bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular picture of folding of a small α/β protein
Authors: Sheinerman Felix B. , Brooks Charles L.
Published: July 2002

We characterize, at the atomic level, the mechanism and thermodynamics of folding of a small α/β protein. The thermodynamically significant states of segment B1 of streptococcal protein G (GB1) are probed by using the statistical mechanical methods of importance sampling and molecular dynamics. From a thermodynamic standpoint, folding commences with overall collapse, accompanied by formation of ∼35% of the native structure. Specific contacts form at the loci experimentally inferred to be structured early in folding kinetics studies. Our study reveals that these initially structured regions are not spatially adjacent. As folding progresses, fluid-like nonlocal native contacts form, with many contacts forming and breaking as the structure searches for the native conformation. Although the α-helix forms early, the β-sheet forms concomitantly with the overall topology. Water is present in the protein core up to a late stage of folding, lubricating conformational transitions during the search process. Once 80% of the native contacts have formed, water is squeezed from the protein interior and the structure descends into the native manifold. Examination of the onset of side-chain mobility within our model indicates side-chain motion is most closely linked to the overall volume of the protein and no sharp order–disorder transition appears to occur. Exploration of models for hydrogen deuterium exchange show qualitative agreement with equilibrium measurement of hydrogen/deuterium protection factors.
Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the
Legionella pneumophila
genome
Authors: Segal Gil , Purcell Mary , Shuman Howard A.
Published: July 2002

A 22-kb DNA locus of
Legionella pneumophila
is described that contains 18 genes, 16 of which are required for macrophage killing (
icm
genes). In this paper two previously described
icm
loci were linked by the discovery of five genes located between the two loci. Four of the newly described genes are required for macrophage killing (
icmMLKE
) and one is dispensable. The 16
icm
genes appeared to be organized as six individual genes (
icmR
,
icmQ
,
icmG
,
icmC
,
icmD,
and
icmF
), and four operons (
icmTS
,
icmPO
,
icmMLKE
, and
icmJB
). Four
icm
genes (
icmP
,
icmO
,
icmL
, and
icmE
) show significant sequence similarity to plasmid genes involved in conjugation, whereas the other
icm
genes were found not to bear any sequence similarity to database entries. We found that
L. pneumophila
can mediate plasmid DNA transfer at a frequency of 10
−3
to 10
−4
per donor. Strains containing null mutations in two
icm
genes (
icmT
and
icmR
) showed a severe reduction in conjugation frequency and macrophage killing. Strains containing an insertion in four other
icm
genes (
icmF, icmE
,
icmC
, and
dotA
) were shown to have a less severe defect in conjugation. Mutations in the other 11
icm
genes had no effect on conjugation frequency. We currently do not know whether conjugation itself plays a role in macrophage killing. It is possible either that small plasmids can take advantage of an existing secretion system to be mobilized or that DNA transfer is required for human macrophage killing by
L. pneumophila
.
Changes in solvation during DNA binding and cleavage are critical to altered specificity of the
Eco
RI endonuclease
Authors: Robinson Clifford R. , Sligar Stephen G.
Published: July 2002

Restriction endonucleases such as
Eco
RI bind and cleave DNA with great specificity and represent a paradigm for protein–DNA interactions and molecular recognition. Using osmotic pressure to induce water release, we demonstrate the participation of bound waters in the sequence discrimination of substrate DNA by
Eco
RI. Changes in solvation can play a critical role in directing sequence-specific DNA binding by
Eco
RI and are also crucial in assisting site discrimination during catalysis. By measuring the volume change for complex formation, we show that at the cognate sequence (GAATTC)
Eco
RI binding releases about 70 fewer water molecules than binding at an alternate DNA sequence (TAATTC), which differs by a single base pair.
Eco
RI complexation with nonspecific DNA releases substantially less water than either of these specific complexes. In cognate substrates (GAATTC)
k
cat
decreases as osmotic pressure is increased, indicating the binding of about 30 water molecules accompanies the cleavage reaction. For the alternate substrate (TAATTC),
release
of about 40 water molecules accompanies the reaction, indicated by a dramatic acceleration of the rate when osmotic pressure is raised. These large differences in solvation effects demonstrate that water molecules can be key players in the molecular recognition process during both association and catalytic phases of the
Eco
RI reaction, acting to change the specificity of the enzyme. For both the protein–DNA complex and the transition state, there may be substantial conformational differences between cognate and alternate sites, accompanied by significant alterations in hydration and solvent accessibility.
Chronic lithium treatment robustly protects neurons in the central nervous system against excitotoxicity by inhibiting
N
-methyl-
d
-aspartate receptor-mediated calcium influx
Authors: Nonaka Shigeyuki , Hough Christopher J. , Chuang De-Maw
Published: July 2002

