bluuebunny/crossref_metadata_2025 huggingface dataset:
Yeast actin: polymerization kinetic studies of wild type and a poorly polymerizing mutant.
Authors: Buzan J M , Frieden C
Published: July 2002

Wild-type actin and a mutant actin were isolated from yeast (Saccharomyces cerevisiae) and the polymerization properties were examined at pH 8.0 and 20 degrees C. The polymerization reaction was followed either by an increase in pyrene-labeled actin fluorescence or by a decrease in intrinsic fluorescence in the absence of pyrene-labeled actin. While similar to the properties of skeletal muscle actin, there are several important differences between the wild-type yeast and muscle actins. First, yeast actin polymerizes more rapidly than muscle actin under the same experimental conditions. The difference in rates may result from a difference in the steps involving formation of the nucleating species. Second, as measured with pyrene-labeled yeast actin, but not with intrinsic fluorescence, there is an overshoot in the fluorescence that has not been observed with skeletal muscle actin under the same conditions. Third, in order to simulate the polymerization process of wild-type yeast actin it is necessary to assume some fragmentation of the filaments. Finally, gelsolin inhibits polymerization of yeast actin but is known to accelerate the polymerization of muscle actin. A mutant actin (R177A/D179A) has also been isolated and studied. The mutations are at a region of contact between monomers across the long axis of the actin filament. This mutant polymerizes more slowly than wild type and filaments do not appear to fragment during polymerization. Elongation rates of the wild type and the mutant differ by only about 3-fold, and the slower polymerization of the mutant appears to result primarily from poorer nucleation.
Mapping leaf surface landscapes.
Authors: Mechaber W L , Marshall D B , Mechaber R A , Jobe R T , Chew F S
Published: July 2002

Leaf surfaces provide the ecologically relevant landscapes to those organisms that encounter or colonize the leaf surface. Leaf surface topography directly affects microhabitat availability for colonizing microbes, microhabitat quality and acceptability for insects, and the efficacy of agricultural spray applications. Prior detailed mechanistic studies that examined particular fungi-plant and pollinator-plant interactions have demonstrated the importance of plant surface topography or roughness in determining the outcome of the interactions. Until now, however, it has not been possible to measure accurately the topography--i.e., the three-dimensional structure--of such leaf surfaces or to record precise changes in patterns of leaf surface elevation over time. Using contact mode atomic force microscopy, we measured three-dimensional coordinates of upper leaf surfaces of Vaccinium macrocarpon (cranberry), a perennial plant, on leaves of two age classes. We then produced topographic maps of these leaf surfaces, which revealed striking differences between age classes of leaves: old leaves have much rougher surfaces than those of young leaves. Atomic force microscope measurements were analyzed by lag (1) autocorrelation estimates of leaf surfaces by age class. We suggest that the changes in topography result from removal of epicuticular lipids and that the changes in leaf surface topography influence phylloplane ecology. Visualizing and mapping leaf surfaces permit detailed investigations into leaf surface-mediated phenomena, improving our understanding of phylloplane interactions.
Bc1-2 protects mice against fatal alphavirus encephalitis.
Authors: Levine B , Goldman J E , Jiang H H , Griffin D E , Hardwick J M
Published: July 2002

Virus-induced apoptosis has been well characterized in vitro, but the role of apoptosis in viral pathogenesis is not well understood. The suicide of a cell in response to viral infection is postulated to be an important host defense for the organism, leading to a reduction in its total viral burden. However, virus-induced death of nonregenerating cells in the central nervous system may be detrimental to the host. Therefore, to investigate the role of apoptosis in the pathogenesis of fatal encephalitis, we constructed a recombinant alphavirus chimera that expresses the antiapoptotic gene, bcl-2, in virally infected neural cells. Infection of neonatal mice with the alphavirus chimera expressing human bcl-2 [Sindbis virus (SIN)/bcl-2] resulted in a significantly lower mortality rate (7.5%) as compared with infection with control chimeric viruses containing a chloramphenicol acetyltransferase (CAT) reporter gene (SIN/CAT) (78.1%) or bcl-2 containing a premature stop codon (SIN/bcl-2stop) (72.1%) (P < 0.001). Viral titers were reduced 5-fold 1 day after infection and 10-fold 6 days after infection in the brains of SIN/bcl-2-infected mice as compared to SIN/CAT or SIN/bcl-2stop-infected mice. In situ end labeling to detect apoptotic nuclei demonstrated a reduction in the number of foci of apoptotic cells in the brains of mice infected with SIN/bcl-2 as compared with SIN/bcl-2stop. The reduction in apoptosis was associated with a reduction in the number of foci of cells expressing alphavirus RNA. Thus, the antiapoptotic gene, bcl-2, suppresses viral replication and protects against a lethal viral disease, suggesting an interaction between cellular genetic control of viral replication and cell death.
Expression of corticotropin-releasing factor in inflamed tissue is required for intrinsic peripheral opioid analgesia.
Authors: Schafer M , Mousa S A , Zhang Q , Carter L , Stein C
Published: July 2002

