bluuebunny/crossref_metadata_2025 huggingface dataset:
Meeting of the minds: Metalloneurochemistry
Authors: Burdette Shawn C. , Lippard Stephen J.
Published: April 2003

Metalloneurochemistry is the study of metal ion function in the brain and nervous system at the molecular level. Research in this area is exemplified through discussion of several forefront areas where significant progress has been made in recent years. The structure and function of ion channels have been elucidated through high-resolution x-ray structural work on the bacterial K
+
ion channel. Selection of potassium over sodium ions is achieved by taking advantage of key principles of coordination chemistry. The role of calcium ions in neuronal signal transduction is effected by several Ca
2+
-binding protein such as calmodulin, calcineurin, and synaptotagmin. Structural changes in response to calcium ion concentrations allow these proteins to function in memory formation and other neurochemical roles. Metallochaperones help to achieve metal ion homeostasis and thus prevent neurological diseases because of metal ion imbalance. Much detailed chemical information about these systems has become available recently. Zinc is another important metal ion in neuroscience. Its concentration in brain is in part controlled by metallothionein, and zinc is released in the hippocampus at glutamatergic synapses. New fluorescent sensors have become available to help track such zinc release.
Mineralized tissue and vertebrate evolution: The secretory calcium-binding phosphoprotein gene cluster
Authors: Kawasaki Kazuhiko , Weiss Kenneth M.
Published: April 2003

Gene duplication creates evolutionary novelties by using older tools in new ways. We have identified evidence that the genes for enamel matrix proteins (EMPs), milk caseins, and salivary proteins comprise a family descended from a common ancestor by tandem gene duplication. These genes remain linked, except for one EMP gene, amelogenin. These genes show common structural features and are expressed in ontogenetically similar tissues. Many of these genes encode secretory Ca-binding phosphoproteins, which regulate the Ca-phosphate concentration of the extracellular environment. By exploiting this fundamental property, these genes have subsequently diversified to serve specialized adaptive functions. Casein makes milk supersaturated with Ca-phosphate, which was critical to the successive mammalian divergence. The innovation of enamel led to mineralized feeding apparatus, which enabled active predation of early vertebrates. The EMP genes comprise a subfamily not identified previously. A set of genes for dentine and bone extracellular matrix proteins constitutes an additional cluster distal to the EMP gene cluster, with similar structural features to EMP genes. The duplication and diversification of the primordial genes for enamel/dentine/bone extracellular matrix may have been important in core vertebrate feeding adaptations, the mineralized skeleton, the evolution of saliva, and, eventually, lactation. The order of duplication events may help delineate early events in mineralized skeletal formation, which is a major characteristic of vertebrates.
The birth and evolution of surface science: Child of the union of science and technology
Authors: Duke C. B.
Published: April 2003

This article is an account of the birth and evolution of surface science as an interdisciplinary research area. Surface science emanated from the confluence of concepts and tools in physics and chemistry with technological innovations that made it possible to determine the structure and properties of surfaces and interfaces and the dynamics of chemical reactions at surfaces. The combination in the 1960s and 1970s of ultra-high-vacuum (i.e.,
P
< 10
−7
Pascal or 10
−9
Torr) technology with the recognition that electrons in the energy range from 50 to 500 eV exhibited inelastic collision mean free paths of the order of a few angstroms fostered an explosion of activity. The results were a reformulation of the theory of electron solid scattering, the nearly universal use of electron spectroscopies for surface characterization, the rise of surface science as an independent interdisciplinary research area, and the emergence of the American Vacuum Society (AVS) as a major international scientific society. The rise of microelectronics in the 1970s and 1980s resulted in huge increases in computational power. These increases enabled more complex experiments and the utilization of density functional theory for the quantitative prediction of surface structure and dynamics. Development of scanning-probe microscopies in the 1990s led to atomic-resolution images of macroscopic surfaces and interfaces as well as videos of atoms moving about on surfaces during growth and diffusion. Scanning probes have since brought solid–liquid interfaces into the realm of atomic-level surface science, expanding its scope to more complex systems, including fragile biological materials and processes.
Biological inorganic chemistry at the beginning of the 21st century
Authors: Gray Harry B.
Published: April 2003

Advances in bioinorganic chemistry since the 1970s have been driven by three factors: rapid determination of high-resolution structures of proteins and other biomolecules, utilization of powerful spectroscopic tools for studies of both structures and dynamics, and the widespread use of macromolecular engineering to create new biologically relevant structures. Today, very large molecules can be manipulated at will, with the result that certain proteins and nucleic acids themselves have become versatile model systems for elucidating biological function.
Misfolded CuZnSOD and amyotrophic lateral sclerosis
Authors: Valentine Joan Selverstone , Hart P. John
Published: April 2003

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease of motor neurons. The inherited form of the disease, familial ALS, represents 5–10% of the total cases, and the best documented of these are due to lesions in SOD1, the gene encoding copper–zinc superoxide dismutase (CuZnSOD). The mechanism by which mutations in SOD1 cause familial ALS is currently unknown. Two hypotheses have dominated recent discussion of the toxicity of ALS mutant CuZnSOD proteins: the oligomerization hypothesis and the oxidative damage hypothesis. The oligomerization hypothesis maintains that mutant CuZnSOD proteins are, or become, misfolded and consequently oligomerize into increasingly high-molecular-weight species that ultimately lead to the death of motor neurons. The oxidative damage hypothesis maintains that ALS mutant CuZnSOD proteins catalyze oxidative reactions that damage substrates critical for viability of the affected cells. This perspective reviews some of the properties of both wild-type and mutant CuZnSOD proteins, suggests how these properties may be relevant to these two hypotheses, and proposes that these two hypotheses are not necessarily mutually exclusive.
Male-biased transmission of deleterious mutations to the progeny in
Arabidopsis
thaliana
Authors: Whittle Carrie-Ann , Johnston Mark O.
Published: April 2003

