifer A. , Leary Julie A.
Published: May 2005

Hepatitis C virus uses an internal ribosome entry site (IRES) in the viral RNA to directly recruit human 40S ribosome subunits during cap‐independent translation initiation. Although IRES‐mediated translation initiation is not subject to many of the regulatory mechanisms that control cap‐dependent translation initiation, it is unknown whether other noncanonical protein factors are involved in this process. Thus, a global protein composition analysis of native and IRES‐bound 40S ribosomal complexes has been conducted to facilitate an understanding of the IRES ribosome recruitment mechanism. A combined top‐down and bottom‐up mass spectrometry approach was used to identify both the proteins and their posttranslational modifications (PTMs) in the native 40S subunit and the IRES recruited translation initiation complex. Thirty‐one out of a possible 32 ribosomal proteins were identified by combining top‐down and bottom‐up mass spectrometry techniques. Proteins were found to contain PTMs, including loss of methionine, acetylation, methylation, and disulfide bond formation. In addition to the 40S ribosomal proteins, RACK1 was consistently identified in the 40S fraction, indicating that this protein is associated with the 40S subunit. Similar methodology was then applied to the hepatitis C virus IRES‐bound 40S complex. Two 40S ribosomal proteins, RS25 and RS29, were found to contain different PTMs than those in the native 40S subunit. In addition, RACK1, eukaryotic initiation factor 3 proteins and nucleolin were identified in the IRES‐mediated translation initiation complex.
Novel and economical purification of recombinant proteins: Intein‐mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association
Authors: Banki Mahmoud Reza , Gerngross Tillman U. , Wood David W.
Published: May 2005

This work combines two well‐established technologies to generate a breakthrough in protein production and purification. The first is the production of polyhydroxybutyrate (PHB) granules in engineered strains of Escherichia coli. The second is a recently developed group of self‐cleaving affinity tags based on protein splicing elements known as inteins. By combining these technologies with a PHB‐specific binding protein, a self‐contained protein expression and purification system has been developed. In this system, the PHB‐binding protein effectively acts as an affinity tag for desired product proteins. The tagged product proteins are expressed in E. coli strains that also produce intracellular PHB granules, where they bind to the granules via the PHB‐binding tag. The granules and attached proteins can then be easily recovered following cell lysis by simple mechanical means. Once purified, the product protein is self‐cleaved from the granules and released into solution in a substantially purified form. This system has been successfully used at laboratory scale to purify several active test proteins at reasonable yield. By allowing the bacterial cells to effectively produce both the affinity resin and tagged target protein, the cost associated with the purification of recombinant proteins could be greatly reduced. It is expected that this combination of improved economics and simplicity will constitute a significant breakthrough in both large‐scale production of purified proteins and enzymes and high‐throughput proteomics studies of peptide libraries.
Domain swapping in the low‐similarity isomerase/hydratase superfamily: The crystal structure of rat mitochondrial Δ3, Δ2‐enoyl‐CoA isomerase
Authors: Hubbard Paul A. , Yu Wenfeng , Schulz Horst , Kim Jung‐Ja P.
Published: May 2005

Two monofunctional Δ3, Δ2‐enoyl‐CoA isomerases, one in mitochondria (mECI) and the other in both mitochondria and peroxisomes (pECI), belong to the low‐similarity isomerase/hydratase superfamily. Both enzymes catalyze the movement of a double bond from C3 to C2 of an unsaturated acyl‐CoA substrate for re‐entry into the β‐oxidation pathway. Mutagenesis has shown that Glu165 of rat mECI is involved in catalysis; however, the putative catalytic residue in yeast pECI, Glu158, is not conserved in mECI. To elucidate whether Glu165 of mECI is correctly positioned for catalysis, the crystal structure of rat mECI has been solved. Crystal packing suggests the enzyme is trimeric, in contrast to other members of the superfamily, which appear crystallographically to be dimers of trimers. The polypeptide fold of mECI, like pECI, belongs to a subset of this superfamily in which the C‐terminal domain of a given monomer interacts with its own N‐terminal domain. This differs from that of crotonase and 1,4‐dihydroxy‐2‐naphtoyl‐CoA synthase, whose C‐terminal domains are involved in domain swapping with an adjacent monomer. The structure confirms Glu165 as the putative catalytic acid/base, positioned to abstract the pro‐R proton from C2 and reprotonate at C4 of the acyl chain. The large tunnel‐shaped active site cavity observed in the mECI structure explains the relative substrate promiscuity in acyl‐chain length and stereochemistry. Comparison with the crystal structure of pECI suggests the catalytic residues from both enzymes are spatially conserved but not in their primary structures, providing a powerful reminder of how catalytic residues cannot be determined solely by sequence alignments.
The solution structure of the oxidative stress‐related protein YggX from Escherichia coli
Authors: Osborne Michael J. , Siddiqui Nadeem , Landgraf Dirk , Pomposiello Pablo J. , Gehring Kalle
Published: May 2005