Lithium is the most commonly used drug for the treatment of manic depressive illness. The precise mechanisms underlying its clinical efficacy remain unknown. We found that long-term exposure to lithium chloride dramatically protects cultured rat cerebellar, cerebral cortical, and hippocampal neurons against glutamate-induced excitotoxicity, which involves apoptosis mediated by
N
-methyl-
d
-aspartate (NMDA) receptors. This neuroprotection is long-lasting, occurs at therapeutically relevant concentrations of lithium with an EC
50
of approximately 1.3 mM, and requires treatment for 6–7 days for complete protection to occur. In contrast, a 24-h treatment with lithium is ineffective. The protection in cerebellar neurons is specific for glutamate-induced excitotoxicity and can be attributed to inhibition of NMDA receptor-mediated calcium influx measured by
45
Ca
2+
uptake studies and fura-2 fluorescence microphotometry. The long-term effects of lithium are not caused by down-regulation of NMDA receptor subunit proteins and are unlikely related to its known ability to block inositol monophosphatase activity. Our results suggest that modulation of glutamate receptor hyperactivity represents at least part of the molecular mechanisms by which lithium alters brain function and exerts its clinical efficacy in the treatment for manic depressive illness. These actions of lithium also suggest that abnormality of glutamatergic neurotransmission as a pathogenic mechanism underlying bipolar illness warrants future investigation.
Specific peptide-activated proteolytic cleavage of
Escherichia coli
elongation factor Tu
Authors: Georgiou T. , Yu Y.-T. N. , Ekunwe S. , Buttner M. J. , Zuurmond A.-M. , Kraal B. , Kleanthous C. , Snyder L.
Published: July 2002

Phage exclusion is a form of programmed cell death in prokaryotes in which death is triggered by infection with phage, a seemingly altruistic response that limits multiplication of the phage and its spread through the population. One of the best-characterized examples of phage exclusion is the exclusion of T-even phages such as T4 by the e14-encoded Lit protein in many
Escherichia coli
K-12 strains. In this exclusion system, transcription and translation of a short region of the major head coat protein gene late in phage infection activates proteolysis of translation elongation factor Tu (EF-Tu), blocking translation and multiplication of the phage. The cleavage occurs between Gly-59 and Ile-60 in the nucleotide-binding domain. In the present work, we show that a 29-residue synthetic peptide spanning the activating region of the major head coat protein can activate the cleavage of GDP-bound EF-Tu in a purified system containing only purified EF-Tu and purified Lit protein. Lit behaves as a
bona fide
enzyme in this system, cleaving EF-Tu to completion when present at substoichiometric amounts. Two mutant peptides with amino acid changes that reduce the activation of cleavage of EF-Tu
in vivo
were also greatly reduced in their ability to activate EF-Tu cleavage
in vitro
but were still able to activate cleavage at a high concentration. Elongation factor G, which has the same sequence at the cleavage site and a nucleotide-binding domain similar to EF-Tu, was not cleaved by this system, and neither was heat-inactivated EF-Tu, suggesting that the structural context of the cleavage site may be important for specificity. This system apparently represents an activation mechanism for proteolysis that targets one of nature’s most evolutionarily conserved proteins for site-specific cleavage.
A large and distinct rotation of the myosin light chain domain occurs upon muscle contraction
Authors: Baker Josh E. , Brust-Mascher Ingrid , Ramachandran Sampath , LaConte Leslie E. W. , Thomas David D.
Published: July 2002

For more than 30 years, the fundamental goal in molecular motility has been to resolve force-generating motor protein structural changes. Although low-resolution structural studies have provided evidence for force-generating myosin rotations upon muscle activation, these studies did not resolve structural states of myosin in contracting muscle. Using electron paramagnetic resonance, we observed two distinct orientations of a spin label attached specifically to a single site on the light chain domain of myosin in relaxed scallop muscle fibers. The two probe orientations, separated by a 36° ± 5° axial rotation, did not change upon muscle activation, but the distribution between them changed substantially, indicating that a fraction (17% ± 2%) of myosin heads undergoes a large (at least 30°) axial rotation of the myosin light chain domain upon force generation and muscle contraction. The resulting model helps explain why this observation has remained so elusive and provides insight into the mechanisms by which motor protein structural transitions drive molecular motility.
Neural precursor differentiation into astrocytes requires signaling through the leukemia inhibitory factor receptor
Authors: Koblar S. A. , Turnley A. M. , Classon B. J. , Reid K. L. , Ware C. B. , Cheema S. S. , Murphy M. , Bartlett P. F.
Published: July 2002

The differentiation of precursor cells into neurons or astrocytes in the developing brain has been thought to be regulated in part by growth factors. We show here that neural precursors isolated from the developing forebrain of mice that are deficient in the gene for the low-affinity leukemia inhibitory factor receptor (LIFR
−/−
) fail to generate astrocytes expressing glial fibrillary acidic protein (GFAP) when cultured
in vitro
. Precursors from mice heterozygous for the null allele show normal levels of GFAP expression. These findings support the
in vivo
findings that show extremely low levels of GFAP mRNA in brains of embryonic day 19 LIFR
−/−
mice. In addition, monolayers of neural cells from LIFR
−/−
mice are far less able to support the neuronal differentiation of normal neural precursors than are monolayers from heterozygous or wild-type animals, indicating that endogenous signaling through the LIFR is required for the expression of both functional and phenotypic markers of astrocyte differentiation. LIFR
−/−
precursors are not irreversibly blocked from differentiating into astrocytes: they express GFAP after long-term passaging or stimulation with bone morphogenetic protein-2. These findings strongly implicate the LIF family of cytokines in the regulation of astrocyte differentiation and indeed the LIF-deficient animals show a significant reduction in the number of GFAP cells in the hippocampus. However, because this reduction is only partial it suggests that LIF may not be the predominant endogenous ligand signaling through the LIFR.
Figure-ground activity in primary visual cortex is suppressed by anesthesia
Authors: Lamme Victor A. F. , Zipser Karl , Spekreijse Henk
Published: July 2002