The extent and cause of male-biased mutation rates, the higher number of mutations in sperm than in eggs, is currently an active and controversial subject. Recent evidence indicates that this male (sperm) bias not only occurs in animals but also in plants. The higher mutation rate in plant sperm was inferred from rates of evolution of neutral DNA regions, and the results were confined to the mitochondria and chloroplasts of gymnosperms. However, the relative transmission rates of deleterious mutations, which have substantial evolutionary consequences, have rarely been studied. Here, an investigation is described by using the hermaphroditic self-compatible flowering plant
Arabidopsis thaliana
, in which we artificially increased the rate of mutation in pollen (i.e., sperm donor) and maternal (i.e., egg donor) parents, by using two kinds of UV irradiation in parallel and separate experiments, and assessed the deleterious effects on fitness of the F
2
generation. The results show that more deleterious induced mutations are transmitted to the progeny by a sperm than by an egg. These findings provide the first experimental evidence that more deleterious mutations are inherited from sperm than from an egg in any organism. Possible causes underlying this male bias are discussed.
Transforming growth factor β1 inhibits expression of NKp30 and NKG2D receptors: Consequences for the NK-mediated killing of dendritic cells
Authors: Castriconi Roberta , Cantoni Claudia , Della Chiesa Mariella , Vitale Massimo , Marcenaro Emanuela , Conte Romana , Biassoni Roberto , Bottino Cristina , Moretta Lorenzo , Moretta Alessandro
Published: April 2003

The surface density of the triggering receptors responsible for the natural killer (NK)-mediated cytotoxicity is crucial for the ability of NK cells to kill susceptible target cells. In this study, we show that transforming growth factor β1 (TGFβ1) down-regulates the surface expression of NKp30 and in part of NKG2D but not that of other triggering receptors such as NKp46. The TGFβ1-mediated inhibition of NKp30 surface expression reflects gene regulation at the transcriptional level. NKp30 has been shown to represent the major receptor involved in the NK-mediated killing of dendritic cells. Accordingly, the TGFβ1-dependent down-regulation of NKp30 expression profoundly inhibited the NK-mediated killing of dendritic cells. On the contrary, killing of different NK-susceptible tumor cell lines was variably affected, reflecting the differential usage of NKp30 and/or NKG2D in the lysis of such tumors. Our present data suggest a possible mechanism by which TGFβ1-producing dendritic cells may acquire resistance to the NK-mediated attack.
Demonstration of functional coupling between γ-aminobutyric acid (GABA) synthesis and vesicular GABA transport into synaptic vesicles
Authors: Jin Hong , Wu Heng , Osterhaus Gregory , Wei Jianning , Davis Kathleen , Sha Di , Floor Eric , Hsu Che-Chang , Kopke Richard D. , Wu Jang-Yen
Published: April 2003

l
-Glutamic acid decarboxylase (GAD) exists as both membrane-associated and soluble forms in the mammalian brain. Here, we propose that there is a functional and structural coupling between the synthesis of γ-aminobutyric acid (GABA) by membrane-associated GAD and its packaging into synaptic vesicles (SVs) by vesicular GABA transporter (VGAT). This notion is supported by the following observations. First, newly synthesized [
3
H]GABA from [
3
H]
l
-glutamate by membrane-associated GAD is taken up preferentially over preexisting GABA by using immunoaffinity-purified GABAergic SVs. Second, the activity of SV-associated GAD and VGAT seems to be coupled because inhibition of GAD also decreases VGAT activity. Third, VGAT and SV-associated Ca
2+
/calmodulin-dependent kinase II have been found to form a protein complex with GAD. A model is also proposed to link the neuronal stimulation to enhanced synthesis and packaging of GABA into SVs.
Distinct roles of dopamine D2L and D2S receptor isoforms in the regulation of protein phosphorylation at presynaptic and postsynaptic sites
Authors: Lindgren Niklas , Usiello Alessandro , Goiny Michel , Haycock John , Erbs Eric , Greengard Paul , Hökfelt Tomas , Borrelli Emiliana , Fisone Gilberto
Published: April 2003