Immune cell-derived opioid peptides can activate opioid receptors on peripheral sensory nerves to inhibit inflammatory pain. The intrinsic mechanisms triggering this neuroimmune interaction are unknown. This study investigates the involvement of endogenous corticotropin-releasing factor (CRF) and interleukin-1beta (IL-1). A specific stress paradigm, cold water swim (CWS), produces potent opioid receptor-specific antinociception in inflamed paws of rats. This effect is dose-dependently attenuated by intraplantar but not by intravenous alpha-helical CRF. IL-1 receptor antagonist is ineffective. Similarly, local injection of antiserum against CRF, but not to IL-1, dose-dependently reverses this effect. Intravenous anti-CRF is only inhibitory at 10(4)-fold higher concentrations and intravenous CRF does not produce analgesia. Pretreatment of inflamed paws with an 18-mer 3'-3'-end inverted CRF-antisense oligodeoxynucleotide abolishes CWS-induced antinociception. The same treatment significantly reduces the amount of CRF extracted from inflamed paws and the number of CRF-immunostained cells without affecting gross inflammatory signs. A mismatch oligodeoxynucleotide alters neither the CWS effect nor CRF immunoreactivity. These findings identify locally expressed CRF as the predominant agent to trigger opioid release within inflamed tissue. Endogenous IL-1, circulating CRF or antiinflammatory effects, are not involved. Thus, an intact immune system plays an essential role in pain control, which is important for the understanding of pain in immunosuppressed patients with cancer or AIDS.
Molecular cloning of violaxanthin de-epoxidase from romaine lettuce and expression in Escherichia coli.
Authors: Bugos R C , Yamamoto H Y
Published: July 2002

Plants need to avoid or dissipate excess light energy to protect photosystem II (PSII) from photoinhibitory damage. Higher plants have a conserved system that dissipates excess energy as heat in the light-harvesting complexes of PSII that depends on the transthylakoid delta pH and violaxanthin de-epoxidase (VDE) activity. To our knowledge, we report the first cloning of a cDNA encoding VDE and expression of functional enzyme in Escherichia coli. VDE is nuclear encoded and has a transit peptide with characteristic features of other lumen-localized proteins. The cDNA encodes a putative polypeptide of 473 aa with a calculated molecular mass of 54,447 Da. Cleavage of the transit peptide results in a mature putative polypeptide of 348 aa with a calculated molecular mass of 39,929 Da, close to the apparent mass of the purified enzyme (43 kDa). The protein has three interesting domains including (i) a cysteine-rich region, (ii) a lipocalin signature, and (iii) a highly charged region. The E. coli expressed enzyme de-epoxidizes violaxanthin sequentially to antheraxanthin and zeaxanthin, and is inhibited by dithiothreitol, similar to VDE purified from chloroplasts. This confirms that the cDNA encodes an authentic VDE of a higher plant and is unequivocal evidence that the same enzyme catalyzes the two-step mono de-epoxidation reaction. The cloning of VDE opens new opportunities for examining the function and evolution of the xanthophyll cycle, and possibly enhancing light-stress tolerance of plants.
Identifying functional domains within terpene cyclases using a domain-swapping strategy.
Authors: Back K , Chappell J
Published: July 2002

Cyclic terpenes and terpenoids are found throughout nature. They comprise an especially important class of compounds from plants that mediate plant- environment interactions, and they serve as pharmaceutical agents with antimicrobial and anti-tumor activities. Molecular comparisons of several terpene cyclases, the key enzymes responsible for the multistep cyclization of C10, C15, and C20 allylic diphosphate substrates, have revealed a striking level of sequence similarity and conservation of exon position and size within the genes. Functional domains responsible for a terminal enzymatic step were identified by swapping regions approximating exons between a Nicotiana tabacum 5-epi-aristolochene synthase (TEAS) gene and a Hyoscyamus muticus vetispiradiene synthase (HVS) gene and by characterization of the resulting chimeric enzymes expressed in bacteria. While exon 4 of the TEAS gene conferred specificity for the predominant reaction products of the tobacco enzyme, exon 6 of the HVS gene conferred specificity for the predominant reaction products of the Hyoscyamus enzyme. Combining these two functional domains of the TEAS and HVS genes resulted in a novel enzyme capable of synthesizing reaction products reflective of both parent enzymes. The relative ratio of the TEAS and HVS reaction products was also influenced by the source of exon 5 present in the new chimeric enzymes. The association of catalytic activities with conserved but separate exonic domains suggests a general means for generating additional novel terpene cyclases.
The cyclin-dependent kinase inhibitor p40SIC1 imposes the requirement for Cln G1 cyclin function at Start.
Authors: Tyers M
Published: July 2002

In yeast, commitment to cell division (Start) is catalyzed by activation of the Cdc28 protein kinase in late G1 phase by the Cln1, Cln2, and Cln3 G1 cyclins. The Clns are essential, rate-limiting activators of Start because cells lacking Cln function (referred to as cln-) arrest at Start and because CLN dosage modulates the timing of Start. At or shortly after Start, the development of B-type cyclin Clb-Cdc28 kinase activity and initiation of DNA replication requires the destruction of p40SIC1, a specific inhibitor of the Clb-Cdc28 kinases. I report here that cln cells are rendered viable by deletion of SIC1. Conversely, in cln1 cln2 cells, which have low CLN activity, modest increases in SIC1 gene dosage cause inviability. Deletion of SIC1 does not cause a general bypass of Start since (cln-)sic1 cells remain sensitive to mating pheromone-induced arrest. Far1, a pheromone-activated inhibitor of Cln-Cdc28 kinases, is dispensable for arrest of (cln-)sic1 cells by pheromone, implying the existence of an alternate Far1-independent arrest pathway. These observations define a pheromone-sensitive activity able to catalyze Start only in the absence of p40SIC1. The existence of this activity means that the B-type cyclin inhibitor p40SIC1 imposes the requirement for Cln function at Start.
Changes in voltage activation, Cs+ sensitivity, and ion permeability in H5 mutants of the plant K+ channel KAT1.
Authors: Becker D , Dreyer I , Hoth S , Reid J D , Busch H , Lehnen M , Palme K , Hedrich R
Published: July 2002