By means of their small receptive fields (RFs), neurons in primary visual cortex perform highly localized analyses of the visual scene, far removed from our normal unified experience of vision. Local image elements coded by the RF are put into more global context, however, by means of modulation of the responses of the V1 neurons. Contextual modulation has been shown to follow closely the perceptual interpretation of the scene as a whole. This would suggest that some aspects of contextual modulation can be recorded only in awake and perceiving animals. In this study, multi-unit activity was recorded with implanted electrodes from primary visual cortex of awake, fixating monkeys viewing textured displays in which figure and ground regions were segregated by differences in either orientation or motion. Contextual modulation was isolated from local RF processing, by keeping RF stimulation identical across trials while sampling responses for various positions of the RF relative to figure and ground. Contextual modulation was observed to unfold spatially and temporally in a way that closely resembles the figure-ground percept. When recording was repeated, but with the animals anesthetized, the figure-ground related modulatory activity was selectively suppressed. RF tuning properties, however, remained unaffected. The results show that the modulatory activity is functionally distinct from the RF properties. V1 thus hosts distinct regimes of activity that are mediated by separate mechanisms and that depend differentially on the animal being awake or anesthetized.
Structure-based prediction of the stability of transmembrane helix–helix interactions: The sequence dependence of glycophorin A dimerization
Authors: MacKenzie Kevin R. , Engelman Donald M.
Published: July 2002

The ability to predict the effects of point mutations on the interaction of α-helices within membranes would represent a significant step toward understanding the folding and stability of membrane proteins. We use structure-based empirical parameters representing steric clashes, favorable van der Waals interactions, and restrictions of side-chain rotamer freedom to explain the relative dimerization propensities of 105 hydrophobic single-point mutants of the glycophorin A (GpA) transmembrane domain. Although the structure at the dimer interface is critical to our model, changes in side-chain hydrophobicity are uncorrelated with dimer stability, indicating that the hydrophobic effect does not influence transmembrane helix–helix association. Our model provides insights into the compensatory effects of multiple mutations and shows that helix–helix interactions dominate the formation of specific structures.
Populations can persist in an environment consisting of sink habitats only
Authors: Jansen Vincent A. A. , Yoshimura Jin
Published: July 2002

Populations that live in environments with different habitats have to distribute their offspring over these habitats. When population densities go to equilibrium, the evolutionary optimum is an ideal free distribution. Under an ideal free distribution, no offspring should be put into sink habitats. However, when the environmental conditions in a habitat are not constant but fluctuate, allocating offspring to sink habitats can increase the long term growth rate of a population. We demonstrate this principle in a simple model for offspring allocation. As a consequence, it is possible that populations persist in environments that only consist of sink habitats.
A stationary phase protein in
Escherichia coli
with binding activity to the major σ subunit of RNA polymerase
Authors: Jishage Miki , Ishihama Akira
Published: July 2002

Switching of the transcription pattern in
Escherichia coli
during the growth transition from exponential to stationary phase is accompanied by the replacement of the RNA polymerase-associated σ
70
subunit (σ
D
) with σ
38
(σ
S
). A fraction of the σ
70
subunit in stationary phase cell extracts was found to exist as a complex with a novel protein, designated Rsd (
R
egulator of
s
igma
D
). The intracellular level of Rsd starts to increase during the transition from growing to stationary phase. The
rsd
gene was identified at 90 min on the
E. coli
chromosome. Overexpressed and purified Rsd protein formed complexes
in vitro
with σ
70
but not with other σ subunits, σ
N
, σ
S
, σ
H
, σ
F
, and σ
E
. Analysis of proteolytic fragments of σ
70
indicated that Rsd binds at or downstream of region 4, the promoter −35 recognition domain. The isolated Rsd inhibited transcription
in vitro
to various extents depending on the promoters used. We propose that Rsd is a stationary phase
E. coli
protein with regulatory activity of the σ
70
function.
A microfabricated device for sizing and sorting DNA molecules
Authors: Chou Hou-Pu , Spence Charles , Scherer Axel , Quake Stephen
Published: July 2002

We have demonstrated a microfabricated single-molecule DNA sizing device. This device does not depend on mobility to measure molecule size, is 100 times faster than pulsed-field gel electrophoresis, and has a resolution that improves with increasing DNA length. It also requires a million times less sample than pulsed-field gel electrophoresis and has comparable resolution for large molecules. Here we describe the fabrication and use of the single-molecule DNA sizing device for sizing and sorting DNA restriction digests and ladders spanning 2–200 kbp.
Mechanism of regulation of Hsp70 chaperones by DnaJ cochaperones
Authors: Laufen Thomas , Mayer Matthias P. , Beisel Christian , Klostermeier Dagmar , Mogk Axel , Reinstein Jochen , Bukau Bernd
Published: July 2002