Dopamine D2 receptors are highly expressed in the dorsal striatum where they participate in the regulation of (
i
) tyrosine hydroxylase (TH), in nigrostriatal nerve terminals, and (
ii
) the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32), in medium spiny neurons. Two isoforms of the D2 receptor are generated by differential splicing of the same gene and are referred to as short (D2S) and long (D2L) dopamine receptors. Here we have used wild-type mice, dopamine D2 receptor knockout mice (D2 KO mice; lacking both D2S and D2L receptors) and D2L receptor-selective knockout mice (D2L KO mice) to evaluate the involvement of each isoform in the regulation of the phosphorylation of TH and DARPP-32. Incubation of striatal slices from wild-type mice with quinpirole, a dopamine D2 receptor agonist, decreased the state of phosphorylation of TH at Ser-40 and its enzymatic activity. Both effects were abolished in D2 KO mice but were still present in D2L KO mice. In wild-type mice, quinpirole inhibits the increase in DARPP-32 phosphorylation at Thr-34 induced by SKF81297, a dopamine D1 receptor agonist. This effect is absent in D2 KO as well as D2L KO mice. The inability of quinpirole to regulate DARPP-32 phosphorylation in D2L KO mice cannot be attributed to decreased coupling of D2S receptors to G proteins, because quinpirole produces a similar stimulation of [
35
S]GTPγS binding in wild-type and D2L KO mice. These results demonstrate that D2S and D2L receptors participate in presynaptic and postsynaptic dopaminergic transmission, respectively.
Structural effects of quinacrine binding in the open channel of the acetylcholine receptor
Authors: Yu Yong , Shi Lei , Karlin Arthur
Published: April 2003

Noncompetitive inhibitors of the nicotinic acetylcholine (ACh) receptors suppress cation flux directly by binding in and blocking the open channel or indirectly by stabilizing closed states of the receptor. The lidocaine derivative QX-314 and the acridine derivative quinacrine act directly as open channel blockers, but can act indirectly as well. The binding site for quinacrine in the open channel of mouse-muscle ACh receptor was mapped in cysteine-substituted mutants of the α subunit expressed with wild-type β, γ, and δ subunits. In the open state, substituted cysteines in the inner half of the second membrane-spanning segment (M2), but not in the outer half, were protected by quinacrine from reaction with 2-aminoethyl methanethiosulfonate. In addition, an alkylating derivative, quinacrine mustard, affinity labeled a subset of the substituted cysteines in M2, but only in the open state. These results, mapped onto a model of the open channel surrounded by five α-helical M2s, imply that quinacrine binds midway down M2 in the same site previously mapped for QX-314. A cysteine substituted for a residue in the outer third of αM1, which reacted with 2-aminoethyl methanethiosulfonate only in the presence of ACh, reacted faster in the additional presence of quinacrine or QX-314. It is proposed that channel opening involves both the opening of the resting gate at the inner end of M2 and the removal of an obstruction formed by the outer end of M1 that retards diffusion of blockers into the closed channel. Blocker binding in the open channel causes a further change in structure.
Preorganization of molecular binding sites in designed diiron proteins
Authors: Maglio Ornella , Nastri Flavia , Pavone Vincenzo , Lombardi Angela , DeGrado William F.
Published: April 2003

De novo
protein design provides an attractive approach to critically test the features that are required for metalloprotein structure and function. Previously we designed and crystallographically characterized an idealized dimeric model for the four-helix bundle class of diiron and dimanganese proteins [Dueferri 1 (DF1)]. Although the protein bound metal ions in the expected manner, access to its active site was blocked by large bulky hydrophobic residues. Subsequently, a substrate-access channel was introduced proximal to the metal-binding center, resulting in a protein with properties more closely resembling those of natural enzymes. Here we delineate the energetic and structural consequences associated with the introduction of these binding sites. To determine the extent to which the binding site was preorganized in the absence of metal ions, the apo structure of DF1 in solution was solved by NMR and compared with the crystal structure of the di-Zn(II) derivative. The overall fold of the apo protein was highly similar to that of the di-Zn(II) derivative, although there was a rotation of one of the helices. We also examined the thermodynamic consequences associated with building a small molecule-binding site within the protein. The protein exists in an equilibrium between folded dimers and unfolded monomers. DF1 is a highly stable protein (
K
diss
= 0.001 fM), but the dissociation constant increases to 0.6 nM (ΔΔ
G
= 5.4 kcal/mol monomer) as the active-site cavity is increased to accommodate small molecules.
Posterior parietal cortex and the filtering of distractors
Authors: Friedman-Hill Stacia R. , Robertson Lynn C. , Desimone Robert , Ungerleider Leslie G.
Published: April 2003

Neural systems for visual processing can focus attention on behaviorally relevant objects, filtering out competing distractors. Neurophysiological studies in animals and brain imaging studies in humans suggest that such filtering depends on top-down inputs to extrastriate visual areas, originating in structures important for attentional control. To test whether the posterior parietal cortex may be a necessary source of signals that filter distractors, we measured the ability of a patient with bilateral parietal lesions to discriminate the features of a target surrounded by distractors of variable contrast. In the presence of distractors, the patient was impaired at discriminating both grating orientation and faces, and the magnitude of the impairment increased with distractor salience. These attentional deficits are remarkably similar to those caused by damage to monkey extrastriate regions V4 and/or TEO, which are thought to be recipients of top-down attentional feedback. In contrast to the effects of V4 and TEO lesions, however, the parietal lesions impaired performance even with widely spaced targets and distractors, a finding consistent with the projections of parietal cortex to visual processing areas covering a wide range of receptive field sizes and eccentricities.
Why the kidney glomerulus does not clog: A gel permeation/diffusion hypothesis of renal function
Authors: Smithies Oliver
Published: April 2003