KAT1 is a voltage-dependent inward rectifying K+ channel cloned from the higher plant Arabidopsis thaliana [Anderson, J. A., Huprikar, S. S., Kochian, L. V., Lucas, W. J. & Gaber, R. F. (1992) Proc. Natl. Acad. Sci. USA 89, 3736-3740]. It is related to the Shaker superfamily of K+ channels characterized by six transmembrane spanning domains (S1-S6) and a putative pore-forming region between S5 and S6 (H5). The 115 region between Pro-247 and Pro-271 in KAT1 contains 14 additional amino acids when compared with Shaker [Aldrich, R. W. (1993) Nature (London) 362, 107-108]. We studied various point mutations introduced into H5 to determine whether voltage-dependent plant and animal K+ channels share similar pore structures. Through heterologous expression in Xenopus oocytes and voltage-clamp analysis combined with phenotypic analysis involving a potassium transport-defective Saccharomyces cerevisiae strain, we investigated the selectivity filter of the mutants and their susceptibility toward inhibition by cesium and calcium ions. With respect to electrophysiological properties, KAT1 mutants segregated into three groups: (i) wild-type-like channels, (ii) channels modified in selectivity and Cs+ or Ca2+ sensitivity, and (iii) a group that was additionally affected in its voltage dependence. Despite the additional 14 amino acids in H5, this motif in KAT1 is also involved in the formation of the ion-conducting pore because amino acid substitutions at Leu-251, Thr-256, Thr-259, and Thr-260 resulted in functional channels with modified ionic selectivity and inhibition. Creation of Ca2+ sensitivity and an increased susceptibility to Cs+ block through mutations within the narrow pore might indicate that both blockers move deeply into the channel. Furthermore, mutations close to the rim of the pore affecting the half-activation potential (U1/2) indicate that amino acids within the pore either interact with the voltage sensor or ion permeation feeds back on gating.
Mutant conformation of p53 translated in vitro or in vivo requires functional HSP90.
Authors: Blagosklonny M V , Toretsky J , Bohen S , Neckers L
Published: July 2002

The p53 mutant, 143ala, was translated in vitro in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE). In RRL, p53-143ala protein of both mutant and wild-type conformation, as detected immunologically with conformation-specific antibodies, was translated. The chaperone protein HSP90, present in RRL, was found to coprecipitate only with the mutated conformation of p53. Geldanamycin, shown previously to bind to HSP90 and destabilize its association with other proteins, decreased the amount of immunologically detectable mutated p53 and increased the amount of detectable wild-type protein, without affecting the total translation of p53. When translated in WGE, known to contain functionally deficient HSP90, p53-143ala produced p53 protein, which was not recognized by a mutated conformation-specific antibody. In contrast, the synthesis of conformationally detectable wild-type p53 in this system was not compromised. Reconstitution of HSP90 function in WGE permitted synthesis of conformationally detectable mutated p53, and this was abrogated by geldanamycin. Finally, when p53-143ala was stably tansfected into yeast engineered to be defective for HSP90 function, conformational recognition of mutated p53 was impaired. When stable transfectants of p53-143ala were prepared in yeast expressing wild-type HSP90, conformational recognition of mutated p53 was antagonized by macbecin I, a geldanamycin analog also known to bind HSP90. Taken together, these data demonstrate a role for HSP90 in the achievement and/or stabilization of the mutated conformation of p53-143ala. Furthermore, we show that the mutated conformation of p53 can be pharmacologically antagonized by drugs targeting HSP90.
Regulation of SOS mutagenesis by proteolysis.
Authors: Frank E G , Ennis D G , Gonzalez M , Levine A S , Woodgate R
Published: July 2002

DNA damage-inducible mutagenesis in Escherichia coli is largely dependent upon the activity of the UmuD (UmuD') and UmuC proteins. The intracellular level of these proteins is tightly regulated at both the transcriptional and the posttranslational levels. Such regulation presumably allows cells to deal with DNA damage via error-free repair pathways before being committed to error-prone pathways. We have recently discovered that as part of this elaborate regulation, both the UmuD and the UmuC proteins are rapidly degraded in vivo. We report here that the enzyme responsible for their degradation is the ATP-dependent serine protease, Lon. In contrast, UmuD' (the posttranslational product and mutagenically active form of UmuD) is degraded at a much reduced rate by Lon, but is instead rapidly degraded by another ATP-dependent protease, ClpXP. Interestingly, UmuD' is rapidly degraded by ClpXP only when it is in a heterodimeric complex with UmuD. Formation of UmuD/UmuD' heterodimers in preference to UmuD' homodimers therefore targets UmuD' protein for proteolysis. Such a mechanism allows cells to reduce the intracellular levels of the mutagenically active Umu proteins and thereby return to a resting state once error-prone DNA repair has occurred. The apparent half-life of the heterodimeric UmuD/D' complex is greatly increased in the clpX::Kan and clpP::Kan strains and these strains are correspondingly rendered virtually UV non-mutable. We believe that these phenotypes are consistent with the suggestion that while the UmuD/D' heterodimer is mutagenically inactive, it still retains the ability to interact with UmuC, and thereby precludes the formation of the mutagenically active UmuD'2C complex.
Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.
Authors: Kaye K M , Devergne O , Harada J N , Izumi K M , Yalamanchili R , Kieff E , Mosialos G
Published: July 2002