Hsp70 chaperones assist a large variety of protein folding processes within the entire lifespan of proteins. Central to these activities is the regulation of Hsp70 by DnaJ cochaperones. DnaJ stimulates Hsp70 to hydrolyze ATP, a key step that closes its substrate-binding cavity and thus allows stable binding of substrate. We show that DnaJ stimulates ATP hydrolysis by
Escherichia coli
Hsp70, DnaK, very efficiently to >1000-fold, but only if present at high (micromolar) concentration. In contrast, the chaperone activity of DnaK in luciferase refolding was maximal at several hundredfold lower concentration of DnaJ. However, DnaJ was capable of maximally stimulating the DnaK ATPase even at this low concentration, provided that protein substrate was present, indicating synergistic action of DnaJ and substrate. Peptide substrates were poorly effective in this synergistic action. DnaJ action required binding of protein substrates to the central hydrophobic pocket of the substrate-binding cavity of DnaK, as evidenced by the reduced ability of DnaJ to stimulate ATP hydrolysis by a DnaK mutant with defects in substrate binding. At high concentrations, DnaJ itself served as substrate for DnaK in a process considered to be unphysiological. Mutant analysis furthermore revealed that DnaJ-mediated stimulation of ATP hydrolysis requires communication between the ATPase and substrate-binding domains of DnaK. This mechanism thus allows DnaJ to tightly couple ATP hydrolysis by DnaK with substrate binding and to avoid jamming of the DnaK chaperone with peptides. It probably is conserved among Hsp70 family members and is proposed to account for their functional diversity.
The role of transient hypermutators in adaptive mutation in
Escherichia coli
Authors: Rosche William A. , Foster Patricia L.
Published: July 2002

Microbial populations under nonlethal selection can give rise to mutations that relieve the selective pressure, a phenomenon that has come to be called “adaptive mutation.” One explanation for adaptive mutation is that a small proportion of the cells experience a period of transient hypermutation, and that these hypermutators account for the mutations that appear. The experiments reported here investigated the contribution that hypermutators make to the mutations occurring in a Lac
−
strain of
Escherichia coli
during selection for lactose utilization. A broad mutational screen, loss of motility, was used to compare the frequency of nonselected mutations in starved Lac
−
cells, in Lac
+
revertants, and in Lac
+
revertants carrying yet another nonselected mutation. These frequencies allowed us to calculate that the hypermutating subpopulation makes up ≈0.06% of the population and that its mutation rate is elevated ≈200-fold. From these numbers we conclude that the hypermutators are responsible for nearly all multiple mutations but produce only ≈10% of the adaptive Lac
+
mutations.
Macaque inferior temporal neurons are selective for disparity-defined three-dimensional shapes
Authors: Janssen Peter , Vogels Rufin , Orban Guy A.
Published: July 2002

Real-world objects are three-dimensional (3D). Yet, it is unknown whether the neurons of the inferior temporal cortex, which is critical for object recognition, are selective for the 3D shape of objects. We tested for such selectivity by comparing responses to stereo-defined curved 3D shapes derived from identical pairs of monocular images. More than one-third of macaque inferior temporal neurons were selective for 3D shape. In the vast majority of those neurons, this selectivity depended on the global binocular disparity gradient and not on the local disparity. Thus, inferior temporal cortex processes not only two-dimensional but also 3D shape information.
Stress-induced behaviors require the corticotropin-releasing hormone (CRH) receptor, but not CRH
Authors: Weninger Stacie C. , Dunn Adrian J. , Muglia Louis J. , Dikkes Pieter , Miczek Klaus A. , Swiergiel Artur H. , Berridge Craig W. , Majzoub Joseph A.
Published: July 2002

Corticotropin-releasing hormone (CRH) is a central regulator of the hormonal stress response, causing stimulation of corticotropin and glucocorticoid secretion. CRH is also widely believed to mediate stress-induced behaviors, implying a broader, integrative role for the hormone in the psychological stress response. Mice lacking the CRH gene exhibit normal stress-induced behavior that is specifically blocked by a CRH type 1 receptor antagonist. The other known mammalian ligand for CRH receptors is urocortin. Normal and CRH-deficient mice have an identical distribution of urocortin mRNA, which is confined to the region of the Edinger–Westphal nucleus, and is absent from regions known to mediate stress-related behaviors. Since the Edinger–Westphal nucleus is not known to project to any brain regions believed to play a role in anxiety-like behavior, an entirely different pathway must be postulated for urocortin in the Edinger–Westphal nucleus to mediate these behaviors in CRH-deficient mice. Alternatively, an unidentified CRH-like molecule other than CRH or urocortin, acting through the CRH receptors in brain regions believed to mediate stress-induced behaviors, may mediate the behavioral response to stress, either alone or in concert with CRH.
Aeolotopic interactions of globular proteins
Authors: Lomakin Aleksey , Asherie Neer , Benedek George B.
Published: July 2002

Protein crystallization, aggregation, liquid–liquid phase separation, and self-assembly are important in protein structure determination in the industrial processing of proteins and in the inhibition of protein condensation diseases. To fully describe such phase transformations in globular protein solutions, it is necessary to account for the strong spatial variation of the interactions on the protein surface. One difficulty is that each globular protein has its own unique surface, which is crucial for its biological function. However, the similarities amongst the macroscopic properties of different protein solutions suggest that there may exist a generic model that is capable of describing the nonuniform interactions between globular proteins. In this paper we present such a model, which includes the short-range interactions that vary from place to place on the surface of the protein. We show that this aeolotopic model [from the Greek
aiolos
(“variable”) and
topos
(“place”)] describes the phase diagram of globular proteins and provides insight into protein aggregation and crystallization.
Phenological changes reflect climate change in Wisconsin
Authors: Bradley Nina L. , Leopold A. Carl , Ross John , Huffaker Wellington
Published: July 2002