Current interpretations of kidney function in terms of a coarse filter followed by a fine filter have difficulty explaining why the glomerulus does not clog. I propose, as an alternative, a semiquantitative hypothesis that assumes that the size-selective property of the glomerulus is a consequence of the limited fraction of space in the glomerular basement membrane (a concentrated gel) into which macromolecules can permeate. The glomerular epithelial cell slits and slit diaphragms are assumed to impose substantial resistance to liquid flow across the glomerulus without acting as a molecular sieve. Calculations based on gel behavior show that proteins cross the glomerular basement membrane mainly by diffusion rather than by liquid flow, whereas water crosses entirely by flow. Thus, diffusion provides most of the protein, whereas flow provides the diluent. As a result, the single-nephron glomerular filtration rate (GFR) becomes a prime factor in (inversely) determining the concentration of proteins in early proximal tubular fluid. Because the reabsorption of proteins from the tubules is a saturable process, the gel permeation/diffusion hypothesis readily accounts for the albuminuria observed when single-nephron GFR is substantially reduced by severe pathological decreases in slit diaphragm length, such as occur in minimal-change nephrotic syndrome in humans, in animals treated with puromycin aminonucleoside, or in humans or animals with mutations in the gene coding for nephrin. My hypothesis predicts that albuminuria will ensue, even with a normal kidney, if the single-nephron GFR falls below ≈50% of normal.
Enhanced oxygen diffusivity in interfaces of nanocrystalline ZrO
2
⋅Y
2
O
3
Authors: Knöner Gregor , Reimann Klaus , Röwer Ralf , Södervall Ulf , Schaefer Hans-Eckhardt
Published: April 2003

First measurements of oxygen grain boundary diffusion coefficients in nanocrystalline yttria-doped ZrO
2
(n-ZrO
2
⋅6.9 mol % Y
2
O
3
) are presented. The
18
O diffusion profiles measured by secondary ion mass spectroscopy are much deeper in the nanocrystalline specimens than in single crystals. An oxygen diffusivity,
D
B
, in the grain boundaries can be deduced, which is ≈3 orders of magnitude higher than in single crystals. From the present data the temperature variation of the oxygen grain boundary diffusivity,
D
B
= 2.0 × 10
−5
exp (−0.91 eV/
k
B
T
) m
2
/s, and the oxygen surface exchange coefficient,
k
= 1.4 × 10
−2
exp (−1.13 eV/
k
B
T
) m/s, are derived.
Evidence against the presence of an anandamide transporter
Authors: Glaser Sherrye T. , Abumrad Nada A. , Fatade Folayan , Kaczocha Martin , Studholme Keith M. , Deutsch Dale G.
Published: April 2003

On the basis of temperature dependency, saturability, selective inhibition, and substrate specificity, it has been proposed that an anandamide transporter exists. However, all of these studies have examined anandamide accumulation at long time points when downstream effects such as metabolism and intracellular sequestration are operative. In the current study, we have investigated the initial rates (<1 min) of anandamide accumulation in neuroblastoma and astrocytoma cells in culture and have determined that uptake is not saturable with increasing concentrations of anandamide. However, anandamide hydrolysis, after uptake in neuroblastoma cells, was saturable at steady-state time points (5 min), suggesting that fatty acid amide hydrolase (FAAH) may be responsible for observed saturation of uptake at long time points. In general, arvanil, olvanil, and
N
-(4-hydroxyphenyl)arachidonylamide (AM404) have been characterized as transport inhibitors in studies using long incubations. However, we found these “transport inhibitors” did not inhibit anandamide uptake in neuroblastoma and astrocytoma cells at short time points (40 sec or less). Furthermore, we confirmed that these inhibitors
in vitro
were actually inhibitors of FAAH. Therefore, the likely mechanism by which the transport inhibitors raise anandamide levels to exert pharmacological effects is by inhibiting FAAH, and they should be reevaluated in this context. Immunofluorescence has indicated that FAAH staining resides mainly on intracellular membranes of neuroblastoma cells, and this finding is consistent with our observed kinetics of anandamide hydrolysis. In summary, these data suggest that anandamide uptake is a process of simple diffusion. This process is driven by metabolism and other downstream events, rather than by a specific membrane-associated anandamide carrier.
Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase
Authors: Bransteitter Ronda , Pham Phuong , Scharff Matthew D. , Goodman Myron F.
Published: April 2003

The expression of activation-induced cytidine deaminase (AID) is prerequisite to a “trifecta” of key molecular events in B cells: class-switch recombination and somatic hypermutation in humans and mice and gene conversion in chickens. Although this critically important enzyme shares common sequence motifs with apolipoprotein B mRNA-editing enzyme, and exhibits deaminase activity on free deoxycytidine in solution, it has not been shown to act on either RNA or DNA. Recent mutagenesis data in
Escherichia coli
suggest that AID may deaminate dC on DNA, but its putative biochemical activities on either DNA or RNA remained a mystery. Here, we show that AID catalyzes deamination of dC residues on single-stranded DNA
in vitro
but not on double-stranded DNA, RNA–DNA hybrids, or RNA. Remarkably, it has no measurable deaminase activity on single-stranded DNA unless pretreated with RNase to remove inhibitory RNA bound to AID. AID catalyzes dC → dU deamination activity most avidly on double-stranded DNA substrates containing a small “transcription-like” single-stranded DNA bubble, suggesting a targeting mechanism for this enigmatic enzyme during somatic hypermutation.
Regulation of hepatic fasting response by PPARγ coactivator-1α (PGC-1): Requirement for hepatocyte nuclear factor 4α in gluconeogenesis
Authors: Rhee James , Inoue Yusuke , Yoon J. Cliff , Puigserver Pere , Fan Melina , Gonzalez Frank J. , Spiegelman Bruce M.
Published: April 2003

