bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct chemical evidence for widespread dairying in prehistoric Britain
Authors: Copley M. S. , Berstan R. , Dudd S. N. , Docherty G. , Mukherjee A. J. , Straker V. , Payne S. , Evershed R. P.
Published: February 2003

Domesticated animals formed an important element of farming practices in prehistoric Britain, a fact revealed through the quantity and variety of animal bone typically found at archaeological sites. However, it is not known whether the ruminant animals were raised purely for their tissues (e.g., meat) or alternatively were exploited principally for their milk. Absorbed organic residues from pottery from 14 British prehistoric sites were investigated for evidence of the processing of dairy products. Our ability to detect dairy fats rests on the observation that the δ13C values of the C18:0fatty acids in ruminant dairy fats are ≈2.3‰ lower than in ruminant adipose fats. This difference can be ascribed to (i) the inability of the mammary gland to biosynthesize C18:0; (ii) the biohydrogenation of dietary unsaturated fatty acids in the rumen; and (iii) differences (i.e., 8.1‰) in the δ13C values of the plant dietary fatty acids and carbohydrates. The lipids from a total of 958 archaeological pottery vessels were extracted, and the compound-specific δ13C values of preserved fatty acids (C16:0and C18:0) were determined via gas chromatography-combustion-isotope ratio mass spectrometry. The results provide direct evidence for the exploitation of domesticated ruminant animals for dairy products at all Neolithic, Bronze Age, and Iron Age settlements in Britain. Most significantly, studies of pottery from a range of key early Neolithic sites confirmed that dairying was a widespread activity in this period and therefore probably well developed when farming was introduced into Britain in the fifth millennium B.C.
Reduced glucose tolerance is associated with poor memory performance and hippocampal atrophy among normal elderly
Authors: Convit Antonio , Wolf Oliver T. , Tarshish Chaim , de Leon Mony J.
Published: February 2003

Poor glucose tolerance and memory deficits, short of dementia, often accompanies aging. The purpose of this study was to ascertain whether, among nondiabetic, nondemented middle-aged and elderly individuals, poorer glucose tolerance is associated with reductions in memory performance and smaller hippocampal volumes. We studied 30 subjects who were evaluated consecutively in an outpatient research setting. The composition of the participant group was 57% female and 68.6 ± 7.5 years of age; the participants had an average education of 16.2 ± 2.3 years, a score on the Mini Mental State Examination of 28.6 ± 1.5, a glycosylated hemoglobin (HbA1C) of 5.88 ± 0.74%, and a body mass index of 24.9 ± 4.1 kg/m
2
. Glucose tolerance was measured by an i.v. glucose tolerance test. Memory was tested by using the Wechsler Paragraphs recall tests at the time of administering the i.v. glucose tolerance test. The hippocampus and other brain volumes were measured by using validated methods on standardized MRIs. Decreased peripheral glucose regulation was associated with decreased general cognitive performance, memory impairments, and atrophy of the hippocampus, a brain area that is key for learning and memory. These associations were independent of age and Mini Mental State Examination scores. Therefore, these data suggest that metabolic substrate delivery may influence hippocampal structure and function. This observation may bring to light a mechanism for aging brain injury that may have substantial medical impact, given the large number of elderly individuals with impaired glucose metabolism.
Crystal structure of a photoactive yellow protein from a sensor histidine kinase: Conformational variability and signal transduction
Authors: Rajagopal Sudarshan , Moffat Keith
Published: February 2003

Photoactive yellow protein (E-PYP) is a blue light photoreceptor, implicated in a negative phototactic response in
Ectothiorhodospira halophila
, that also serves as a model for the Per–Arnt–Sim superfamily of signaling molecules. Because no biological signaling partner for E-PYP has been identified, it has not been possible to correlate any of its photocycle intermediates with a relevant signaling state. However, the PYP domain (Ppr-PYP) from the sensor histidine kinase Ppr in
Rhodospirillum centenum
, which regulates the catalytic activity of Ppr by blue light absorption, may allow such issues to be addressed. Here we report the crystal structure of Ppr-PYP at 2 Å resolution. This domain has the same absorption spectrum and similar photocycle kinetics as full length Ppr, but a blue-shifted absorbance and considerably slower photocycle than E-PYP. Although the overall fold of Ppr-PYP resembles that of E-PYP, a novel conformation of the β4–β5 loop results in inaccessibility of Met-100, thought to catalyze chromophore reisomerization, to the chromophore. This conformation also exposes a highly conserved molecular surface that could interact with downstream signaling partners. Other structural differences in the α3–α4 and β4–β5 loops are consistent with these regions playing significant roles in the control of photocycle dynamics and, by comparison to other sensory Per–Arnt–Sim domains, in signal transduction. Because of its direct linkage to a measurable biological output, Ppr-PYP serves as an excellent system for understanding how changes in photocycle dynamics affect signaling by PYPs.
Deletion of the p66
Shc
longevity gene reduces systemic and tissue oxidative stress, vascular cell apoptosis, and early atherogenesis in mice fed a high-fat diet
Authors: Napoli Claudio , Martin-Padura Ines , de Nigris Filomena , Giorgio Marco , Mansueto Gelsomina , Somma Pasquale , Condorelli Mario , Sica Giacomo , De Rosa Gaetano , Pelicci PierGiuseppe
Published: February 2003