YggX is a highly conserved protein found only in eubacteria and is proposed to be involved in the bacterial response to oxidative stress. Here we report the solution structure of YggX from Escherichia coli determined by nuclear magnetic resonance spectroscopy. The structure of YggX displays a fold consisting of two N‐terminal antiparallel β‐sheets and three α‐helices, which shares significant structural similarity to the crystal structure of a hypothetical protein PA5148 from Pseudomonas aeruginosa. Previous studies propose YggX as an iron binding protein that is involved in cellular iron trafficking. Our data indicate that the protein alone does not bind iron in vitro, suggesting other cofactors or different conditions may be necessary for metal binding.
Asymmetric effect of domain interactions on the kinetics of folding in yeast phosphoglycerate kinase
Authors: Osváth Szabolcs , Köhler Gottfried , Závodszky Péter , Fidy Judit
Published: May 2005

The aim of this work is to shed more light on the effect of domain–domain interactions on the kinetics and the pathway of protein folding. A model protein system consisting of several single‐tryptophan variants of the two‐domain yeast phosphoglycerate kinase (PGK) and its individual domains was studied. Refolding was initiated from the guanidine‐unfolded state by stopped‐flow or manual mixing and monitored by tryptophan fluorescence from 1 msec to 1000 sec. Denaturant titrations of both individual domains showed apparent two‐state unfolding transitions. Refolding kinetics of the individual domains from different denaturant concentrations, however, revealed the presence of intermediate structures during titration for both domains. Refolding of the same domains within the complete protein showed that domain–domain interactions direct the folding of both domains, but in an asymmetric way. Folding of the N domain was already altered within 1 msec, while detectable changes in the folding of the C domain occurred only 60–100 msec after initiating refolding. All mutants showed a hyperfluorescent kinetic intermediate. Both the disappearance of this intermediate and the completion of the folding were significantly faster in the individual N domain than in the complete protein. On the contrary, folding of the individual C domain was slower than in the complete protein. The presence of the C domain directs the refolding of the N domain along a completely different pathway than that of the individual N domain, while folding of the individual C domain follows the same path as within the complete protein.
Strain relief at the active site of phosphoserine aminotransferase induced by radiation damage
Authors: Dubnovitsky Anatoly P. , Ravelli Raimond B.G. , Popov Alexander N. , Papageorgiou Anastassios C.
Published: May 2005

The X‐ray susceptibility of the lysine‐pyridoxal‐5′‐phosphate Schiff base in Bacillus alcalophilus phosphoserine aminotransferase has been investigated using crystallographic data collected at 100 K to 1.3 Å resolution, complemented by on‐line spectroscopic studies. X‐rays induce deprotonation of the internal aldimine, changes in the Schiff base conformation, displacement of the cofactor molecule, and disruption of the Schiff base linkage between pyridoxal‐5′‐phosphate and the Lys residue. Analysis of the “undamaged” structure reveals a significant chemical strain on the internal aldimine bond that leads to a pronounced geometrical distortion of the cofactor. However, upon crystal exposure to the X‐rays, the strain and distortion are relaxed and eventually diminished when the total absorbed dose has exceeded 4.7 × 106 Gγ. Our data provide new insights into the enzymatic activation of pyridoxal‐5′‐phosphate and suggest that special care should be taken while using macromolecular crystallography to study details in strained active sites.
T cells in aging mice: genetic, developmental, and biochemical analyses
Authors: Miller Richard A. , Berger Scott B. , Burke David T. , Galecki Andrzej , Garcia Gonzalo G. , Harper James M. , Sadighi Akha Amir A.
Published: May 2005

Summary:  A combination of approaches – gene mapping, biomarker analysis, and studies of signal transduction – has helped to clarify the mechanisms of age‐related change in mouse immune status and the implications of immune aging for late‐life disease. Mapping studies have documented multiple quantitative trait loci (QTL) that influence the levels of age‐sensitive T‐cell subsets. Some of these QTL have effects that are demonstrable in young‐adult mice (8 months of age) and others demonstrable only in middle‐aged mice (18 months). Biomarker studies show that T‐cell subset levels measured at 8 or 18 months are significant predictors of lifespan for mice dying of lymphoma, fibrosarcoma, mammary adenocarcinoma, or all causes combined. Mice whose immune systems resemble that of young animals, i.e. with low levels of CD4+ and CD8+ memory T cells and relatively high levels of CD4+ T cells, tend to outlive their siblings with the opposite subset pattern. Biochemical analyses show that T cells from aged mice show defects in the activation process within a few minutes of encountering a stimulus and that the defects precede the recognition by the T‐cell receptor of agonist peptides on the antigen‐presenting cell. Defective assembly of cytoskeletal fibers and hyperglycosylation of T‐cell surface glycoproteins contribute to the immunodeficiency state, and indeed treatment with a sialylglycoprotein endopeptidase can restore full function to CD4+ T cells from aged donors in vitro.
The effect of age on the cognate function of CD4+ T cells
Authors: Haynes Laura , Eaton Sheri M.
Published: May 2005