The liver plays several critical roles in the metabolic adaptation to fasting. We have shown previously that the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in fasted or diabetic liver and activates the entire program of gluconeogenesis. PGC-1α interacts with several nuclear receptors known to bind gluconeogenic promoters including the glucocorticoid receptor, hepatocyte nuclear factor 4α (HNF4α), and the peroxisome proliferator-activated receptors. However, the genetic requirement for any of these interactions has not been determined. Using hepatocytes from mice lacking HNF4α in the liver, we show here that PGC-1α completely loses its ability to activate key genes of gluconeogenesis such as phospho
enol
pyruvate carboxykinase and glucose-6-phosphatase when HNF4α is absent. It is also shown that PGC-1α can induce genes of β-oxidation and ketogenesis in hepatocytes, but these effects do not require HNF4α. Analysis of the glucose-6-phosphatase promoter indicates a key role for HNF4α-binding sites that function robustly only when HNF4α is coactivated by PGC-1α. These data illustrate the involvement of PGC-1α in several aspects of the hepatic fasting response and show that HNF4α is a critical component of PGC-1α-mediated gluconeogenesis.
The structural basis for biphasic kinetics in the folding of the WW domain from a formin-binding protein: Lessons for protein design?
Authors: Karanicolas John , Brooks Charles L.
Published: April 2003

The mechanism of formation of β-sheets is of great importance because of the significant role of such structures in the initiation and propagation of amyloid diseases. In this study we examine the folding of a series of three-stranded antiparallel β-sheets known as WW domains. Whereas other WW domains have been shown to fold with single-exponential kinetics, the WW domain from murine formin-binding protein 28 has recently been shown to fold with biphasic kinetics. By using a combination of kinetics and thermodynamics to characterize a simple model for this protein, the origins of the biphasic kinetics is found to lie in the fact that most of the protein is able to fold without requiring one of the β-hairpins to be correctly registered. The correct register of this hairpin is enforced by a surface-exposed hydrophobic contact, which is not present in other WW domains. This finding suggests the use of judiciously chosen surface-exposed hydrophobic pairs as a protein design strategy for enforcing the desired strand registry.
Somatic hypermutation of the B cell receptor genesB29(Igβ, CD79b) andmb1(Igα, CD79a)
Authors: Gordon Melinda S. , Kanegai Cindy M. , Doerr Jeanette R. , Wall Randolph
Published: April 2003

Somatic hypermutation (SHM), coupled to selection by antigen, generates high-affinity antibodies during germinal center (GC) B cell maturation. SHM is known to affectBcl6, four additional oncogenes in diffuse large B cell lymphoma, and theCD95/Fasgene and is regarded as a major mechanism of B cell tumorigenesis. We find that mutations in the genes encoding the B cell receptor (BCR) accessory proteins B29 (Igβ, CD79b) and mb1 (Igα, CD79a) occur as often asIggenes in a broad spectrum of GC- and post-GC-derived malignant B cell lines, as well as in normal peripheral B cells. TheseB29andmb1mutations are typical SHM consisting largely of single nucleotide substitutions targeted to hotspots. TheB29andmb1mutations appear at frequencies similar to those of othernon-Iggenes but lower thanIggenes. The distribution ofmb1mutations followed the characteristic pattern found inIgand mostnon-Iggenes. In contrast,B29mutations displayed a bimodal distribution resembling theCD95/Fasgene, in which promoter distal mutations conferred resistance to apoptosis. DistalB29mutations in the cytoplasmic domain may contribute to B cell survival by limiting BCR signaling.B29andmb1are mutated in a much broader spectrum of GC-derived B cells than any other known somatically hypermutatednon-Iggene. This may be caused by the commoncis-acting regulatory sequences that control the requisite coexpression of the B29, mb1, and Ig chains in the BCR.
The history of effective population size and genetic diversity in the Yellowstone grizzly (
Ursus arctos
): Implications for conservation
Authors: Miller Craig R. , Waits Lisette P.
Published: April 2003

Protein, mtDNA, and nuclear microsatellite DNA analyses have demonstrated that the Yellowstone grizzly bear has low levels of genetic variability compared with other
Ursus arctos
populations. Researchers have attributed this difference to inbreeding during a century of anthropogenic isolation and population size reduction. We test this hypothesis and assess the seriousness of genetic threats by generating microsatellite data for 110 museum specimens collected between 1912 and 1981. A loss of variability is detected, but it is much less severe than hypothesized. Variance in allele frequencies over time is used to estimate an effective population size of ≈80 across the 20th century and >100 currently. The viability of the population is unlikely to be substantially reduced by genetic factors in the next several generations. However, gene flow from outside populations will be beneficial in avoiding inbreeding and the erosion of genetic diversity in the future.
The LIM-only protein FHL2 is a serum-inducible transcriptional coactivator of AP-1
Authors: Morlon Aurore , Sassone-Corsi Paolo
Published: April 2003