Several experimental and clinical studies have shown that oxidized low-density lipoprotein and oxidation-sensitive mechanisms are central in the pathogenesis of vascular dysfunction and atherogenesis. Here, we have used p66
Shc−/−
and WT mice to investigate the effects of high-fat diet on both systemic and tissue oxidative stress and the development of early vascular lesions. To date, the p66
Shc−/−
mouse is the unique genetic model of increased resistance to oxidative stress and prolonged life span in mammals. Computer-assisted image analysis revealed that chronic 21% high-fat treatment increased the aortic cumulative early lesion area by ≈21% in WT mice and only by 3% in p66
Shc−/−
mice. Early lesions from p66
Shc−/−
mice had less content of macrophage-derived foam cells and apoptotic vascular cells, in comparison to the WT. Furthermore, in p66
Shc−/−
mice, but not WT mice, we found a significant reduction of systemic and tissue oxidative stress (assessed by isoprostanes, plasma low-density lipoprotein oxidizability, and the formation of arterial oxidation-specific epitopes). These results support the concept that p66
Shc−/−
may play a pivotal role in controlling systemic oxidative stress and vascular diseases. Therefore, p66
Shc
might represent a molecular target for therapies against vascular diseases.
Matchmaking and species marriage: A game-theory model of community assembly
Authors: Uriarte María , Reeve Hudson Kern
Published: February 2003

Theories developed to explain the processes that govern the assembly and composition of natural plant communities can be divided into two broad categories. Niche-based theories propose that coevolutionary changes among species lead to character divergence (displacement), which allows for coexistence by partitioning resources among species. In contrast, ecological-drift theories propose that species diversity results from a balance of migration, speciation, and extinction, with little microevolutionary change. We use a game-theory model to reconcile drift and niche perspectives by developing a theory of species “marriage.” Initially, ecological drift may determine which species encounter each other in a competitive arena. Once species come into contact, historical niche development as a result of prior coevolutionary molding of competitive ability determines which species may coexist. The model shows that only species that display the well-known tradeoff between seed size and competitive ability as a result of past competitive histories comply with the requisite for mutual evolutionary stability. Mutual evolutionary stability of competitive ability tends to make reproductive outputs more similar between species, increasing the chances of ecological equilibrium, i.e., the coexistence of species competing for a single resource. Moreover, mutual evolutionary stability guarantees that such an ecological equilibrium will be stable. The species-marriage model predicts that two or more plant species will coexist indefinitely (i.e., “marry”) when their difference in seed size, their densities, and the resource availability obey a specific quantitative relation. For example, when resource availability is high, married species should be characterized by a greater asymmetry in seed size than when resource availability is low. Thus, in the species-marriage model, competition can shape the detailed properties of communities without violating the postulates of ecological-drift theory.
Genome-wide protective response used by group AStreptococcusto evade destruction by human polymorphonuclear leukocytes
Authors: Voyich Jovanka M. , Sturdevant Daniel E. , Braughton Kevin R. , Kobayashi Scott D. , Lei Benfang , Virtaneva Kimmo , Dorward David W. , Musser James M. , DeLeo Frank R.
Published: February 2003

Group AStreptococcus(GAS) evades polymorphonuclear leukocyte (PMN) phagocytosis and killing to cause human disease, including pharyngitis and necrotizing fasciitis (flesh-eating syndrome). We show that GAS genes differentially regulated during phagocytic interaction with human PMNs comprise a global pathogen-protective response to innate immunity. GAS prophage genes and genes involved in virulence, oxidative stress, cell wall biosynthesis, and gene regulation were up-regulated during PMN phagocytosis. Genes encoding novel secreted proteins were up-regulated, and the proteins were produced during human GAS infections. We discovered an essential role for the Ihk-Irr two-component regulatory system in evading PMN-mediated killing and promoting host–cell lysis, processes that would facilitate GAS pathogenesis. Importantly, theirrgene was highly expressed during human GAS pharyngitis. We conclude that a complex pathogen genetic program circumvents human innate immunity to promote disease. The gene regulatory program revealed by our studies identifies previously undescribed potential vaccine antigens and targets for therapeutic interventions designed to control GAS infections.
Transfer of a passive additive in a turbulent boundary layer at very large Reynolds numbers
Authors: Barenblatt G. I.
Published: February 2003

We formulate the mass transfer problem for a passive additive in a turbulent boundary layer based on the recently proposed model of the turbulent boundary layer at very large Reynolds numbers. The solutions of three basic problems are obtained. These solutions are self-similar asymptotics describing the mass exchange at its initial stages. The solutions obtained can be used for the construction (in particular, the numerical construction) of the solution to the more general problems of passive admixture transfer in the developed turbulent wall-bounded shear flows.
Uptake and antifungal activity of oligonucleotides in
Candida albicans
Authors: Disney Matthew D. , Haidaris Constantine G. , Turner Douglas H.
Published: February 2003

