bluuebunny/crossref_metadata_2025 huggingface dataset:
Leptin activates distinct projections from the dorsomedial and ventromedial hypothalamic nuclei
Authors: Elmquist Joel K. , Ahima Rexford S. , Elias Carol F. , Flier Jeffrey S. , Saper Clifford B.
Published: July 2002

Leptin has profound effects on feeding, metabolism, and neuroendocrine status. Evidence indicates that the hypothalamus coordinates these responses, though the specific brain pathways engaged by leptin remain obscure. The paraventricular nucleus of the hypothalamus (PVH) regulates pituitary gland function and feeding, and innervates autonomic preganglionic neurons, making it a candidate to regulate many of the responses to leptin. The subparaventricular zone, an anterior hypothalamic region receiving dense innervation from the suprachiasmatic nucleus, is thought to integrate circadian and metabolic information. We investigated the distribution of neurons in the rat brain activated by leptin administration that also project to the PVH or the subparaventricular zone by coupling immunohistochemistry for Fos with retrograde transport of cholera toxin-b. Intravenous leptin characteristically activated several cell groups including the ventromedial hypothalamic nucleus, the dorsomedial hypothalamic nucleus (DMH), and the PVH. When tracer injections were centered in the subparaventricular zone, many double-labeled cells were observed in the dorsomedial subdivision of the ventromedial hypothalamic nucleus. This projection may provide an anatomic substrate for integration of metabolic and circadian information to regulate the hypothalamo-pituitary axis. When cholera toxin-b injections were centered in the PVH, many double-labeled cells were found within the caudal DMH. Hence, activation of specific neuroendocrine and autonomic elements of the PVH may be triggered by leptin-activated afferents arising in the DMH. Our results demonstrate that a discrete set of hypothalamic pathways may underlie leptin’s autonomic, endocrine, and behavioral effects.
Elevated anxiety and antidepressant-like responses in serotonin 5-HT
1A
receptor mutant mice
Authors: Heisler Lora K. , Chu Hung-Ming , Brennan Thomas J. , Danao Jean A. , Bajwa Preetpaul , Parsons Loren H. , Tecott Laurence H.
Published: July 2002

The brain serotonin (5-hydroxytryptamine; 5-HT) system is a powerful modulator of emotional processes and a target of medications used in the treatment of psychiatric disorders. To evaluate the contribution of serotonin 5-HT
1A
receptors to the regulation of these processes, we have used gene-targeting technology to generate 5-HT
1A
receptor-mutant mice. These animals lack functional 5-HT
1A
receptors as indicated by receptor autoradiography and by resistance to the hypothermic effects of the 5-HT
1A
receptor agonist 8-hydroxy-2-(di-
n
-propylamino)tetralin (8-OH-DPAT). Homozygous mutants display a consistent pattern of responses indicative of elevated anxiety levels in open-field, elevated-zero maze, and novel-object assays. Moreover, they exhibit antidepressant-like responses in a tail-suspension assay. These results indicate that the targeted disruption of the 5-HT
1A
receptor gene leads to heritable perturbations in the serotonergic regulation of emotional state. 5-HT
1A
receptor-null mutant mice have potential as a model for investigating mechanisms through which serotonergic systems modulate affective state and mediate the actions of psychiatric drugs.
Structural code for DNA recognition revealed in crystal structures of papillomavirus E2-DNA targets
Authors: Rozenberg Haim , Rabinovich Dov , Frolow Felix , Hegde Rashmi S. , Shakked Zippora
Published: July 2002

Transcriptional regulation in papillomaviruses depends on sequence-specific binding of the regulatory protein E2 to several sites in the viral genome. Crystal structures of bovine papillomavirus E2 DNA targets reveal a conformational variant of B-DNA characterized by a roll-induced writhe and helical repeat of 10.5 bp per turn. A comparison between the free and the protein-bound DNA demonstrates that the intrinsic structure of the DNA regions contacted directly by the protein and the deformability of the DNA region that is not contacted by the protein are critical for sequence-specific protein/DNA recognition and hence for gene-regulatory signals in the viral system. We show that the selection of dinucleotide or longer segments with appropriate conformational characteristics, when positioned at correct intervals along the DNA helix, can constitute a structural code for DNA recognition by regulatory proteins. This structural code facilitates the formation of a complementary protein–DNA interface that can be further specified by hydrogen bonds and nonpolar interactions between the protein amino acids and the DNA bases.
Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia
Authors: Yrjänheikki Juha , Keinänen Riitta , Pellikka Milla , Hökfelt Tomas , Koistinaho Jari
Published: July 2002