Proteins with LIM domains have been implicated in transcriptional regulation. The four and half LIM domain (FHL) group of LIM-only proteins is composed of five members, some of which have been shown to have intrinsic activation function. Here we show that FHL2 is the only member of the family whose expression is inducible upon serum stimulation in cultured cells. Induction of FHL2 is coordinated in time with the increased levels of two early-response products, the oncoproteins Fos and Jun. FHL2 associates with both Jun and Fos,
in vitro
and
in vivo
. The FHL2-Jun interaction requires the Ser-63-Ser-73 JNK phosphoacceptor sites in c-Jun, but not their phosphorylation. FHL2 powerfully stimulates Fos- and Jun-dependent transcription, thereby acting as an inducible coactivator of AP-1 function. Moreover, we show that intracellular localization of FHL2 is controlled by signaling events and a Crm1-dependent active nuclear export mechanism. Thus, FHL2, as an inducible coactivator of AP-1, coordinately participates with Fos and Jun in the early transcriptional response to serum factors.
Visualizing the Ca
2+
-dependent activation of gelsolin by using synchrotron footprinting
Authors: Kiselar Janna G. , Janmey Paul A. , Almo Steven C. , Chance Mark R.
Published: April 2003

Radiolytic protein footprinting with a synchrotron source is used to reveal detailed structural changes that occur in the Ca
2+
-dependent activation of gelsolin. More than 80 discrete peptides segments within the structure, covering 95% of the sequence in the molecule, were examined by footprinting and mass spectrometry for their solvent accessibility as a function of Ca
2+
concentration in solution. Twenty-two of the peptides exhibited detectable oxidation; for seven the oxidation extent was seen to be Ca
2+
sensitive. Ca
2+
titration isotherms monitoring the oxidation within residues 49–72 (within subdomain S1), 121–135 (S1), 162–166 (S2), and 722–748 (S6) indicate a three-state activation process with a intermediate that was populated at a Ca
2+
concentration of 1–5 μM that is competent for capping and severing activity. A second structural transition with a midpoint of ≈60–100 μM, where the accessibility of the above four peptides is further increased, is also observed. Tandem mass spectrometry showed that buried residues within the helical “latch” of S6 (including Pro-745) that contact an F-actin-binding site on S2 and buried F-actin-binding residues within S2 (including Phe-163) are unmasked in the submicromolar Ca
2+
transition. However, residues within S4 that are part of an extended β-sheet with S6 (including Tyr-453) are revealed only in the subsequent transition at higher Ca
2+
concentrations; the disruption of this extended contact between S4 and S6 (and likely the analogous contact between S1 and S3) likely results in an extended structure permitting additional functions consistent with the fully activated gelsolin molecule.
Yeast genetic selections to optimize RNA decoys for transcription factor NF-κB
Authors: Cassiday Laura A. , Maher L. James
Published: April 2003

In vitro
-selected RNA aptamers are potential inhibitors of disease-related proteins. Our laboratory previously isolated an RNA aptamer that binds with high affinity to human transcription factor NF-κB. This RNA aptamer competitively inhibits DNA binding by NF-κB
in vitro
and is recognized by its target protein
in vivo
in a yeast three-hybrid system. In the present study, yeast genetic selections were used to optimize the RNA aptamer for binding to NF-κB in the eukaryotic nucleus. Selection for improved binding to NF-κB from RNA libraries encoding (
i
) degenerate aptamer variants and (
ii
) sequences present at round 8 of 14 total rounds of
in vitro
selection yielded RNA aptamers with dramatically improved
in vivo
activity. Furthermore, we show that an
in vivo
-optimized RNA aptamer exhibits specific “decoy” activity, inhibiting transcriptional activation by its NF-κB target protein in a yeast one-hybrid assay. This decoy activity is enhanced by the expression of a bivalent aptamer. The combination of
in vitro
and
in vivo
genetic selections was crucial for obtaining RNA aptamers with
in vivo
decoy activity.
Importance of the
CNGA4
channel gene for odor discrimination and adaptation in behaving mice
Authors: Kelliher Kevin R. , Ziesmann Jürgen , Munger Steven D. , Reed Randall R. , Zufall Frank
Published: April 2003

Odor stimulation of olfactory sensory neurons (OSNs) leads to both the activation and subsequent desensitization of a heteromultimeric cyclic-nucleotide-gated (CNG) channel present in these cells. The native olfactory CNG channel consists of three distinct subunits: CNGA2, CNGA4, and CNGB1b. Mice in which the
CNGA4
gene has been deleted display defective Ca
2+
/calmodulin-dependent inhibition of the CNG channel, resulting in a striking reduction in adaptation of the odor-induced electrophysiological response in the OSNs. These mutants therefore afford an excellent opportunity to assess the importance of Ca
2+
-mediated CNG channel desensitization for odor discrimination and adaptation in behaving animals. By using an operant conditioning paradigm, we show that
CNGA4
-null mice are profoundly impaired in the detection and discrimination of olfactory stimuli in the presence of an adapting background odor. The extent of this impairment depends on both the concentration and the molecular identity of the adapting stimulus. Thus, Ca
2+
-dependent desensitization of the odor response in the OSNs mediated by the CNGA4 subunit is essential for normal odor sensation and adaptation of freely behaving mice, preventing saturation of the olfactory signal transduction machinery and extending the range of odor detection and discrimination.
Macula densa cell signaling involves ATP release through a maxi anion channel
Authors: Bell Phillip Darwin , Lapointe Jean-Yves , Sabirov Ravshan , Hayashi Seiji , Peti-Peterdi Janos , Manabe Ken-ichi , Kovacs Gergely , Okada Yasunobu
Published: April 2003