Candida albicans
is a significant cause of disease in immunocompromised humans. Because the number of people infected by fungal pathogens is increasing, strategies are being developed to target RNAs in fungi. This work shows that oligonucleotides can serve as therapeutics against
C. albicans
. In particular, oligonucleotides are taken up from cell culture medium in an energy-dependent process. After uptake, oligonucleotides, including RNA, remain mostly intact after 12 h in culture. For culture conditions designed for mammalian cells, intracellular concentrations of oligonucleotides in
C. albicans
exceed those in COS-7 mammalian cells, suggesting that uptake can provide selective targeting of fungi over human cells. A 19-mer 2′OMe (oligonucleotide with a 2′-
O
-methyl backbone) hairpin is described that inhibits growth of a
C. albicans
strain at pH < 4.0. This pH is easily tolerated in some parts of the body subject to
C. albicans
infections.
In vivo
dimethyl sulfate modification of ribosomal RNA and the decreased rate of protein synthesis suggest that this hairpin's activity may be due to targeting the ribosome in a way that does not depend on base pairing. Addition of anti-
C. albicans
oligonucleotides to COS-7 mammalian cells has no effect on cell growth. Evidently, oligonucleotides can selectively serve as therapeutics toward
C. albicans
and, presumably, other pathogens. Information from genome sequencing and functional genomics studies on
C. albicans
and other pathogens should allow rapid design and testing of other approaches for oligonucleotide therapies.
Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells
Authors: Taraska Justin W. , Perrais David , Ohara-Imaizumi Mica , Nagamatsu Shinya , Almers Wolfhard
Published: February 2003

Classical cell biology teaches that exocytosis causes the membrane of exocytic vesicles to disperse into the cell surface and that a cell must later retrieve by molecular sorting whatever membrane components it wishes to keep inside. We have tested whether this view applies to secretory granules in intact PC-12 cells. Three granule proteins were labeled with fluorescent proteins in different colors, and two-color evanescent-field microscopy was used to view single granules during and after exocytosis. Whereas neuro-peptide Y was lost from granules in seconds, tissue plasminogen activator (tPA) and the membrane protein phogrin remained at the granule site for over 1 min, thus providing markers for postexocytic granules. When tPA was imaged simultaneously with cyan fluorescent protein (CFP) as a cytosolic marker, the volume occupied by the granule appeared as a dark spot where it excluded CFP. The spot remained even after tPA reported exocytosis, indicating that granules failed to flatten into the cell surface. Phogrin was labeled with GFP at its luminal end and used to sense the pH in granules. When exocytosis caused the acidic granule interior to neutralize, GFP–phogrin at first brightened and later dimmed again as the interior separated from the extracellular space and reacidified. Reacidification and dimming could be reversed by application of NH
4
Cl. We conclude that most granules reseal in <10 s after releasing cargo, and that these empty or partially empty granules are recaptured otherwise intact.
Phenotype variation in two-locus mouse models of Hirschsprung disease: Tissue-specific interaction between
Ret
and
Ednrb
Authors: McCallion Andrew S. , Stames Erine , Conlon Ronald A. , Chakravarti Aravinda
Published: February 2003

Clinical expression of Hirschsprung disease (HSCR) requires the interaction of multiple susceptibility genes. Molecular genetic analyses have revealed that interactions between mutations in the genes encoding the RET receptor tyrosine kinase and the endothelin receptor type B (EDNRB) are central to the genesis of HSCR. We have established two locus noncomplementation assays in mice, using allelic series at
Ednrb
in the context of
Ret
kinase-null heterozygotes, to understand the clinical presentation, incomplete penetrance, variation in length of aganglionic segment, and sex bias observed in human HSCR patients. Titration of
Ednrb
in the presence of half the genetic dose of
Ret
determines the presentation of an enteric phenotype in these strains, revealing or abrogating a sex bias in disease expression depending on the genotype at
Ednrb
. RET and EDNRB signaling pathways are also critical for the normal development of other tissues, including the kidneys and neural crest-derived melanocytes. Our data demonstrate that interaction between these genes is restricted to the enteric nervous system and does not affect renal, coat color, and retinal choroid development.
PCR-targeted
Streptomyces
gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin
Authors: Gust Bertolt , Challis Greg L. , Fowler Kay , Kieser Tobias , Chater Keith F.
Published: February 2003

Streptomycetes are high G+C Gram-positive, antibiotic-producing, mycelial soil bacteria. The 8.7-Mb
Streptomyces coelicolor
genome was previously sequenced by using an ordered library of Supercos-1 clones. Here, we describe an efficient procedure for creating precise gene replacements in the cosmid clones by using PCR targeting and λ-Red-mediated recombination. The cloned
Streptomyces
genes are replaced with a cassette containing a selectable antibiotic resistance and
oriT
RK2
for efficient transfer to
Streptomyces
by RP4-mediated intergeneric conjugation. Supercos-1 does not replicate in
Streptomyces
, but the clones readily undergo double-crossover recombination, thus creating gene replacements. The antibiotic resistance cassettes are flanked by yeast FLP recombinase target sequences for removal of the antibiotic resistance and
oriT
RK2
to generate unmarked, nonpolar mutations. The technique has been used successfully by >20 researchers to mutate around 100
Streptomyces
genes. As an example, we describe its application to the discovery of a gene involved in the production of geosmin, the ubiquitous odor of soil. The gene, Sco6073 (
cyc2
), codes for a protein with two sesquiterpene synthase domains, only one of which is required for geosmin biosynthesis, probably via a germacra-1 (10)
E,5E
-dien-11-ol intermediate generated by the sesquiterpene synthase from farnesyl pyrophosphate.
Gain control of firing rate by shunting inhibition: Roles of synaptic noise and dendritic saturation
Authors: Prescott Steven A. , De Koninck Yves
Published: February 2003