Ischemic stroke is the most common life-threatening neurological disease and has limited therapeutic options. One component of ischemic neuronal death is inflammation. Here we show that doxycycline and minocycline, which are broad-spectrum antibiotics and have antiinflammatory effects independent of their antimicrobial activity, protect hippocampal neurons against global ischemia in gerbils. Minocycline increased the survival of CA1 pyramidal neurons from 10.5% to 77% when the treatment was started 12 h before ischemia and to 71% when the treatment was started 30 min after ischemia. The survival with corresponding pre- and posttreatment with doxycycline was 57% and 47%, respectively. Minocycline prevented completely the ischemia-induced activation of microglia and the appearance of NADPH-diaphorase reactive cells, but did not affect induction of glial acidic fibrillary protein, a marker of astrogliosis. Minocycline treatment for 4 days resulted in a 70% reduction in mRNA induction of interleukin-1β-converting enzyme, a caspase that is induced in microglia after ischemia. Likewise, expression of inducible nitric oxide synthase mRNA was attenuated by 30% in minocycline-treated animals. Our results suggest that lipid-soluble tetracyclines, doxycycline and minocycline, inhibit inflammation and are neuroprotective against ischemic stroke, even when administered after the insult. Tetracycline derivatives may have a potential use also as antiischemic compounds in humans.
A null mutation in the photoreceptor guanylate cyclase gene causes the
retinal degeneration
chicken phenotype
Authors: Semple-Rowland Susan L. , Lee Nancy R. , Van Hooser J. Preston , Palczewski Krzysztof , Baehr Wolfgang
Published: July 2002

The retinas of the
retinal degeneration
(
rd
) chicken are fully developed and possess normal morphology at hatching but fail to respond to light stimulation. Analyses of retinal cGMP, the internal messenger of phototransduction, show that the amount of cGMP in predegenerate, fully developed
rd/rd
photoreceptors is 5–10 times less than that seen in normal photoreceptor cells. We show that the low levels of cGMP in
rd
chicken retina are a consequence of a null mutation in the photoreceptor guanylate cyclase (GC1) gene. Thus, the
rd
chicken is a model for human Leber’s congenital amaurosis. Absence of GC1 in
rd
retina prevents phototransduction and affects survival of rods and cones but does not interfere with normal photoreceptor development.
Spatially independent activity patterns in functional MRI data during the Stroop color-naming task
Authors: McKeown Martin J. , Jung Tzyy-Ping , Makeig Scott , Brown Greg , Kindermann Sandra S. , Lee Te-Won , Sejnowski Terrence J.
Published: July 2002

A method is given for determining the time course and spatial
extent of consistently and transiently task-related activations from
other physiological and artifactual components that contribute to
functional MRI (fMRI) recordings. Independent component analysis (ICA)
was used to analyze two fMRI data sets from a subject performing 6-min
trials composed of alternating 40-sec Stroop color-naming and control
task blocks. Each component consisted of a fixed three-dimensional
spatial distribution of brain voxel values (a “map”) and an
associated time course of activation. For each trial, the algorithm
detected, without
a priori
knowledge of their spatial or
temporal structure, one consistently task-related component activated
during each Stroop task block, plus several transiently task-related
components activated at the onset of one or two of the Stroop task
blocks only. Activation patterns occurring during only part of the fMRI
trial are not observed with other techniques, because their time
courses cannot easily be known in advance. Other ICA components were
related to physiological pulsations, head movements, or machine noise.
By using higher-order statistics to specify stricter criteria for
spatial independence between component maps, ICA produced improved
estimates of the temporal and spatial extent of task-related activation
in our data compared with principal component analysis (PCA). ICA
appears to be a promising tool for exploratory analysis of fMRI data,
particularly when the time courses of activation are not known in
advance.
Components of verbal working memory: Evidence from neuroimaging
Authors: Smith Edward E. , Jonides John , Marshuetz Christy , Koeppe Robert A.
Published: July 2002