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-kappa B activation, we have evaluated the role of TRAF2 in LMP1-mediated NF-kappa B activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF/ delta 6-86) inhibits NF-kappa B activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2 delta 6-86 inhibits NF-kappa B activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-kappa B activation through the last 55 amino acids of the CT by less than 40%. A TRAF interacting protein, TANK, inhibits NF-kappa B activation by more than 70% from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-kappa B activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-kappa B activation by the last 55 amino acids of the LMP1 CT.
The role of apolipoprotein AI domains in lipid binding
Authors: Davidson W. Sean , Hazlett Theodore , Mantulin William W. , Jonas Ana
Published: July 2002

Apolipoprotein AI (apoAI) is the principal protein constituent of
high density lipoproteins and it plays a key role in human cholesterol
homeostasis; however, the structure of apoAI is not clearly understood.
To test the hypothesis that apoAI is organized into domains, three
deletion mutants of human apoAI expressed in
Escherichia
coli
were studied in solution and in reconstituted high density
lipoprotein particles. Each mutant lacked one of three specific regions
that together encompass almost the entire 243 aa sequence of native
apoAI (
apoAI Δ44-126
,
apoAI Δ139-170
,
and
apoAI Δ190-243
). Circular dichroism spectroscopy
showed that the α-helical content of lipid-free apoAI Δ44-126 was
27% while the other mutants and native apoAI averaged 55 ± 2%,
suggesting that the missing N-terminal portion contains most of the
α-helical structure of lipid-free apoAI. ApoAI Δ44-126 exhibited
the largest increase in α-helix upon lipid binding (125% increase
versus an average of 25% for the others), confirming the importance of
the C-terminal half of apoAI in lipid binding. Denaturation studies
showed that the N-terminal half of apoAI is primarily responsible for
α-helix stability in the lipid-free state, whereas the C terminus is
required for α-helix stability when lipid-bound. We conclude that the
N-terminal half (aa 44–126) of apoAI is responsible for most of the
α-helical structure and the marginal stability of lipid-free apoAI
while the C terminus (aa 139–243) is less organized. The increase in
α-helical content observed when native apoAI binds lipid results from
the formation of α-helix primarily in the C-terminal half of the
molecule.
Blockade of plasma membrane calcium pumping ATPase isoform I impairs nerve growth factor-induced neurite extension in pheochromocytoma cells
Authors: Brandt Paul C. , Sisken Jesse E. , Neve Rachael L. , Vanaman Thomas C.
Published: July 2002

Numerous lines of evidence indicate that calcium signaling is
essential for nerve growth factor (NGF)-directed neuronal cell
differentiation. We report here that blocking production of the plasma
membrane Ca
2+
-ATPase isoform 1 (PMCA1) in PC6 cells with
antisense RNA impairs their ability to extend normal neurites in
response to NGF. This result does not appear to be due to loss in NGF
signaling as NGF-dependent tyrosine phosphorylation of erk1 and erk2,
as well as expression of the NGF-inducible immediate early gene,
NGFI-A, was observed in these cells. Resting cytosolic calcium levels
did not appear to be altered in the antisense transfectants and release
of calcium from internal bradykinin-sensitive calcium pools was
unchanged. However, the rate of removal of free cytosolic calcium
following this release was reduced in the antisense-transfected cells
compared with controls. It is concluded that PMCA1 is involved in
neurite extension and/or stabilization either through moderation of
local calcium levels, or by some other mechanism.
The change in hydrogen bond strength accompanying charge rearrangement: Implications for enzymatic catalysis
Authors: Shan Shu-ou , Herschlag Daniel
Published: July 2002

The equilibrium for formation of the intramolecular hydrogen bond
(K
HB
) in a series of substituted salicylate monoanions was
investigated as a function of ΔpK
a
, the difference
between the pK
a
values of the hydrogen bond donor and
acceptor, in both water and dimethyl sulfoxide. The dependence of log
K
HB
upon ΔpK
a
is linear in both solvents, but
is steeper in dimethyl sulfoxide (slope = 0.73) than in water
(slope = 0.05). Thus, hydrogen bond strength can undergo
substantially larger increases in nonaqueous media than aqueous
solutions as the charge density on the donor or acceptor atom
increases. These results support a general mechanism for enzymatic
catalysis, in which hydrogen bonding to a substrate is strengthened as
charge rearranges in going from the ground state to the transition
state; the strengthening of the hydrogen bond would be greater in a
nonaqueous enzymatic active site than in water, thus providing a rate
enhancement for an enzymatic reaction relative to the solution
reaction. We suggest that binding energy of an enzyme is used to fix
the substrate in the low-dielectric active site, where the
strengthening of the hydrogen bond in the course of a reaction is
increased.
The multiple phenotypes of allosteric receptor mutants.
Authors: Galzi J L , Edelstein S J , Changeux J
Published: July 2002