Macula densa cells are unique renal biosensor cells that detect changes in luminal NaCl concentration ([NaCl]
L
) and transmit signals to the mesangial cell/afferent arteriolar complex. They are the critical link between renal salt and water excretion and glomerular hemodynamics, thus playing a key role in regulation of body fluid volume. Since identification of these cells in the early 1900s, the nature of the signaling process from macula densa cells to the glomerular contractile elements has remained unknown. In patch–clamp studies of macula densa cells, we identified an [NaCl]
L
-sensitive ATP-permeable large-conductance (380 pS) anion channel. Also, we directly demonstrated the release of ATP (up to 10 μM) at the basolateral membrane of macula densa cells, in a manner dependent on [NaCl]
L
, by using an ATP bioassay technique. Furthermore, we found that glomerular mesangial cells respond with elevations in cytosolic Ca
2+
concentration to extracellular application of ATP (EC
50
0.8 μM). Importantly, we also found increases in cytosolic Ca
2+
concentration with elevations in [NaCl]
L
, when fura-2-loaded mesangial cells were placed close to the basolateral membrane of macula densa cells. Thus, cell-to-cell communication between macula densa cells and mesangial cells, which express P2Y
2
receptors, involves the release of ATP from macula densa cells via maxi anion channels at the basolateral membrane. This mechanism may represent a new paradigm in cell-to-cell signal transduction mediated by ATP.
A metal-binding site in the catalytic subunit of anaerobic ribonucleotide reductase
Authors: Logan Derek T. , Mulliez Etienne , Larsson Karl-Magnus , Bodevin Sabrina , Atta Mohamed , Garnaud Pierre Emmanuel , Sjöberg Britt-Marie , Fontecave Marc
Published: April 2003

A Zn(Cys)
4
center has been found in the C-terminal region of the crystal structure of the anaerobic class III ribonucleotide reductase (RNR) from bacteriophage T4. The metal center is structurally related to the zinc ribbon motif and to rubredoxin and rubrerythrin. Mutant enzymes of the homologous RNR from
Escherichia coli
, in which the coordinating cysteines, conserved in almost all known class III RNR sequences, have been mutated into alanines, are shown to be inactive as the result of their inability to generate the catalytically essential glycyl radical. The possible roles of the metal center are discussed in relationship to the currently proposed reaction mechanism for generation of the glycyl radical in class III RNRs.
Anatomical organization of forward fiber projections from area TE to perirhinal neurons representing visual long-term memory in monkeys
Authors: Yoshida Masatoshi , Naya Yuji , Miyashita Yasushi
Published: April 2003

A number of studies have shown that the perirhinal (PRh) cortex, which is part of the medial temporal lobe memory system, plays an important role in declarative long-term memory. The PRh cortex contains neurons that represent visual long-term memory. The aim of the present study is to characterize the anatomical organization of forward projections that mediate information flow from visual area TE to memory neurons in the PRh cortex. In monkeys performing a visual pair-association memory task, we conducted an extensive mapping of neuronal responses in the anteroventral part of area TE (TEav) and area 36 (A36) of the PRh cortex. Then, three retrograde tracers were separately injected into A36 and the distribution of retrograde labels in TEav was analyzed. We focused on the degree of divergent projections from TEav to memory neurons in A36, because the highly divergent nature of these forward fiber projections has been implicated in memory function. We found that the degree of divergent projection to memory neurons in A36 was smaller from the TEav neurons selective to learned pictures than from the nonselective TEav neurons. This result demonstrates that the anatomical difference (the divergence) correlates with the physiological difference (selectivity of TEav neurons to the learned pictures). Because the physiological difference is attributed to whether the projections are involved in information transmission required for memory neurons in A36, it can be speculated that the reduced divergent projection resulted from acquisition of visual long-term memory, possibly through retraction of the projecting axon collaterals.
Toll-like receptor 4 on stromal and hematopoietic cells mediates innate resistance to uropathogenic
Escherichia
coli
Authors: Schilling Joel D. , Martin Steven M. , Hung Chia S. , Lorenz Robin G. , Hultgren Scott J.
Published: April 2003

Innate host defenses at mucosal surfaces are critical in the early stages of many bacterial infections. In addition to cells of the traditional innate immune system, epithelial cells can also produce inflammatory mediators during an infection. However, the role of the epithelium in innate host defense
in vivo
is unclear. Recent studies have shown that lipopolysaccharide (LPS) recognition is critical for bladder epithelial cells to recognize and respond to
Escherichia coli
. Moreover, the LPS-nonresponsive mouse strain C3H/HeJ, which has a mutation in the primary LPS receptor, Toll-like receptor 4 (TLR4), is extremely susceptible to infection with uropathogenic strains of
E. coli
. In this study, a bone marrow transplant approach was used to investigate the specific contributions of the bladder epithelium (and other stromal cells) in the TLR4-mediated innate immune response to the invading
E. coli
pathogen. Mice expressing the mutant TLR4 in the epithelial/stromal compartment were not able to mount a protective inflammatory response to control the early infection even when their hematopoietic cells expressed wild-type TLR4. However, the presence of TLR4
+
epithelial/stromal cells was not sufficient to activate an acute inflammatory response unless the hematopoietic cells were also TLR4
+
. These results demonstrated that bladder epithelial cells play a critical role in TLR4-mediated innate immunity
in vivo
during a mucosal bacterial infection.
Promyogenic members of the Ig and cadherin families associate to positively regulate differentiation
Authors: Kang Jong-Sun , Feinleib Jessica L. , Knox Sarah , Ketteringham Michael A. , Krauss Robert S.
Published: April 2003