We review research on the neural bases of verbal working
memory, focusing on human neuroimaging studies. We first consider
experiments that indicate that verbal working memory is composed of
multiple components. One component involves the subvocal rehearsal of
phonological information and is neurally implemented by left-hemisphere
speech areas, including Broca’s area, the premotor area, and the
supplementary motor area. Other components of verbal working memory may
be devoted to pure storage and to executive processing of the contents
of memory. These studies rest on a subtraction logic, in which two
tasks are imaged, differing only in that one task presumably has an
extra process, and the difference image is taken to reflect that
process. We then review studies that show that the previous results can
be obtained with experimental methods other than subtraction. We focus
on the method of parametric variation, in which a parameter that
presumably reflects a single process is varied. In the last section, we
consider the distinction between working memory tasks that require only
storage of information vs. those that require that the stored items be
processed in some way. These experiments provide some support for the
hypothesis that, when a task requires processing the contents of
working memory, the dorsolateral prefrontal cortex is
disproportionately activated.
Nuclear and cytoplasmic free calcium level changes induced by elastin peptides in human endothelial cells
Authors: Faury Gilles , Usson Yves , Robert-Nicoud Michel , Robert Ladislas , Verdetti Jean
Published: July 2002

The extracellular matrix protein “elastin” is the major component of elastic fibers present in the arterial wall. Physiological degradation of elastic fibers, enhanced in vascular pathologies, leads to the presence of circulating elastin peptides (EP). EP have been demonstrated to influence cell migration and proliferation. EP also induce, at circulating pathophysiological concentrations (and not below), an endothelium- and NO- dependent vasorelaxation mediated by the 67-kDa subunit of the elastin-laminin receptor. Here, by using the techniques of patch-clamp, spectrofluorimetry and confocal microscopy, we demonstrate that circulating concentrations of EP activate low specificity calcium channels on human umbilical venous endothelial cells, resulting in increase in cytoplasmic and nuclear free calcium concentrations. This action is independent of phosphoinositide metabolism. Furthermore, these effects are inhibited by lactose, an antagonist of the elastin-laminin receptor, and by cytochalasin D, an actin microfilament depolymerizer. These observations suggest that EP-induced signal transduction is mediated by the elastin-laminin receptor via coupling of cytoskeletal actin microfilaments to membrane channels and to the nucleus. Because vascular remodeling and carcinogenesis are accompanied by extracellular matrix modifications involving elastin, the processes here described could play a role in the elastin-laminin receptor-mediated cellular migration, differentiation, proliferation, as in atherogenesis, and metastasis formation.
Glutathione levels in antigen-presenting cells modulate Th1 versus Th2 response patterns
Authors: Peterson Jeffrey D. , Herzenberg Leonore A. , Vasquez Kristine , Waltenbaugh Carl
Published: July 2002

Current thinking attributes the balance between T helper 1 (Th1) and Th2 cytokine response patterns in immune responses to the nature of the antigen, the genetic composition of the host, and the cytokines involved in the early interaction between T cells and antigen-presenting cells. Here we introduce glutathione, a tripeptide that regulates intracellular redox and other aspects of cell physiology, as a key regulatory element in this process. By using three different methods to deplete glutathione from T cell receptor transgenic and conventional mice and studyingin vivoand/orin vitroresponses to three distinct antigens, we show that glutathione levels in antigen-presenting cells determine whether Th1 or Th2 response patterns predominate. These findings present new insights into immune response alterations in HIV and other diseases. Further, they potentially offer an explanation for the well known differences in immune responses in “Th1” and “Th2” mouse strains.
NF-κB-dependent inhibition of apoptosis is essential for host cell survival during
Rickettsia rickettsii
infection
Authors: Clifton Dawn R. , Goss Rachel A. , Sahni Sanjeev K. , van Antwerp Daniel , Baggs Raymond B. , Marder Victor J. , Silverman David J. , Sporn Lee Ann
Published: July 2002

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using
Rickettsia rickettsii
, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during
in vivo
infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that
R. rickettsii
infection activates the transcription factor NF-κB and alters expression of several genes under its control. However, when
R. rickettsii-
induced activation of NF-κB was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit IκB that rendered NF-κB inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived.
R. rickettsii
, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-κB activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-κB activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell’s apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection.
A third member of the synapsin gene family
Authors: Kao Hung-Teh , Porton Barbara , Czernik Andrew J. , Feng Jian , Yiu Glenn , Häring Monika , Benfenati Fabio , Greengard Paul
Published: July 2002