Channel-linked neurotransmitter receptors are membrane-bound heterooligomers made up of distinct, although homologous, subunits. They mediate chemo-electrical signal transduction and its regulation via interconversion between multiple conformations that exhibit distinct pharmacological properties and biological activities. The large diversity of functional properties and the widely pleiotropic phenotypes, which arise from point mutations in their subunits (or from subunit substitutions), are interpreted in terms of an allosteric model that incorporates multiple discrete conformational states. The model predicts that three main categories of phenotypes may result from point mutations, altering selectively one (or more) of the following features: (i) the properties of individual binding sites (K phenotype), (ii) the biological activity of the ion channel (gamma phenotype) of individual conformations, or (iii) the isomerization constants between receptor conformations (L phenotype). Several nicotinic acetylcholine and glycine receptor mutants with complex phenotypes are quantitatively analyzed in terms of the model, and the analogies among phenotypes are discussed.
The effects of sequence context on DNA curvature.
Authors: Dlakić M , Harrington R E
Published: July 2002

Recent experiments have exposed significant discrepancies between experimental data and predictive models for DNA structure. These results strongly suggest that DNA structural parameters incorporated in the models are not always sufficient to account for the influence of sequence context and of specific ion effects. In an attempt to evaluate these two effects, we have investigated repetitive DNA sequences with the sequence motif GAGAG.CTCTC located in different helical phasing arrangements with respect to poly(A) tracts and GGGCCC.GGGCCC sequence motifs. Methods used are ligase-mediated cyclization and gel mobility experiments along with DNase I cutting and chemical probe studies. The results provide new evidence for curvature in poly(A) tracts. They also show that the sequence context in which bending and flexible sequence elements are found is an important aspect of sequence-dependent DNA conformation. Although dinucleotide models generally have good predictive power, this work demonstrates that in some instances sequence elements larger than the dinucleotide must be taken into account, and hence it provides a starting point for the appropriate modification and refinement of existing structural models for DNA.
Constitutive signaling by the phototaxis receptor sensory rhodopsin II from disruption of its protonated Schiff base–Asp-73 interhelical salt bridge
Authors: Spudich Elena N. , Zhang Weisheng , Alam Maqsudul , Spudich John L.
Published: July 2002

Sensory rhodopsin II (SRII) is a repellent phototaxis receptor in the archaeon
Halobacterium salinarum
, similar to visual pigments in its seven-helix structure and linkage of retinal to the protein by a protonated Schiff base in helix G. Asp-73 in helix C is shown by spectroscopic analysis to be a counterion to the protonated Schiff base in the unphotolyzed SRII and to be the proton acceptor from the Schiff base during photoconversion to the receptor signaling state. Coexpression of the genes encoding mutated SRII with Asn substituted for Asp-73 (D73N) and the SRII transducer HtrII in
H. salinarum
cells results in a 3-fold higher swimming reversal frequency accompanied by demethylation of HtrII in the dark, showing that D73N SRII produces repellent signals in its unphotostimulated state. Analogous constitutive signaling has been shown to be produced by the similar neutral residue substitution of the Schiff base counterion and proton acceptor Glu-113 in human rod rhodopsin. The interpretation for both seven-helix receptors is that light activation of the wild-type protein is caused primarily by photoisomerization-induced transfer of the Schiff base proton on helix G to its primary carboxylate counterion on helix C. Therefore receptor activation by helix C–G salt-bridge disruption in the photoactive site is a general mechanism in retinylidene proteins spanning the vast evolutionary distance between archaea and humans.
Phenotypic alterations in insulin-deficient mutant mice
Authors: Duvillié Bertrand , Cordonnier Nathalie , Deltour Louise , Dandoy-Dron Françoise , Itier Jean-Michel , Monthioux Eliane , Jami Jacques , Joshi Rajiv L. , Bucchini Danielle
Published: July 2002

Two mouse insulin genes,
Ins1
and
Ins2
, were disrupted and
lacZ
was inserted at the
Ins2
locus by gene targeting. Double nullizygous insulin-deficient pups were growth-retarded. They did not show any glycosuria at birth but soon after suckling developed diabetes mellitus with ketoacidosis and liver steatosis and died within 48 h. Interestingly, insulin deficiency did not preclude pancreas organogenesis and the appearance of the various cell types of the endocrine pancreas. The presence of
lacZ
expressing β cells and glucagon-positive α cells was demonstrated by cytochemistry and immunocytochemistry. Reverse transcription-coupled PCR analysis showed that somatostatin and pancreatic polypeptide mRNAs were present, although at reduced levels, accounting for the presence also of δ and pancreatic polypeptide cells, respectively. Morphometric analysis revealed enlarged islets of Langherans in the pancreas from insulin-deficient pups, suggesting that insulin might function as a negative regulator of islet cell growth. Whether insulin controls the growth of specific islet cell types and the molecular basis for this action remain to be elucidated.
Folding funnels and energy landscapes of larger proteins within the capillarity approximation
Authors: Wolynes Peter G.
Published: July 2002