Summary:  With increasing age, the ability to produce protective antibodies in response to immunization declines, resulting in reduced efficacy of vaccination. We have examined how reductions in CD4+ T‐cell function contribute to reduced humoral responses, using a model that allows us to compare identical numbers of antigen‐specific naive T cells from young and aged T‐cell receptor transgenic mice. Naive cells from aged mice exhibit reduced responses, both in vitro and in vivo. In vitro, responses of aged T cells can be enhanced by addition of interleukin (IL)‐2. In vivo, using an adoptive transfer model with young hosts, naive cells from aged mice exhibit significant reductions in cognate helper function, leading to reduced B‐cell expansion and differentiation. These age‐related defects could be overcome by prior in vitro T helper 2 effector generation with aged T cells. This improvement in cognate function of the aged effectors may be related to the enhancement of CD154 expression, which occurs on aged T cells in the presence of exogenous IL‐2. We also found no difference in B‐cell expansion and differentiation when young cells were transferred to young or aged hosts. Our results indicate that age‐related reductions in humoral responses are mainly due to defects in the cognate helper function of naive CD4+ T cells from aged individuals.
CD28 extinction in human T cells: altered functions and the program of T‐cell senescence
Authors: Vallejo Abbe N.
Published: May 2005

Summary:  The loss of CD28 expression on T cells is the most consistent biological indicator of aging in the human immune system, and the frequency of CD28null T cells is a key predictor of immune incompetence in the elderly. There is also mounting evidence for the high frequency of these unusual T cells among patients with inflammatory syndromes or with chronic infections disproportionate with their age. In these pathological states, CD28null T cells likely represent prematurely senescent lymphocytes due to persistent immune activation. Unlike the situation in CD28 gene knockout mice that have anergic CD280/0 T cells, human CD28null T cells are functionally active, long‐lived, oligoclonal lymphocytes that lack or have limited proliferative capacity. Results of replicative senescence studies show that CD28null T cells are derived from CD28+ precursors that have undergone repeated stimulation, indicating that CD28 silencing underlies the program of T‐cell aging. Dissection of the machinery regulating CD28 expression is paving the way in elucidating the molecular events leading to immune senescence as well as providing clues into the functional rejuvenation of senescent T cells.
The influence of age on immunity to infection with Mycobacterium tuberculosis
Authors: Vesosky Bridget , Turner Joanne
Published: May 2005

Summary:  Changes within the immune system during aging lead to an elderly population that is both highly susceptible to infectious diseases and unresponsive to typical vaccine protocols. Using the murine model of tuberculosis, we have identified key differences in the generation of T‐helper 1 cell immunity between old and young mice, and this information may be important for the design of new vaccines or post exposure therapies to protect the elderly against infectious diseases. In response to infection with Mycobacterium tuberculosis, it has been shown that the generation of antigen‐specific CD4+ T‐cell immunity is impaired in old mice. In contrast, recent findings document that old mice display a transient enhanced resistance that occurs within the first 3 weeks of infection. Early resistance was associated with the presence of CD8+ T cells and their ability to produce interferon‐γ (IFN‐γ) well before their young counterparts. Further investigation into the mechanism by which CD8+ T cells are induced to secrete IFN‐γin vivo could provide an approach to enhance the effector function of these cells and subsequently protect elderly individuals from respiratory pathogens such as M. tuberculosis.
Molecular mechanisms of apoptosis in the cells of the immune system in human aging
Authors: Gupta Sudhir
Published: May 2005

Summary:  Aging is associated with progressive decline in immune functions and increased frequency of infections, autoimmunity, and cancer. Among immune functions, a decline in T‐cell functions during aging predominates. In this review, I discuss the molecular signaling of three distinct pathways of apoptosis, namely the death receptor pathway, the mitochondrial pathway, and the most recently described endoplasmic reticulum stress pathway, and the relative sensitivity of naïve, central memory, and effector memory CD8+ T‐cell subsets to apoptosis. In addition, I review apoptosis, especially via death receptor pathway, in naïve and various memory subsets of CD4+ and CD8+ T cells (with primary emphasis on CD8+ naïve and memory subsets) in human aging and discuss the role of apoptosis in immune senescence.