Synapsins are a family of neuron-specific synaptic vesicle-associated phosphoproteins that have been implicated in synaptogenesis and in the modulation of neurotransmitter release. In mammals, distinct genes for synapsins I and II have been identified, each of which gives rise to two alternatively spliced isoforms. We have now cloned and characterized a third member of the synapsin gene family, synapsin III, from human DNA. Synapsin III gives rise to at least one protein isoform, designated synapsin IIIa, in several mammalian species. Synapsin IIIa is associated with synaptic vesicles, and its expression appears to be neuron-specific. The primary structure of synapsin IIIa conforms to the domain model previously described for the synapsin family, with domains A, C, and E exhibiting the highest degree of conservation. Synapsin IIIa contains a novel domain, termed domain J, located between domains C and E. The similarities among synapsins I, II, and III in domain organization, neuron-specific expression, and subcellular localization suggest a possible role for synapsin III in the regulation of neurotransmitter release and synaptogenesis. The human synapsin III gene is located on chromosome 22q12–13, which has been identified as a possible schizophrenia susceptibility locus. On the basis of this localization and the well established neurobiological roles of the synapsins, synapsin III represents a candidate gene for schizophrenia.
Crystal structure of troponin C in complex with troponin I fragment at 2.3-Å resolution
Authors: Vassylyev Dmitry G. , Takeda Soichi , Wakatsuki Soichi , Maeda Kayo , Maéda Yuichiro
Published: July 2002

Troponin (Tn), the complex of three subunits (TnC, TnI, and TnT), plays a key role in Ca
2+
-dependent regulation of muscle contraction. To elucidate the interactions between the Tn subunits and the conformation of TnC in the Tn complex, we have determined the crystal structure of TnC (two Ca
2+
bound state) in complex with the N-terminal fragment of TnI (TnI
1–47
). The structure was solved by the single isomorphous replacement method in combination with multiple wavelength anomalous dispersion data. The refinement converged to a crystallographic
R
factor of 22.2% (
R
free
= 32.6%). The central, connecting α-helix observed in the structure of uncomplexed TnC (TnC
free
) is unwound at the center (residues Ala-87, Lys-88, Gly-89, Lys-90, and Ser-91) and bent by 90°. As a result, TnC in the complex has a compact globular shape with direct interactions between the N- and C-terminal lobes, in contrast to the elongated dumb-bell shaped molecule of uncomplexed TnC. The 31-residue long TnI
1–47
α-helix stretches on the surface of TnC and stabilizes its compact conformation by multiple contacts with both TnC lobes. The amphiphilic C-end of the TnI
1–47
α-helix is bound in the hydrophobic pocket of the TnC C-lobe through 38 van der Waals interactions. The results indicate the major difference between Ca
2+
receptors integrated with the other proteins (TnC in Tn) and isolated in the cytosol (calmodulin). The TnC/TnI
1–47
structure implies a mechanism of how Tn regulates the muscle contraction and suggests a unique α-helical regulatory TnI segment, which binds to the N-lobe of TnC in its Ca
2+
bound conformation.
Crystal structures of two tropinone reductases: Different reaction stereospecificities in the same protein fold
Authors: Nakajima Keiji , Yamashita Atsuko , Akama Hiroyuki , Nakatsu Toru , Kato Hiroaki , Hashimoto Takashi , Oda Jun’ichi , Yamada Yasuyuki
Published: July 2002