A phenological study of springtime events was made over a 61-year
period at one site in southern Wisconsin. The records over this long
period show that several phenological events have been increasing in
earliness; we discuss evidence indicating that these changes reflect
climate change. The mean of regressions for the 55 phenophases studied
was −0.12 day per year, an overall increase in phenological earliness
at this site during the period. Some phenophases have not increased in
earliness, as would be expected for phenophases that are regulated by
photoperiod or by a physiological signal other than local temperature.
Emergence of homeostasis and “noise imprinting” in an evolution model
Authors: Stern Michael D.
Published: July 2002

Homeostasis, the creation of a stabilized internal milieu, is ubiquitous in biological evolution, despite the entropic cost of excluding noise information from a region. The advantages of stability seem self evident, but the alternatives are not so clear. This issue was studied by means of numerical experiments on a simple evolution model: a population of Boolean network “organisms” selected for performance of a curve-fitting task while subjected to noise. During evolution, noise sensitivityincreasedwith fitness. Noise exclusion evolved spontaneously, but only if the noise was sufficiently unpredictable. Noise that was limited to one or a few stereotyped patterns caused symmetry breaking that prevented noise exclusion. Instead, the organisms incorporated the noise into their function at little cost in ultimate fitness and became totally noise dependent. This “noise imprinting” suggests caution when interpreting apparent adaptations seen in nature. If the noise was totally random from generation to generation, noise exclusion evolved reliably and irreversibly, but if the noise was correlated over several generations, maladaptive selection of noise-dependent traits could reverse noise exclusion, with catastrophic effect on population fitness. Noise entering the selection process rather than the organism had a different effect: adaptive evolution was totally abolished above a critical noise amplitude, in a manner resembling a thermodynamic phase transition. Evolutionary adaptation to noise involves the creation of a subsystem screened from noise information but increasingly vulnerable to its effects. Similar considerations may apply to information channeling in human cultural evolution.
A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood
Authors: Brown Michael S. , Goldstein Joseph L.
Published: July 2002

The integrity of cell membranes is maintained by a balance between
the amount of cholesterol and the amounts of unsaturated and saturated
fatty acids in phospholipids. This balance is maintained by
membrane-bound transcription factors called sterol regulatory
element-binding proteins (SREBPs) that activate genes encoding enzymes
of cholesterol and fatty acid biosynthesis. To enhance transcription,
the active NH
2
-terminal domains of SREBPs are released from
endoplasmic reticulum membranes by two sequential cleavages. The first
is catalyzed by Site-1 protease (S1P), a membrane-bound
subtilisin-related serine protease that cleaves the hydrophilic loop of
SREBP that projects into the endoplasmic reticulum lumen. The second
cleavage, at Site-2, requires the action of S2P, a hydrophobic protein
that appears to be a zinc metalloprotease. This cleavage is
unusual because it occurs within a membrane-spanning domain of SREBP.
Sterols block SREBP processing by inhibiting S1P. This response is
mediated by SREBP cleavage-activating protein (SCAP), a regulatory
protein that activates S1P and also serves as a sterol sensor, losing
its activity when sterols overaccumulate in cells. These regulated
proteolytic cleavage reactions are ultimately responsible for
controlling the level of cholesterol in membranes, cells, and blood.
Content mixing and membrane integrity during membrane fusion driven by pairing of isolated v-SNAREs and t-SNAREs
Authors: Nickel Walter , Weber Thomas , McNew James A. , Parlati Francesco , Söllner Thomas H. , Rothman James E.
Published: July 2002

Membrane bilayer fusion has been shown to be mediated by v- and t-SNAREs initially present in separate populations of liposomes and to occur with high efficiency at a physiologically meaningful rate. Lipid mixing was demonstrated to involve both the inner and the outer leaflets of the membrane bilayer. Here, we use a fusion assay that relies on duplex formation of oligonucleotides introduced in separate liposome populations and report that SNARE proteins suffice to mediate complete membrane fusion accompanied by mixing of luminal content. We also find that SNARE-mediated membrane fusion does not compromise the integrity of liposomes.
Pituitary adenylyl cyclase-activating peptide: A pivotal modulator of glutamatergic regulation of the suprachiasmatic circadian clock
Authors: Chen Dong , Buchanan Gordon F. , Ding Jian M. , Hannibal Jens , Gillette Martha U.
Published: July 2002

The circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus organizes behavioral rhythms, such as the sleep–wake cycle, on a near 24-h time base and synchronizes them to environmental day and night. Light information is transmitted to the SCN by direct retinal projections via the retinohypothalamic tract (RHT). Both glutamate (Glu) and pituitary adenylyl cyclase-activating peptide (PACAP) are localized within the RHT. Whereas Glu is an established mediator of light entrainment, the role of PACAP is unknown. To understand the functional significance of this colocalization, we assessed the effects of nocturnal Glu and PACAP on phasing of the circadian rhythm of neuronal firing in slices of rat SCN. When coadministered, PACAP blocked the phase advance normally induced by Glu during late night. Surprisingly, blocking PACAP neurotransmission, with either PACAP6–38, a specific PACAP receptor antagonist, or anti-PACAP antibodies, augmented the Glu-induced phase advance. Blocking PACAP
in vivo
also potentiated the light-induced phase advance of the rhythm of hamster wheel-running activity. Conversely, PACAP enhanced the Glu-induced delay in the early night, whereas PACAP6–38 inhibited it. These results reveal that PACAP is a significant component of the Glu-mediated light-entrainment pathway. When Glu activates the system, PACAP receptor-mediated processes can provide gain control that generates graded phase shifts. The relative strengths of the Glu and PACAP signals together may encode the amplitude of adaptive circadian behavioral responses to the natural range of intensities of nocturnal light.
Interspecific and intraspecific competition as causes of direct and delayed density dependence in a fluctuating vole population
Authors: Hansen Thomas F. , Stenseth Nils C. , Henttonen Heikki , Tast Johan
Published: July 2002

A 3- to 5-year cycle of vole abundances is a characteristic phenomenon in the ecology of northern regions, and their explanation stands as a central theoretical challenge in population ecology. Although many species of voles usually coexist and are in severe competition for food and breeding space, the role of interspecific competition in vole cycles has never been evaluated statistically. After studying community effects on the population dynamics of the gray-sided vole (Clethrionomys rufocanus) in the subarctic birch forest at Kilpisjärvi, Finland, we report statistical results showing that both interspecific and intraspecific effects are important in the direct year-to-year density dependence. However, interspecific effects are not detectable in the 2-year delayed density dependence that is crucial for generating the characteristic cycles. Furthermore, we show that most of the competition takes place during the winter. The results are evaluated against two models of community dynamics. One assumes that the delayed effects are caused by an interaction with a specialist predator, and the other assumes that they are caused by overgrazing food plants. These statistical results show that vole cycles may be generated by a species-specific trophic interaction. The results also suggest that the gray-sided vole may be the focal species in the birch-forest community, as field voles may be in the taiga and as lemmings may be on the tundra.
Structure, thermostability, and conformational flexibility of hen egg-white lysozyme dissolved in glycerol
Authors: Knubovets Tatyana , Osterhout John J. , Connolly Peter J. , Klibanov Alexander M.
Published: July 2002

Hen egg-white lysozyme dissolved in glycerol containing 1% water
was studied by using CD and amide proton exchange monitored by
two-dimensional
1
H NMR. The far- and near-UV CD spectra of
the protein showed that the secondary and tertiary structures of
lysozyme in glycerol were similar to those in water. Thermal melting of
lysozyme in glycerol followed by CD spectral changes indicated
unfolding of the tertiary structure with a
T
m
of 76.0 ± 0.2°C and no appreciable loss of the secondary
structure up to 85°C. This is in contrast to the coincident
denaturation of both tertiary and secondary structures with
T
m
values of 74.8 ± 0.4°C and 74.3
± 0.7°C, respectively, under analogous conditions in water. Quenched
amide proton exchange experiments revealed a greater structural
protection of amide protons in glycerol than in water for a majority of
the slowly exchanging protons. The results point to a highly ordered,
native-like structure of lysozyme in glycerol, with the stability
exceeding that in water.
The neurotransmitter receptor-anchoring protein gephyrin reconstitutes molybdenum cofactor biosynthesis in bacteria, plants, and mammalian cells
Authors: Stallmeyer B. , Schwarz G. , Schulze J. , Nerlich A. , Reiss J. , Kirsch J. , Mendel R. R.
Published: July 2002

The molybdenum cofactor (Moco), a highly conserved pterin
compound complexing molybdenum, is required for the enzymatic
activities of all molybdenum enzymes except nitrogenase. Moco is
synthesized by a unique and evolutionarily old pathway that requires
the activities of at least six gene products. Some of the proteins
involved in bacterial, plant, and invertebrate Moco biosynthesis show
striking homologies to the primary structure of gephyrin, a polypeptide
required for the clustering of inhibitory glycine receptors in
postsynaptic membranes in the rat central nervous system. Here, we show
that gephyrin binds with high affinity to molybdopterin, the metabolic
precursor of Moco. Furthermore, gephyrin expression can reconstitute
Moco biosynthesis in Moco-deficient bacteria, a molybdenum-dependent
mouse cell line, and a Moco-deficient plant mutant. Conversely,
inhibition of gephyrin expression by antisense RNA expression in
cultured murine cells reduces their Moco content significantly. These
data indicate that in addition to clustering glycine receptors,
gephyrin also is involved in Moco biosynthesis and illustrate the
remarkable conservation of its function in Moco biosynthesis throughout
phylogeny.
Trapping cDNAs encoding secreted proteins from the salivary glands of the malaria vector
Anopheles
gambiae
Authors: Arcà Bruno , Lombardo Fabrizio , de Lara Capurro Margareth , della Torre Alessandra , Dimopoulos George , James Anthony A. , Coluzzi Mario
Published: July 2002