Adjusting input–output gain is crucial for information processing by the brain. Gain control of subthreshold depolarization is commonly ascribed to increased membrane conductance caused by shunting inhibition. But contrary to its divisive effect on depolarization, shunting inhibition on its own fails to divisively modulate firing rate, apparently upsetting a critical tenet of neural models that use shunting inhibition to achieve gain control. Using a biophysically realistic neuron model, we show that divisive modulation of firing rate by shunting inhibition requires synaptic noise to smooth the relation between firing rate and somatic depolarization; although necessary, noise alone endows shunting inhibition with only a modest divisive effect on firing rate. In addition to introducing noise, synaptic input is associated with a nonlinear relation between somatic depolarization and excitation because of dendritic saturation; this nonlinearity dramatically enhances divisive modulation of firing rate by shunting inhibition under noisy conditions. Thus, shunting inhibition can act as a mechanism for firing rate gain control, but its modulatory effects (which include both divisive and subtractive components) are fully explained only when both synaptic noise and dendritic saturation are taken into account.
GDE1/MIR16 is a glycerophosphoinositol phosphodiesterase regulated by stimulation of G protein-coupled receptors
Authors: Zheng Bin , Berrie Christopher P. , Corda Daniela , Farquhar Marilyn G.
Published: February 2003

Previously we identified MIR16 (membrane interacting protein of RGS16) as an integral membrane glycoprotein that interacts with regulator of G protein signaling proteins and shares significant sequence homology with bacterial glycerophosphodiester phosphodiesterases (GDEs), suggesting that it is a putative mammalian GDE. Here we show that MIR16 belongs to a large, evolutionarily conserved family of GDEs with a characteristic putative catalytic domain that shares a common motif (amino acids 92–116) with the catalytic domains of mammalian phosphoinositide phospholipases C. Expression of wild-type MIR16 (renamed GDE1), but not two catalytic domain mutants (E97A/D99A and H112A), leads to a dramatic increase in glycerophosphoinositol phosphodiesterase (GPI-PDE) activity in HEK 293T cells. Analysis of substrate specificity shows that GDE1/MIR16 selectively hydrolyzes GPI over glycerophosphocholine. The GPI-PDE activity of GDE1/MIR16 expressed in HEK 293T cells can be regulated by stimulation of G protein-coupled, α/β-adrenergic, and lysophospholipid receptors. Membrane topology studies suggest a model in which the catalytic GDE domain faces the lumen/extracellular space and the C terminus faces the cytoplasm. Our results suggest that by serving as a PDE for GPI with its activity regulated by G protein signaling, GDE1/MIR16 provides a link between phosphoinositide metabolism and G protein signal transduction.
Expression and mechanistic analysis of a germacradienol synthase from
Streptomyces coelicolor
implicated in geosmin biosynthesis
Authors: Cane David E. , Watt Rory M.
Published: February 2003

The PCR has been used to amplify a 2,181-bp ORF from
Streptomyces coelicolor
A3(2), designated SC9B1.20 (= SCO6073), encoding a protein of 726 amino acids and showing significant sequence similarity at the deduced amino acid level in both the N-terminal and C-terminal halves to the known sesquiterpene synthase pentalenene synthase. The full-length recombinant protein was expressed at high levels in
Escherichia coli
and shown to catalyze the Mg
2+
-dependent conversion of farnesyl diphosphate to the sesquiterpene alcohol (4
S
, 7
R
)-germacra-1 (10)
E
, 5
E
-diene-11-ol. The enzymatic cyclization had a
k
cat
of 6.2 ± 0.5 × 10
−3
s
−1
and a
K
m
for farnesyl diphosphate of 62 ± 8 nM. Expression of the N-terminal (366 amino acids) domain of the SC9B1.20 protein also gave a fully functional cyclase which converted farnesyl diphosphate to the identical sesquiterpene alcohol with a slightly lower
k
cat
of 3.2 ± 0.4 × 10
−3
s
−1
and a twofold greater
k
m
of 115 ± 14 nM. By contrast, the expressed C-terminal domain of SC9B1.20 had no farnesyl diphosphate cyclase activity. The formation of the germacradienol seems to be the committed step in the formation of geosmin, the characteristic odoriferous constituent of
Streptomyces
species.
Tracking an embryonic L1 retrotransposition event
Authors: Prak Eline T. Luning , Dodson Allen W. , Farkash Evan A. , Kazazian Haig H.
Published: February 2003

Long interspersed nuclear elements 1 (L1) are active retrotransposons that reside in many species, including humans and rodents. L1 elements produce an RNA intermediate that is reverse transcribed to DNA and inserted in a new genomic location. We have tagged an active human L1 element (L1
RP
) with a gene encoding enhanced GFP (EGFP). Expression of GFP occurs only if L1-EGFP has undergone a cycle of transcription, reverse transcription, and integration into a transcriptionally permissive genomic region. We show here that L1-EGFP can undergo retrotransposition
in vivo
and produce fluorescence in mouse testis. The retrotransposition event characterized here has occurred at a very early stage in the development of an L1-EGFP transgenic founder mouse.
In socially isolated mice, the reversal of brain allopregnanolone down-regulation mediates the anti-aggressive action of fluoxetine
Authors: Pinna Graziano , Dong Erbo , Matsumoto Kinzo , Costa Erminio , Guidotti Alessandro
Published: February 2003