A pair of tropinone reductases (TRs) share 64% of the same amino acid residues and belong to the short-chain dehydrogenase/reductase family. In the synthesis of tropane alkaloids in several medicinal plants, the TRs reduce a carbonyl group of an alkaloid intermediate, tropinone, to hydroxy groups with different diastereomeric configurations. To clarify the structural basis for their different reaction stereospecificities, we determined the crystal structures of the two enzymes at 2.4- and 2.3-Å resolutions. The overall folding of the two enzymes was almost identical. The conservation was not confined within the core domains that are conserved within the protein family but extended outside the core domain where each family member has its characteristic structure. The binding sites for the cofactor and the positions of the active site residues were well conserved between the two TRs. The substrate binding site was composed mostly of hydrophobic amino acids in both TRs, but the presence of different charged residues conferred different electrostatic environments on the two enzymes. A modeling study indicated that these charged residues play a major role in controlling the binding orientation of tropinone within the substrate binding site, thereby determining the stereospecificity of the reaction product. The results obtained herein raise the possibility that in certain cases different stereospecificities can be acquired in enzymes by changing a few amino acid residues within substrate binding sites.
An engineered closterovirus RNA replicon and analysis of heterologous terminal sequences for replication
Authors: Satyanarayana T. , Gowda S. , Boyko V. P. , Albiach-Marti M. R. , Mawassi M. , Navas-Castillo J. , Karasev A. V. , Dolja V. , Hilf M. E. , Lewandowski D. J. , Moreno P. , Bar-Joseph M. , Garnsey S. M. , Dawson W. O.
Published: July 2002

Citrus tristeza virus (CTV) populations in citrus trees are unusually complex mixtures of viral genotypes and defective RNAs developed during the long-term vegetative propagation of the virus and by additional mixing by aphid transmission. The viral replication process allows the maintenance of minor amounts of disparate genotypes and defective RNAs in these populations. CTV is a member of the
Closteroviridae
possessing a positive-stranded RNA genome of ≈20 kilobases that expresses the replicase-associated genes as an ≈400-kDa polyprotein and the remaining 10 3′ genes through subgenomic mRNAs. A full-length cDNA clone of CTV was generated from which RNA transcripts capable of replication in protoplasts were derived. The large size of cDNA hampered its use as a genetic system. Deletion of 10 3′ genes resulted in an efficient RNA replicon that was easy to manipulate. To investigate the origin and maintenance of the genotypes in CTV populations, we tested the CTV replicase for its acceptance of divergent sequences by creating chimeric replicons with heterologous termini and examining their ability to replicate. Exchange of the similar 3′ termini resulted in efficient replication whereas substitution of the divergent (up to 58% difference in sequence) 5′ termini resulted in reduced but significant replication, generally in proportion to the extent of sequence divergence.
Selective loss of cone function in mice lacking the cyclic nucleotide-gated channel CNG3
Authors: Biel Martin , Seeliger Mathias , Pfeifer Alexander , Kohler Konrad , Gerstner Andrea , Ludwig Andreas , Jaissle Gesine , Fauser Sascha , Zrenner Eberhart , Hofmann Franz
Published: July 2002

Two types of photoreceptors, rods and cones, coexist in the vertebrate retina. An in-depth analysis of the retinal circuitry that transmits rod and cone signals has been hampered by the presence of intimate physical and functional connections between rod and cone pathways. By deleting the cyclic nucleotide-gated channel CNG3 we have generated a mouse lacking any cone-mediated photoresponse. In contrast, the rod pathway is completely intact in CNG3-deficient mice. The functional loss of cone function correlates with a progressive degeneration of cone photoreceptors but not of other retinal cell types. CNG3-deficient mice provide an animal model to dissect unequivocally the contribution of rod and cone pathways for normal retinal function.
Cellular mechanisms of neuropathic pain, morphine tolerance, and their interactions
Authors: Mayer David J. , Mao Jianren , Holt Jason , Price Donald D.
Published: July 2002

Compelling evidence has accumulated over the last several
years from our laboratory, as well as others, indicating that central
hyperactive states resulting from neuronal plastic changes within the
spinal cord play a critical role in hyperalgesia associated with nerve
injury and inflammation. In our laboratory, chronic constriction injury
of the common sciatic nerve, a rat model of neuropathic pain, has been
shown to result in activation of central nervous system excitatory
amino acid receptors and subsequent intracellular cascades including
protein kinase C translocation and activation, nitric oxide production,
and nitric oxide-activated poly(ADP ribose) synthetase activation.
Similar cellular mechanisms also have been implicated in the
development of tolerance to the analgesic effects of morphine. A
recently observed phenomenon, the development of “dark neurons,”
is associated with both chronic constriction injury and morphine
tolerance. A site of action involved in both hyperalgesia and morphine
tolerance is in the superficial laminae of the spinal cord dorsal horn.
These observations suggest that hyperalgesia and morphine tolerance may
be interrelated at the level of the superficial laminae of the dorsal
horn by common neural substrates that interact at the level of
excitatory amino acid receptor activation and subsequent intracellular
events. The demonstration of interrelationships between neural
mechanisms underlying hyperalgesia and morphine tolerance may lead to a
better understanding of the neurobiology of these two phenomena in
particular and pain in general. This knowledge may also provide a
scientific basis for improved pain management with opiate analgesics.
Selective expression of the large neutral amino acid transporter at the blood–brain barrier
Authors: Boado Ruben J. , Li Jian Yi , Nagaya Marie , Zhang Crystal , Pardridge William M.
Published: July 2002