Determination and differentiation of cells in the skeletal muscle lineage is positively regulated by cell–cell contact. Cell-surface proteins proposed to mediate this effect include both classical cadherins and Ig superfamily members; potential interactions between the promyogenic activities of these classes of protein, however, are unknown. We show here that CDO and BOC, two promyogenic Ig superfamily members that bind to each other in a
cis
fashion, form complexes with N- and M-cadherin. These complexes contain β-catenin and are enriched at sites of cell–cell contact between myoblasts. In transient expression assays, the ectodomains and intracellular regions of CDO, BOC, and N-cadherin each interact independently, suggesting that the interactions occur in a
cis
fashion; consistent with this conclusion, cadherin-mediated cell adhesion is not required for them to occur. Stable expression in myoblasts of a CDO deletion mutant deficient in its ability to associate with N-cadherin interferes with differentiation as assessed by biochemical, morphological, and reporter gene assays, suggesting that this interaction is functionally important in myogenesis. Thus, some of the cell–cell contact-mediated activities that are required for myogenesis seem to be based on interdependent activities of promyogenic classical cadherins and Ig superfamily members.
Bioinorganic chemistry in the postgenomic era
Authors: Bertini Ivano , Rosato Antonio
Published: April 2003

Genome sequencing has revolutionized all fields of life sciences. Bioinorganic chemistry is certainly not immune to this influence, which is presenting unprecedented challenges. A new goal for bioinorganic chemistry is the investigation of the linkages between inorganic elements and genomic information. This requires new advancements and/or the development of new expertise in fields such as bioinformatics and genetics but also provides a driving force to push forward the exploitation of traditional analytical techniques and spectroscopic tools. The “case study” of metal homeostasis in cells is discussed to provide a flavor of the current evolution of the field.
Gapped DNA is anisotropically bent
Authors: Guo Hong , Tullius Thomas D.
Published: April 2003

Ionizing radiation damages DNA in several ways, including through formation of a single-nucleoside gap in one DNA strand. We have developed a two-dimensional gel electrophoresis method to investigate the effect of a strand gap on DNA structure. We generate a library of gapped DNA molecules by treating a DNA restriction fragment with the hydroxyl radical, generated by the reaction of Fe(II) EDTA with hydrogen peroxide. The DNA molecule studied contains a fixed bend produced by a set of phased adenine tracts. The A-tract bend serves as a reference bend for investigating the conformational nature of a strand gap. In the first electrophoretic dimension, a bent DNA molecule that has been treated with the hydroxyl radical is electrophoresed on a native gel. Smearing of the band on the native gel indicates that the library of gapped DNA molecules contains a variety of DNA conformations. In the second electrophoretic dimension, gapped DNA molecules having different native gel mobilities are electrophoresed on separate lanes of a denaturing gel to reveal how each strand gap affects the native gel mobility (and thus shape) of the DNA. Our results demonstrate that a single-nucleoside gap in a DNA duplex leads to an anisotropic, directional bend in the DNA helix axis. The implications of our findings for recognition of this lesion by DNA repair proteins are discussed.
Neutral thiol as a proximal ligand to ferrous heme iron: Implications for heme proteins that lose cysteine thiolate ligation on reduction
Authors: Perera Roshan , Sono Masanori , Sigman Jeffrey A. , Pfister Thomas D. , Lu Yi , Dawson John H.
Published: April 2003

Cysteine plays a key role as a metal ligand in metalloproteins. In all well-recognized cases, however, it is the anionic cysteinate that coordinates. Several cysteinate-ligated heme proteins are known, but some fail to retain thiolate ligation in the ferrous state, possibly following protonation to form neutral cysteine. Ligation by cysteine thiol in ferrous heme proteins has not been documented. To establish spectroscopic signatures for such systems, we have prepared five-coordinate adducts of the ferrous myoglobin H94G cavity mutant with neutral thiol and thioether sulfur donors as well as six-coordinate derivatives such as with CO and, when possible, with NO and O
2
. A thiol-ligated oxyferrous complex is reported, to our knowledge for the first time. Further, a bis-thioether ferrous H93G model for bis-methionine ligation, as found in
Pseudomonas aeruginosa
bacterioferritin heme protein, is described. Magnetic CD spectroscopy has been used due to its established ability in axial ligand identification. The magnetic CD spectra of the H93G complexes have been compared with those of ferrous H175C/D235L cytochrome
c
peroxidase to show that its proximal ligand is neutral cysteine. We had previously reported this cytochrome
c
peroxidase mutant to be cysteinate-ligated in the ferric state, but the ferrous ligand was undetermined. The spectral properties of ferrous liver microsomal cytochrome P420 (inactive P450) are also consistent with thiol ligation. This study establishes that neutral cysteine can serve as a ligand in ferrous heme iron proteins, and that ferric cysteinate-ligated heme proteins that fail to retain such ligation on reduction may simply be ligated by neutral cysteine.