Social isolation (
SI
) of male mice lasting >4 weeks is associated with aggression toward intruders and a down-regulation of brain allopregnanolone (Allo) content.
SI
of female mice fails to down-regulate brain Allo content or to induce aggressiveness. Fluoxetine (Prozac in clinical use) is an S- and R-fluoxetine (FLX) mixture, which in mammals is metabolized into S- and R-norfluoxetine (NFLX). The S isomers of FLX and NFLX are more active than their respective R isomers in normalizing brain Allo down-regulation and in reducing the aggressiveness induced by
SI
. Thus, FLX stereospecifically reduces brain Allo down-regulation and the aggressiveness induced by
SI
, whereas serotonin (5-HT) uptake inhibition lacks stereospecificity. The doses of S-FLX and S-NFLX that reduce aggressiveness and Allo brain content down-regulation induced by
SI
are at least one order of magnitude lower than the doses that block 5-HT reuptake. Doses of imipramine that inhibit 5-HT uptake neither reduce aggressiveness nor normalize brain Allo down-regulation. We conclude that Allo brain content normalization is a better candidate than 5-HT reuptake inhibition to explain the reduction of aggressiveness elicited by S-FLX and S-NFLX.
Contribution of transplanted bone marrow cells to Purkinje neurons in human adult brains
Authors: Weimann James M. , Charlton Carol A. , Brazelton Timothy R. , Hackman Robert C. , Blau Helen M.
Published: February 2003

We show here that cells within human adult bone marrow can contribute to cells in the adult human brain. Cerebellar tissues from female patients with hematologic malignancies, who had received chemotherapy, radiation, and a bone marrow transplant, were analyzed. Brain samples were obtained at autopsy from female patients who received male (sex-mismatched) or female (sex-matched, control) bone marrow transplants. Cerebella were evaluated in 10-μm-thick, formaldehyde-fixed, paraffin-embedded sections that encompassed up to ≈50% of a human Purkinje nucleus. A total of 5,860 Purkinje cells from sex-mismatched females and 3,202 Purkinje cells from sex-matched females were screened for Y chromosomes by epifluorescence. Confocal laser scanning microscopy allowed definitive identification of the sex chromosomes within the morphologically distinct Purkinje cells. In the brains of females who received male bone marrow, four Purkinje neurons were found that contained an X and a Y chromosome and two other Purkinje neurons contained more than a diploid number of sex chromosomes. No Y chromosomes were detected in the brains of sex-matched controls. The total frequency of male bone marrow contribution to female Purkinje cells approximated 0.1%. This study demonstrates that although during human development Purkinje neurons are no longer generated after birth, cells within the bone marrow can contribute to these CNS neurons even in adulthood. The underlying mechanism may be caused either by generation
de novo
of Purkinje neurons from bone marrow-derived cells or by fusion of marrow-derived cells with existing recipient Purkinje neurons.
Histidine-rich amphipathic peptide antibiotics promote efficient delivery of DNA into mammalian cells
Authors: Kichler Antoine , Leborgne Christian , März Josefine , Danos Olivier , Bechinger Burkhard
Published: February 2003

Gene delivery has shown potential in a wide variety of applications, including basic research, therapies for genetic and acquired diseases, and vaccination. Most available nonviral systems have serious drawbacks such as the inability to control and scale the production process in a reproducible manner. Here, we demonstrate a biotechnologically feasible approach for gene delivery, using synthetic cationic amphipathic peptides containing a variable number of histidine residues. Gene transfer to different cell lines
in vitro
was achieved with an efficiency comparable to commercially available reagents. We provide evidence that the transfection efficiency depends on the number and positioning of histidine residues in the peptide as well as on the pH at which the in-plane to transmembrane transition takes place. Endosomal acidification is also required. Interestingly, even when complexed to DNA these peptides maintain a high level of antibacterial activity, opening the possibility of treating the genetic defect and the bacterial infections associated with cystic fibrosis with a single compound. Thus, this family of peptides represents a new class of agents that may have broad utility for gene transfer and gene therapy applications.
Protein kinase A-anchoring (AKAP) domains in brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2)
Authors: Li Hewang , Adamik Ronald , Pacheco-Rodriguez Gustavo , Moss Joel , Vaughan Martha
Published: February 2003