The characterization of protein-folding kinetics with increasing chain length under various thermodynamic conditions is addressed using the capillarity picture in which distinct spatial regions of the protein are imagined to be folded or trapped and separated by interfaces. The quantitative capillarity theory is based on the nucleation theory of first-order transitions and the droplet analysis of glasses and random magnets. The concepts of folding funnels and rugged energy landscapes are shown to be applicable in the large size limit just as for smaller proteins. An ideal asymptotic free-energy profile as a function of a reaction coordinate measuring progress down the funnel is shown to be quite broad. This renders traditional transition state theory generally inapplicable but allows a diffusive picture with a transition-state region to be used. The analysis unifies several scaling arguments proposed earlier. The importance of fluctuational fine structure both to the free-energy profile and to the glassy dynamics is highlighted. The fluctuation effects lead to a very broad trapping-time distribution. Considerations necessary for understanding the crossover between the mean field and capillarity pictures of the energy landscapes are discussed. A variety of mechanisms that may roughen the interfaces and may lead to a complex structure of the transition-state ensemble are proposed.
Three-dimensional diffuse x-ray scattering from crystals of
Staphylococcal
nuclease
Authors: Wall Michael E. , Ealick Steven E. , Gruner Sol M.
Published: July 2002

We have developed methods for obtaining and characterizing three-dimensional maps of the reciprocal-space distribution of diffuse x-ray scattering from protein crystals, and have used the methods to study the nature of disorder in crystals of
Staphylococcal
nuclease. Experimentally obtained maps are 99.5% complete in the reciprocal-space resolution range of 10 Å–2.5 Å, show symmetry consistent with the P4
1
space group of the unit cell, and are highly reproducible. Quantitative comparisons of the data with three-dimensional simulations imply liquid-like motions of the protein [Caspar, D. L. D., Clarage, J., Salunke, D. M. & Clarage, M. (1988)
Nature (London)
332, 659–662], with a correlation length of 10 Å and a root-mean-square displacement of 0.36 Å.
d
-Ala-
d
-Ala ligases from glycopeptide antibiotic-producing organisms are highly homologous to the enterococcal vancomycin-resistance ligases VanA and VanB
Authors: Marshall C. G. , Broadhead G. , Leskiw B. K. , Wright G. D.
Published: July 2002

The crisis in antibiotic resistance has resulted in an increasing fear of the emergence of untreatable organisms. Resistance to the glycopeptide antibiotic vancomycin in the enterococci, and the spread of these pathogens throughout the environment, has shown that this scenario is a matter of fact rather than fiction. The basis for vancomycin resistance is the manufacture of the depsipeptide
d
-Ala-
d
-lactate, which is incorporated into the peptidoglycan cell wall in place of the vancomycin target
d
-Ala-
d
-Ala. Pivotal to the resistance mechanism is the production of a
d
-Ala-
d
-Ala ligase capable of ester formation. Two highly efficient depsipeptide ligases have been cloned from vancomycin-resistant enterococci: VanA and VanB. These ligases show high amino acid sequence similarity to each other (≈75%), but less so to other
d
-Ala-
d
-X ligases (<30%). We have cloned
ddls
from two glycopeptide-producing organisms, the vancomycin producer
Amycolatopsis orientalis
and the A47934 producer
Streptomyces toyocaensis
. These ligases show strong predicted amino acid homology to VanA and VanB (>60%) but not to other
d
-Ala-
d
-X ligases (<35%). The
d
-Ala-
d
-Ala ligase from
S. toyocaensis
shows
d
-Ala-
d
-lactate synthase activity in cell-free extracts of
S. lividans
transformed with the
ddl
gene and confirms the predicted enzymatic activity. These results imply a close evolutionary relationship between resistance mechanisms in the clinics and in drug-producing bacteria.
Activation of heteromeric G protein-gated inward rectifier K
+
channels overexpressed by adenovirus gene transfer inhibits the excitability of hippocampal neurons
Authors: Ehrengruber Markus U. , Doupnik Craig A. , Xu Youfeng , Garvey Justine , Jasek Mark C. , Lester Henry A. , Davidson Norman
Published: July 2002

G protein-gated inward rectifier K
+
channel subunits 1–4 (GIRK1–4) have been cloned from neuronal and atrial tissue and function as heterotetramers. To examine the inhibition of neuronal excitation by GIRKs, we overexpressed GIRKs in cultured hippocampal neurons from 18 day rat embryos, which normally lack or show low amounts of GIRK protein and currents. Adenoviral recombinants containing the cDNAs for GIRK1, GIRK2, GIRK4, and the serotonin 1A receptor were constructed. Typical GIRK currents could be activated by endogenous GABA
B
, serotonin 5-HT
1A
, and adenosine A1 receptors in neurons coinfected with GIRK1+2 or GIRK1+4. Under current clamp, GIRK activation increased the cell membrane conductance by 1- to 2-fold, hyperpolarized the cell by 11–14 mV, and inhibited action potential firing by increasing the threshold current for firing by 2- to 3-fold. These effects were not found in non- and mock-infected neurons, and were similar to the effects of muscarinic stimulation of native GIRK currents in atrial myocytes. Two inhibitory effects of GIRK activation, hyperpolarization and diminution of depolarizing pulses, were simulated from the experimental data. These inhibitory effects are physiologically important in the voltage range between the resting membrane potential and the potential where voltage-gated Na
+
and K
+
currents are activated; that is where GIRK currents are
outward
.
Elevated free nitrotyrosine levels, but not protein-bound nitrotyrosine or hydroxyl radicals, throughout amyotrophic lateral sclerosis (ALS)-like disease implicate tyrosine nitration as an aberrant
in vivo
property of one familial ALS-linked superoxide dismutase 1 mutant
Authors: Bruijn L. I. , Beal M. F. , Becher M. W. , Schulz J. B. , Wong P. C. , Price D. L. , Cleveland D. W.
Published: July 2002