The signal sequence trap method was used to isolate cDNAs
corresponding to proteins containing secretory leader peptides and
whose genes are expressed specifically in the salivary glands of the
malaria vector
Anopheles gambiae
. Fifteen unique cDNA
fragments, ranging in size from 150 to 550 bp, were isolated and
sequenced in a first round of immunoscreening in COS-7 cells. All but
one of the cDNAs contained putative signal sequences at their 5′ ends,
suggesting that they were likely to encode secreted or transmembrane
proteins. Expression analysis by reverse transcription–PCR showed that
at least six cDNA fragments were expressed specifically in the salivary
glands. Fragments showing a high degree of similarity to D7 and
apyrase, two salivary gland-specific genes previously found in
Aedes aegypti
, were identified. Of interest, three
different D7-related cDNAs that are likely to represent a new gene
family were found in
An. gambiae
. Moreover, three salivary
gland-specific cDNA fragments that do not show similarity to known
proteins in the databases were identified, and the corresponding full
length cDNAs were cloned and sequenced. RNA
in situ
hybridization to whole female salivary glands showed patterns of
expression that overlap only in part those observed in the culicine
mosquito
A. aegypti
.
Increased anxiety and altered responses to anxiolytics in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase
Authors: Kash Shera F. , Tecott Laurence H. , Hodge Clyde , Baekkeskov Steinunn
Published: July 2002

The larger isoform of the enzyme glutamate decarboxylase, GAD67,
synthesizes >90% of basal levels of γ-aminobutyric acid (GABA)
in the brain. In contrast, the smaller isoform, GAD65, has been
implicated in the fine-tuning of inhibitory neurotransmission. Mice
deficient in GAD65 exhibit increased anxiety-like responses in both the
open field and elevated zero maze assays. Additionally, GAD65-deficient
mice have a diminished response to the anxiolytics diazepam and
pentobarbital, both of which interact with GABA-A receptors in a
GABA-dependent fashion to facilitate GABAergic neurotransmission. Loss
of GAD65-generated GABA does not appear to result in compensatory
postsynaptic GABA-A receptor changes based on radioligand receptor
binding studies, which revealed no change in the postsynaptic GABA-A
receptor density. Furthermore, mutant and wild-type animals do not
differ in their behavioral response to muscimol, which acts
independently of the presence of GABA. We propose that stress-induced
GABA release is impaired in GAD65-deficient mice, resulting in
increased anxiety-like responses and a diminished response to the acute
effects of drugs that facilitate the actions of released GABA.
Specificity of native-like interhelical hydrophobic contacts in the apomyoglobin intermediate
Authors: Kay Michael S. , Ramos Carlos H. I. , Baldwin Robert L.
Published: July 2002

On exposure to mildly acidic conditions, apomyoglobin forms a partially folded intermediate, I. The A, B, G, and H helices are significantly structured in this equilibrium intermediate, whereas the remainder of the protein is largely unfolded. We report here the effects of mutations at helix pairing sites on the stability of I in three classes of mutants that: (
i
) truncate hydrophobic side chains in native helix packing sites, (
ii
) truncate hydrophobic side chains not involved in interhelical contacts, and (
iii
) extend hydrophobic side chains at residues not involved in interhelical contacts. Class I mutants significantly decrease the stability and cooperativity of folding of the intermediate. Class II and III mutants show smaller effects on stability and have little effect on cooperativity. Qualitatively similar results to those found in I were obtained for all three classes of mutants in native myoglobin (N), demonstrating that hydrophobic burial is fairly specific to native helix packing sites in I as well as in N. These results suggest that hydrophobic burial along native-like interhelical contacts is important for the formation of the cooperatively folded intermediate.
Conservation of the expression and function of
apterous
orthologs in
Drosophila
and mammals
Authors: Rincón-Limas Diego E. , Lu Cheng-Hsin , Canal Inmaculada , Calleja Manuel , Rodríguez-Esteban Concepción , Izpisúa-Belmonte Juan Carlos , Botas Juan
Published: July 2002

The
Drosophila apterous
(
ap
) gene encodes a protein of the LIM-homeodomain family. Many transcription factors of this class have been conserved during evolution; however, the functional significance of their structural conservation is generally not known.
ap
is best known for its fundamental role as a dorsal selector gene required for patterning and growth of the wing, but it also has other important functions required for neuronal fasciculation, fertility, and normal viability. We isolated mouse (
mLhx2
) and human (
hLhx2
)
ap
orthologs, and we used transgenic animals and rescue assays to investigate the conservation of the Ap protein during evolution. We found that the human protein LHX2 is able to regulate correctly
ap
target genes in the fly, causes the same phenotypes as Ap when ectopically produced, and most importantly rescues
ap
mutant phenotypes as efficiently as the fly protein. In addition, we found striking similarities in the expression patterns of the
Drosophila
and murine genes. Both
mLhx2
and
ap
are expressed in the respective nerve cords, eyes, olfactory organs, brain, and limbs. These results demonstrate the conservation of Ap protein function across phyla and argue that aspects of its expression pattern have also been conserved from a common ancestor of insects and vertebrates.
Spontaneous heterosis in larval life-history traits of hemiclonal frog hybrids
Authors: Hotz Hansjürg , Semlitsch Raymond D. , Gutmann Eva , Guex Gaston-Denis , Beerli Peter
Published: July 2002