Like other guanine nucleotide-exchange proteins (GEPs) that activate ADP-ribosylation factor (ARF) GTPases, brefeldin A-inhibited GEP2, BIG2, contains an ≈200-aa Sec7 domain that is responsible for this catalytic activity and its inhibition by brefeldin A. The Sec7 domain is located near the center of the molecule and serves to accelerate replacement of GDP bound to ARF with GTP. To explore possible functions of the N-terminal region of BIG2 (1–832), we used three coding-region constructs as bait to screen a human heart cDNA library in a yeast two-hybrid system, retrieving two unique clones that encode a type I protein kinase A (PKA) regulatory subunit, RIα. Coimmunoprecipitation experiments confirmed interaction of
in vitro
translated BIG2 and RIα, as well as of the endogenous proteins in cytosol of cultured HepG2 cells. Using 28 deletion mutants, we found three regions of BIG2 that interacted with R subunits of PKA. Residues 27–48 (domain A) interacted with RIα and RIβ, 284–301 (domain B) interacted with RIIα and RIIβ, and 517–538 (domain C) interacted with RIα, RIIα, and RIIβ. Sequence analysis and helical wheel projection of amino acids in the three domains revealed potential amphipathic wheel structures characteristic for binding of PKA R subunits. Western blot analysis of subcellular fractions demonstrated translocation of BIG2 (and BIG1) from cytosol to the Golgi and other membrane structures after incubation of cells with 8-Br-cAMP or forskolin. All findings are consistent with a role for BIG2 as an A kinase-anchoring protein (or AKAP) that could coordinate cAMP and ARF regulatory pathways.
Designed to be stable: Crystal structure of a consensus ankyrin repeat protein
Authors: Kohl Andreas , Binz H. Kaspar , Forrer Patrik , Stumpp Michael T. , Plückthun Andreas , Grütter Markus G.
Published: February 2003

Ankyrin repeat (AR) proteins mediate innumerable protein–protein interactions in virtually all phyla. This finding suggested the use of AR proteins as designed binding molecules. Based on sequence and structural analyses, we designed a consensus AR with fixed framework and randomized interacting residues. We generated several combinatorial libraries of AR proteins consisting of defined numbers of this repeat. Randomly chosen library members are expressed in soluble form in the cytoplasm ofEscherichia coliconstituting up to 30% of total cellular protein and show high thermodynamic stability. We determined the crystal structure of one of those library members to 2.0-Å resolution, providing insight into the consensus AR fold. Besides the highly complementary hydrophobic repeat–repeat interfaces and the absence of structural irregularities in the consensus AR protein, the regular and extended hydrogen bond networks in the β-turn and loop regions are noteworthy. Furthermore, all residues found in the turn region of the Ramachandran plot are glycines. Many of these features also occur in natural AR proteins, but not in this rigorous and standardized fashion. We conclude that the AR domain fold is an intrinsically very stable and well-expressed scaffold, able to display randomized interacting residues. This scaffold represents an excellent basis for the design of novel binding molecules.
Short-term administration of omega 3 fatty acids from fish oil results in increased transthyretin transcription in old rat hippocampus
Authors: Puskás László G. , Kitajka Klára , Nyakas Csaba , Barcelo-Coblijn Gwendolyn , Farkas Tibor
Published: February 2003

Reduced brain levels of long chain polyunsaturated fatty acids [arachidonic acid and docosahexanoic acid (DHA)] are observed in elderly subjects and patients with Alzheimer's disease. To determine the effects of n-3 fatty acids on aged rat brain, 2-year-old rats were fed fish oil (27% DHA content) for 1 month, and gene expression analysis and fatty acid and molecular species composition of the major phospholipid species were assessed. No significant alteration could be observed in the fatty acid composition of ethanolamine phosphoglycerides and phosphatidylserines with the exception of DHA, which was slightly higher in brains of rats receiving fish oil. However, a drastic reduction in arachidonic acid in phosphatidylinositoles was observed. The expression of 23 genes was altered in response to fish oil feeding in the hippocampus. The transcription of transthyretin (TTR) was induced by 10-fold as evidenced by microarray analysis and confirmed by real-time quantitative RT-PCR. Expression of IL-1 and NO synthase, which has been implicated in the prevention of neurological diseases, was unaltered. TTR is an amyloid β protein scavenger, so an increase in its expression could prevent amyloid aggregate formation. We believe the beneficial effects of fish oil might be common to other agents, i.e., induce TTR expression, like nicotine and
Ginkgo biloba
extract.
Dosage of
Fgf8
determines whether cell survival is positively or negatively regulated in the developing forebrain
Authors: Storm Elaine E. , Rubenstein John L. R. , Martin Gail R.
Published: February 2003

FGF8 is known to be an important regulator of forebrain development. Here, we investigated the effects of varying the level of
Fgf8
expression in the mouse forebrain. We detected two distinct responses, one that was proportionate with
Fgf8
expression and another that was not. The latter response, which led to effects on cell survival, displayed a paradoxical relationship to
Fgf8
dosage. Either eliminating or increasing
Fgf8
expression increased apoptosis, whereas reducing
Fgf8
expression had the opposite effect. To explain these counterintuitive observations, we suggest that an FGF8-dependent cell-survival pathway is negatively regulated by intracellular inhibitors produced in proportion to FGF8 concentration. Our data provide insight into the function of FGF8 in forebrain development and underscore the value of using multiple alleles and different experimental approaches to unravel the complexities of gene function in vertebrate development.
The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: Studies on the mechanisms of the reactions catalyzed by IspG and IspH protein
Authors: Rohdich Felix , Zepeck Ferdinand , Adam Petra , Hecht Stefan , Kaiser Johannes , Laupitz Ralf , Gräwert Tobias , Amslinger Sabine , Eisenreich Wolfgang , Bacher Adelbert , Arigoni Duilio
Published: February 2003