Mutations in superoxide dismutase 1 (SOD1; EC
1.15.1.1
) are responsible for a proportion of familial amyotrophic lateral sclerosis (ALS) through acquisition of an as-yet-unidentified toxic property or properties. Two proposed possibilities are that toxicity may arise from imperfectly folded mutant SOD1 catalyzing the nitration of tyrosines [Beckman, J. S., Carson, M., Smith, C. D. & Koppenol, W. H. (1993)
Nature (London)
364, 584] through use of peroxynitrite or from peroxidation arising from elevated production of hydroxyl radicals through use of hydrogen peroxide as a substrate [Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Gralla, E. D., Roe, J. A., Valentine, J. S. & Bredesen, D. E. (1996)
Science
271, 515–518]. To test these possibilities, levels of nitrotyrosine and markers for hydroxyl radical formation were measured in two lines of transgenic mice that develop progressive motor neuron disease from expressing human familial ALS-linked SOD1 mutation G37R. Relative to normal mice or mice expressing high levels of wild-type human SOD1, 3-nitrotyrosine levels were elevated by 2- to 3-fold in spinal cords coincident with the earliest pathological abnormalities and remained elevated in spinal cord throughout progression of disease. However, no increases in protein-bound nitrotyrosine were found during any stage of SOD1-mutant-mediated disease in mice or at end stage of sporadic or SOD1-mediated familial human ALS. When salicylate trapping of hydroxyl radicals and measurement of levels of malondialdehyde were used, there was no evidence throughout disease progression in mice for enhanced production of hydroxyl radicals or lipid peroxidation, respectively. The presence of elevated nitrotyrosine levels beginning at the earliest stages of cellular pathology and continuing throughout progression of disease demonstrates that tyrosine nitration is one
in vivo
aberrant property of this ALS-linked SOD1 mutant.
The subunits MECL-1 and LMP2 are mutually required for incorporation into the 20S proteasome
Authors: Groettrup Marcus , Standera Sybille , Stohwasser Ralf , Kloetzel Peter M.
Published: July 2002

Processing of antigens for presentation by major histocompatibility complex (MHC) class I molecules requires the activity of the proteasome. The 20S proteasome complex is composed of 14 different subunits, 2 of which can be substituted by the interferon γ (IFN-γ)-inducible and MHC-encoded subunits LMP2 and LMP7 (low molecular mass poylpeptides 2 and 7). A third subunit, MECL-1, is inducible by IFN-γ but is encoded outside the MHC. Here we show by cotransfection experiments that the incorporation of MECL-1 into the 20S proteasome is directly dependent on the expression of LMP2 but independent of LMP7. Conversely, the uptake of LMP2 is strongly enhanced by MECL-1 expression. The expression of MECL-1 caused a replacement of the homologous subunit Z in the 20S proteasome complex. LMP2 is required for MECL-1 incorporation at the level of proteasome precursor formation that guarantees the concerted incorporation of two IFN-γ-inducible proteasome subunits encoded inside and outside the MHC. The obligatory coincorporation of MECL-1 and LMP2 is an important parameter for the interpretation of results obtained with LMP2-deficient cell lines and mice as well as for the design of experiments addressing the function of MECL-1 in antigen presentation.
Desynchronization of cells on the developmental path triggers the formation of spiral waves of cAMP during
Dictyostelium
aggregation
Authors: Lauzeral Jacques , Halloy José , Goldbeter Albert
Published: July 2002

Whereas it is relatively easy to account for the formation of concentric (target) waves of cAMP in the course of
Dictyostelium discoideum
aggregation after starvation, the origin of spiral waves remains obscure. We investigate a physiologically plausible mechanism for the spontaneous formation of spiral waves of cAMP in
D. discoideum
. The scenario relies on the developmental path associated with the continuous changes in the activity of enzymes such as adenylate cyclase and phosphodiesterase observed during the hours that follow starvation. These changes bring the cells successively from a nonexcitable state to an excitable state in which they relay suprathreshold cAMP pulses, and then to autonomous oscillations of cAMP, before the system returns to an excitable state. By analyzing a model for cAMP signaling based on receptor desensitization, we show that the desynchronization of cells on this developmental path triggers the formation of fully developed spirals of cAMP. Developmental paths that do not correspond to the sequence of dynamic transitions no relay-relay-oscillations-relay are less able or fail to give rise to the formation of spirals.
Phylogeny of mRNA capping enzymes
Authors: Wang Shuang Ping , Deng Liang , Ho C. Kiong , Shuman Stewart
Published: July 2002