European water frog hybrids
Rana esculenta
(
Rana ridibunda
×
Rana lessonae
) reproduce hemiclonally, transmitting only their
ridibunda
genome to gametes. We compared fitness-related larval life-history traits of natural
R. esculenta
from Poland with those of the two sympatric parental species and of newly generated F
1
hybrids. Compared with either parental species, F
1
hybrid offspring had higher survival, higher early growth rates, a more advanced developmental stage by day 49, and earlier metamorphosis, but similar mass at metamorphosis.
R. esculenta
from natural lineages had trait values intermediate between those of F
1
offspring and of the two parental species. The data support earlier observations on natural
R. esculenta
that had faster larval growth, earlier metamorphosis, and higher resistance to hypoxic conditions compared with either parental species. Observing larval heterosis in F
1
hybrids in survival, growth rate, and time to metamorphosis, however, at an even higher degree than in hybrids from natural lineages, demonstrates that heterosis is spontaneous and results from hybridity
per se
rather than from subsequent interclonal selection; in natural lineages the effects of hybridity and of clonal history are confounded. This is compelling evidence for spontaneous heterosis in hybrid clonals. Results on hemiclonal fish hybrids (
Poeciliopsis
) showed no spontaneous heterosis; thus, our frog data are not applicable to all hybrid clonals. Our data do show, however, that heterosis is an important potential source for the extensively observed ecological success of hybrid clonals. We suggest that heterosis and interclonal selection together shape fitness of natural
R. esculenta
lineages.
Protein folding at the air–water interface studied with x-ray reflectivity
Authors: Gidalevitz David , Huang Zhengqing , Rice Stuart A.
Published: July 2002

We report the results of x-ray reflectivity measurements of thin films formed by different water-soluble proteins at the air–aqueous solution interface. It is demonstrated that glucose oxidase, alcohol dehydrogenase, and urease molecules denaturate at the air–aqueous solution interface to form 8- to 14-Å-thick peptide sheets. X-ray reflectivity data indicate that the spreading of a lipid monolayer at the aqueous solution surface before protein injection does not prevent proteins from unfolding. On the other hand, crosslinking of proteins results in intact enzyme layers at the subphase surface. A model that involves interaction of glucose oxidase molecules with a phospholipid monolayer is proposed. In this model, an observed decrease of the lipid electron density in the protein presence is explained in terms of “holes” in the monolayer film caused by protein molecule adsorption.
A novel lipopolysaccharide-induced transcription factor regulating tumor necrosis factor α gene expression: Molecular cloning, sequencing, characterization, and chromosomal assignment
Authors: Myokai Fumio , Takashiba Shogo , Lebo Roger , Amar Salomon
Published: July 2002

Lipopolysaccharide (LPS) is a potent stimulator of monocytes and macrophages, causing secretion of tumor necrosis factor α (TNF-α) and other inflammatory mediators. Given the deleterious effects to the host of TNF-α, it has been postulated that TNF-α gene expression must be tightly regulated. The nature of the nuclear factor(s) that control TNF-α gene transcription in humans remains obscure, although NF-κB has been suggested. Our previous studies pertaining to macrophage response to LPS identified a novel DNA-binding domain located from −550 to −487 in the human TNF-α promoter that contains transcriptional activity, but lacks any known NF-κB-binding sites. We have used this DNA fragment to isolate and purify a 60-kDa protein binding to this fragment and obtained its amino-terminal sequence, which was used to design degenerate probes to screen a cDNA library from THP-1 cells. A novel cDNA clone (1.8 kb) was isolated and fully sequenced. Characterization of this cDNA clone revealed that its induction was dependent on LPS activation of THP-1 cells; hence, the name LPS-induced TNF-alpha factor (LITAF). Inhibition of LITAF mRNA expression in THP-1 cells resulted in a reduction of TNF-α transcripts. In addition, high level of expression of LITAF mRNA was observed predominantly in the placenta, peripheral blood leukocytes, lymph nodes, and the spleen. Finally, chromosomal localization using fluorescencein situhybridization revealed that LITAF mapped to chromosome 16p12–16p13.3. Together, these findings suggest that LITAF plays an important role in the activation of the human TNF-α gene and proposes a new mechanism to control TNF-α gene expression.
Transglutaminase-catalyzed crosslinking of the Aα and γ constituent chains in fibrinogen
Authors: Murthy S. N. Prasanna , Wilson James H. , Lukas Thomas J. , Veklich Yuri , Weisel John W. , Lorand Laszlo
Published: July 2002

Studies on transglutaminases usually focus on the polymerization of protein substrates by intermolecular N
ɛ
(γ-glutamyl)lysine bridges, without considering the possibility that the monomeric protein units, themselves, could also become crosslinked internally. Both types of crosslinks are produced in the reaction of fibrinogen with red cell transglutaminase. We isolated the transglutaminase-modified, mostly monomeric form (92–96%) of fibrinogen with a N
ɛ
(γ-glutamyl)lysine content of ≈1.6 moles/mole of fibrinogen. The preparation was fully clottable by thrombin, but the rates of release of fibrinopeptides and clotting times were delayed compared with control. Hybrid Aα⋅γ type of crosslinking, the hallmark of the reaction of the transglutaminase with fibrinogen, occurred by bridging the Aα(408–421) chain segment of the protein to that of γ(392–406). Rotary shadowed electron microscope images showed many monomers to be bent, and the crosslinks seemed to bind the otherwise flexible αC domain closer to the backbone of fibrinogen.