Earlier
in vivo
studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2
C
-methyl-
d
-erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-(
E
)-butenyl 4-diphosphate. A recombinant fusion protein comprising maltose binding protein and IspG protein domains was purified from a recombinant
Escherichia coli
strain. The purified protein failed to transform 2
C
-methyl-
d
-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(
E
)-butenyl 4-diphosphate, but catalytic activity could be restored by the addition of crude cell extract from an
ispG
-deficient
E. coli
mutant. This indicates that auxiliary proteins are required, probably as shuttles for redox equivalents. On activation by photoreduced 10-methyl-5-deaza-isoalloxazine, the recombinant protein catalyzed the formation of 1-hydroxy-2-methyl-2-(
E
)-butenyl 4-diphosphate from 2
C
-methyl-
d
-erythritol 2,4-cyclodiphosphate at a rate of 1 nmol⋅min
−1
⋅mg
−1
. Similarly, activation by photoreduced 10-methyl-5-deaza-isoalloxazine enabled purified IspH protein to catalyze the conversion of 1-hydroxy-2-methyl-2-(
E
)-butenyl 4-diphosphate into a 6:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate at a rate of 0.4 μmol⋅min
−1
⋅mg
−1
. IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol⋅min
−1
⋅mg
−1
. The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions.
Assembly of core helices and rapid tertiary folding of a small bacterial group I ribozyme
Authors: Rangan Prashanth , Masquida Benoît , Westhof Eric , Woodson Sarah A.
Published: February 2003

Compact but non-native intermediates have been implicated in the hierarchical folding of several large RNAs, but there is little information on their structure. In this article, ribonuclease and hydroxyl radical cleavage protection assays showed that base pairing of core helices stabilize a compact state of a small group I ribozyme from
Azoarcus
pre-tRNA
ile
. Base pairing of the ribozyme core requires 10-fold less Mg
2+
than stable tertiary interactions, indicating that assembly of helices in the catalytic core represents a distinct phase that precedes the formation of native tertiary structure. Tertiary folding occurs in <100 ms at 37°C. Such rapid folding is unprecedented among group I ribozymes and illustrates the association between structural complexity and folding time. A 3D model of the
Azoarcus
ribozyme was constructed by identifying homologous sequence motifs in rRNA. The model reveals distinct structural features, such as a large interface between the P4–P6 and P3–P9 domains, that may explain the unusual stability of the
Azoarcus
ribozyme and the cooperativity of folding.
Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes
Authors: Rothwell P. J. , Berger S. , Kensch O. , Felekyan S. , Antonik M. , Wöhrl B. M. , Restle T. , Goody R. S. , Seidel C. A. M.
Published: February 2003

By using single-molecule multiparameter fluorescence detection, fluorescence resonance energy transfer experiments, and newly developed data analysis methods, this study demonstrates directly the existence of three structurally distinct forms of reverse transcriptase (RT):nucleic acid complexes in solution. Single-molecule multiparameter fluorescence detection also provides first information on the structure of a complex not observed by x-ray crystallography. This species did not incorporate nucleotides and is structurally distinct from the other two observed species. We determined that the nucleic acid substrate is bound at a site far removed from the nucleic acid-binding tract observed by crystallography. In contrast, the other two states are identified as being similar to the x-ray crystal structure and represent distinct enzymatically productive stages in DNA polymerization. These species differ by only a 5-Å shift in the position of the nucleic acid. Addition of nucleoside triphosphate or of inorganic pyrophosphate allowed us to assign them as the educt and product state in the polymerization reaction cycle; i.e., the educt state is a complex in which the nucleic acid is positioned to allow nucleotide incorporation. The second RT:nucleic acid complex is the product state, which is formed immediately after nucleotide incorporation, but before RT translates to the next nucleotide.
Histone deacetylation by Sir2 generates a transcriptionally repressed nucleoprotein complex
Authors: Parsons Xuejun Huang , Garcia Sandra N. , Pillus Lorraine , Kadonaga James T.
Published: February 2003

Sir2 is an NAD-dependent histone deacetylase required for transcriptional silencing. To study the mechanism of Sir2 function, we examined the biochemical properties of purified recombinant
Drosophila
Sir2 (dSir2). First, we performed histone deacetylation assays and found that dSir2 deacetylates a broad range of acetylated lysine residues. We then carried out
in vitro
transcription experiments and observed that dSir2 does not repress transcription with either naked DNA templates or chromatin assembled from native (and mostly unacetylated) histones. It was possible, however, that repression by dSir2 requires an acetylated histone substrate. We therefore tested the transcriptional effects of dSir2 with native histones that were hyperacetylated by treatment with acetic anhydride. Assembly of the hyperacetylated histones onto DNA yields a soluble histone–DNA complex that differs from canonical nucleosomal chromatin. With this hyperacetylated histone–DNA complex, we observed potent (50- to 100-fold) NAD-dependent transcriptional repression by purified dSir2. In contrast, repression by dSir2 was not observed in parallel experiments in which histones were hyperpropionylated with propionic anhydride. We also found that dSir2 mediates the formation of a nuclease-resistant fast-sedimenting histone–DNA complex in an NAD-dependent manner. Unlike dSir2, the dHDAC1 deacetylase does not strongly repress transcription or generate a nuclease-resistant histone–DNA complex. Furthermore, with yeast Sir2, the transcriptional repression we observe correlates with deacetylation activity
in vitro
and silencing activity
in vivo
. These findings suggest that deacetylation by Sir2 causes a conformational change or rearrangement of histones into a transcriptionally repressive chromatin structure.
Requirement of subunit expression for cAMP-mediated regulation of a heart potassium channel
Authors: Kurokawa Junko , Chen Lei , Kass Robert S.
Published: February 2003