The m
7
GpppN cap structure of eukaryotic mRNA is formed cotranscriptionally by the sequential action of three enzymes: RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase. A multifunctional polypeptide containing all three active sites is encoded by vaccinia virus. In contrast, fungi and
Chlorella
virus encode monofunctional guanylyltransferase polypeptides that lack triphosphatase and methyltransferase activities. Transguanylylation is a two-stage reaction involving a covalent enzyme-GMP intermediate. The active site is composed of six protein motifs that are conserved in order and spacing among yeast and DNA virus capping enzymes. We performed a structure–function analysis of the six motifs by targeted mutagenesis of Ceg1, the
Saccharomyces cerevisiae
guanylyltransferase. Essential acidic, basic, and aromatic functional groups were identified. The structural basis for covalent catalysis was illuminated by comparing the mutational results with the crystal structure of the
Chlorella
virus capping enzyme. The results also allowed us to identify the capping enzyme of
Caenorhabditis elegans
. The 573-amino acid nematode protein consists of a C-terminal guanylyltransferase domain, which is homologous to Ceg1 and is strictly conserved with respect to all 16 amino acids that are essential for Ceg1 function, and an N-terminal phosphatase domain that bears no resemblance to the vaccinia triphosphatase domain but, instead, has strong similarity to the superfamily of protein phosphatases that act via a covalent phosphocysteine intermediate.
Chloroplast DNA footprints of postglacial recolonization by oaks
Authors: Petit Rémy J. , Pineau Emmanuel , Demesure Brigitte , Bacilieri Roberto , Ducousso Alexis , Kremer Antoine
Published: July 2002

Recolonization of Europe by forest tree species after the last glaciation is well documented in the fossil pollen record. This spread may have been achieved at low densities by rare events of long-distance dispersal, rather than by a compact wave of advance, generating a patchy genetic structure through founder effects. In long-lived oak species, this structure could still be discernible by using maternally transmitted genetic markers. To test this hypothesis, a fine-scale study of chloroplast DNA (cpDNA) variability of two sympatric oak species was carried out in western France. The distributions of six cpDNA length variants were analyzed at 188 localities over a 200 × 300 km area. A cpDNA map was obtained by applying geostatistics methods to the complete data set. Patches of several hundred square kilometers exist which are virtually fixed for a single haplotype for both oak species. This local systematic interspecific sharing of the maternal genome strongly suggests that long-distance seed dispersal events followed by interspecific exchanges were involved at the time of colonization, about 10,000 years ago.
Peptide analogue studies of the hypothalamic neuropeptide Y receptor mediating pituitary adrenocorticotrophic hormone release
Authors: Small C. J. , Morgan D. G. A. , Meeran K. , Heath M. M. , Gunn I. , Edwards C. M. B. , Gardiner J. , Taylor G. M. , Hurley J. D. , Rossi M. , Goldstone A. P. , O’Shea D. , Smith D. M. , Ghatei M. A. , Bloom S. R.
Published: July 2002

Hypothalamic neuropeptide Y (NPY) is thought to be important in the regulation of feeding and also in the release of Adrenocorticotrophic hormone (ACTH). Intracerebroventricular administration of NPY to male rats significantly increased plasma ACTH 10 min after injection and stimulated 2-h food intake. A series of analogues of NPY that have a greatly reduced affinity for the Y1 [human pancreatic polypeptide (human PP), NPY(3–36)], the Y2 ([Pro
34
]NPY, human PP), the Y3 (peptide YY), and the Y6 (human PP) receptor, all markedly stimulated ACTH release. Rat PP, which binds with high affinity to the Y4 receptor, was unable to stimulate ACTH release. A novel analogue fragment [Pro
34
]NPY(13–36) was synthesized as a ligand with low Y1 and Y2 receptor affinity. Interestingly, neither [Pro
34
]NPY(13–36) nor the selective Y5 receptor agonist [
d
-Trp
32
]NPY stimulated food intake, whereas both significantly increased plasma ACTH. Thus the hypothalamic NPY receptor mediating increases in plasma ACTH has a fragment activation profile unlike the Y1–Y4 or Y6 receptors and appears distinct from the NPY receptor controlling food intake.
Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: Role in restricted conformational mobility and compaction of native state
Authors: Kendrick Brent S. , Chang Byeong S. , Arakawa Tsutomu , Peterson Brian , Randolph Theodore W. , Manning Mark C. , Carpenter John F.
Published: July 2002

Understanding the mechanism for sucrose-induced protein stabilization is important in many diverse fields, ranging from biochemistry and environmental physiology to pharmaceutical science. Timasheff and Lee [Lee, J. C. & Timasheff, S. N. (1981)
J. Biol. Chem.
256, 7193–7201] have established that thermodynamic stabilization of proteins by sucrose is due to preferential exclusion of the sugar from the protein’s surface, which increases protein chemical potential. The current study measures the preferential exclusion of 1 M sucrose from a protein drug, recombinant interleukin 1 receptor antagonist (rhIL-1ra). It is proposed that the degree of preferential exclusion and increase in chemical potential are directly proportional to the protein surface area and that, hence, the system will favor the protein state with the smallest surface area. This mechanism explains the observed sucrose-induced restriction of rhIL-1ra conformational fluctuations, which were studied by hydrogen–deuterium exchange and cysteine reactivity measurements. Furthermore, infrared spectroscopy of rhlL-1ra suggested that a more ordered native conformation is induced by sucrose. Electron paramagnetic resonance spectroscopy demonstrated that in the presence of sucrose, spin-labeled cysteine 116 becomes more buried in the protein’s interior and that the hydrodynamic diameter of the protein is reduced. The preferential exclusion of sucrose from the protein and the resulting shift in the equilibrium between protein states toward the most compact conformation account for sucrose-induced effects on rhIL-1ra.