β-Adrenergic receptor stimulation increases heart rate and shortens ventricular action-potential duration, the latter effect due in part to a cAMP-dependent increase in the slow outward potassium current (I
Ks
). Mutations in either
KCNQ1
or
KCNE1
, the I
Ks
subunits, are associated with variants (LQT-1 and LQT-5) of the congenital long QT syndrome. We now show that cAMP-mediated functional regulation of KCNQ1/KCNE1 channels, a consequence of cAMP-dependent protein kinase A phosphorylation of the KCNQ1 N terminus, requires coexpression of KCNQ1 with KCNE1, its auxiliary subunit. Further, at least two KCNE1 mutations linked to LQT-5 (D76N and W87R) cause functional disruption of cAMP-mediated KCNQ1/KCNE1-channel regulation despite the response of the substrate protein (KCNQ1) to protein kinase A phosphorylation. Transduction of protein phosphorylation into physiologically necessary channel function represents a previously uncharacterized role for the KCNE1 auxiliary subunit, which can be disrupted in LQT-5.
The
Escherichia coli
multidrug transporter MdfA catalyzes both electrogenic and electroneutral transport reactions
Authors: Lewinson Oded , Adler Julia , Poelarends Gerrit J. , Mazurkiewicz Piotr , Driessen Arnold J. M. , Bibi Eitan
Published: February 2003

The resistance of cells to many drugs simultaneously (multidrug resistance) often involves the expression of membrane transporters (Mdrs); each recognizes and expels a broad spectrum of chemically unrelated drugs from the cell. The
Escherichia coli
Mdr transporter MdfA is able to transport differentially charged substrates in exchange for protons. This includes neutral compounds, namely chloramphenicol and thiamphenicol, and lipophilic cations such as tetraphenylphosphonium and ethidium. Here we show that the chloramphenicol and thiamphenicol transport reactions are electrogenic, whereas the transport of several monovalent cationic substrates is electroneutral. Therefore, unlike with positively charged substrates, the transmembrane electrical potential (negative inside) constitutes a major part of the driving force for the transport of electroneutral substrates by MdfA. These results demonstrate an unprecedented ability of a single secondary transporter to catalyze discrete transport reactions that differ in their electrogenicity and are governed by different components of the proton motive force.
Visualization of synaptotagmin I oligomers assembled onto lipid monolayers
Authors: Wu Yi , He Yuhong , Bai Jihong , Ji Shang-Rong , Tucker Ward C. , Chapman Edwin R. , Sui Sen-Fang
Published: February 2003

Neuronal exocytosis is mediated by Ca
2+
-triggered rearrangements between proteins and lipids that result in the opening and dilation of fusion pores. Synaptotagmin I (syt I) is a Ca
2+
-sensing protein proposed to regulate fusion pore dynamics via Ca
2+
-promoted binding of its cytoplasmic domain (C2A-C2B) to effector molecules, including anionic phospholipids and other copies of syt. Functional studies indicate that Ca
2+
-triggered oligomerization of syt is a critical step in excitation–secretion coupling; however, this activity has recently been called into question. Here, we show that Ca
2+
does not drive the oligomerization of C2A-C2B in solution. However, analysis of Ca
2+
⋅C2A-C2B bound to lipid monolayers, using electron microscopy, revealed the formation of ring-like heptameric oligomers that are ≈11 nm long and ≈11 nm in diameter. In some cases, C2A-C2B also assembled into long filaments. Oligomerization, but not membrane binding, was disrupted by neutralization of two lysine residues (K326,327) within the C2B domain of syt. These data indicate that Ca
2+
first drives C2A-C2B⋅membrane interactions, resulting in conformational changes that trigger a subsequent C2B-mediated oligomerization step. Ca
2+
-mediated rearrangements between syt subunits may regulate the opening or dilation kinetics of fusion pores or may play a role in endocytosis after fusion.
Cell transfection
in vitro
and
in vivo
with nontoxic TAT peptide-liposome–DNA complexes
Authors: Torchilin Vladimir P. , Levchenko Tatyana S. , Rammohan Ram , Volodina Natalia , Papahadjopoulos-Sternberg Brigitte , D'Souza Gerard G. M.
Published: February 2003

Liposomes modified with TAT peptide (TATp-liposomes) showed fast and efficient translocation into the cell cytoplasm with subsequent migration into the perinuclear zone. TATp-liposomes containing a small quantity (≤10 mol %) of a cationic lipid formed firm noncovalent complexes with DNA. Here, we present results demonstrating both
in vitro
and
in vivo
transfection with TATp-liposome–DNA complexes. Mouse NIH/3T3 fibroblasts and rat H9C2 cardiomyocytes were transfected with such complexes
in vitro
. The transfection with the TATp-liposome-associated pEGFP-N1 plasmid encoding for the green fluorescent protein (GFP) was high, whereas the cytotoxicity was lower than that of commonly used cationic lipid-based gene-delivery systems. Intratumoral injection of TATp-liposome–DNA complexes into the Lewis lung carcinoma tumor of mice also resulted in an expression of GFP in tumor cells. This transfection system should be useful for various protocols of cell treatment
in vitro
or
ex vivo
as well as for localized
in vivo
gene therapy.