Amino acid supply in brain is regulated by the activity of the large neutral amino acid transporter (LAT) at the brain capillary endothelial cell, which forms the blood–brain barrier (BBB)
in vivo
. Bovine BBB poly(A)
+
RNA was isolated from 2.0 kg of fresh bovine brain and size fractionated on a sucrose density gradient, and a size-fractionated bovine BBB cDNA library in the pSPORT vector was prepared. The full-length cDNA encoding the bovine BBB LAT was isolated from this library, and the predicted amino acid sequence was 89–92% identical to the LAT1 isoform. The bovine BBB LAT1 mRNA produced a 10-fold enhancement in tryptophan transport into frog oocytes coinjected with bovine BBB LAT1 mRNA and the mRNA for 4F2hc, which encodes the heavy chain of the heterodimer. Tryptophan transport into the mRNA-injected oocytes was sodium independent and was specifically inhibited by other large neutral amino acids, and the
K
m
of tryptophan transport was 31.5 ± 5.5 μM. Northern blotting with the bovine BBB LAT1 cDNA showed that the LAT1 mRNA is 100-fold higher in isolated bovine brain capillaries compared with C6 rat glioma cells or rat brain, and the LAT1 mRNA was not detected in rat liver, heart, lung, or kidney. These studies show that the LAT1 transcript is selectively expressed at the BBB compared with other tissues, and the abundance of the LAT1 mRNA at the BBB is manyfold higher than that of transcripts such as the 4F2hc antigen, actin, or the
Glut1
glucose transporter.
A structural view of evolutionary divergence
Authors: Spiller Ben , Gershenson Anne , Arnold Frances H. , Stevens Raymond C.
Published: July 2002

Two directed evolution experiments on
p
-nitrobenzyl esterase yielded one enzyme with a 100-fold increased activity in aqueous-organic solvents and another with a 17°C increase in thermostability. Structures of the wild type and its organophilic and thermophilic counterparts are presented at resolutions of 1.5 Å, 1.6 Å, and 2.0 Å, respectively. These structures identify groups of interacting mutations and demonstrate how directed evolution can traverse complex fitness landscapes. Early-generation mutations stabilize flexible loops not visible in the wild-type structure and set the stage for further beneficial mutations in later generations. The mutations exert their influence on the esterase structure over large distances, in a manner that would be difficult to predict. The loops with the largest structural changes generally are not the sites of mutations. Similarly, none of the seven amino acid substitutions in the organophile are in the active site, even though the enzyme experiences significant changes in the organization of this site. In addition to reduction of surface loop flexibility, thermostability in the evolved esterase results from altered core packing, helix stabilization, and the acquisition of surface salt bridges, in agreement with other comparative studies of mesophilic and thermophilic enzymes. Crystallographic analysis of the wild type and its evolved counterparts reveals networks of mutations that collectively reorganize the active site. Interestingly, the changes that led to diversity within the α/β hydrolase enzyme family and the reorganization seen in this study result from main-chain movements.
Transfection of rat kidney with human 15-lipoxygenase suppresses inflammation and preserves function in experimental glomerulonephritis
Authors: Munger Karen A. , Montero Angel , Fukunaga Megumu , Uda Susumu , Yura Takafumi , Imai Enyu , Kaneda Yasufumi , Valdivielso José M. , Badr Kamal F.
Published: July 2002

The human 15-lipoxygenase (15-LO) gene was transfected into rat kidneysin vivovia intra-renal arterial injection. Three days later, acute (passive) or accelerated forms of antiglomerular basement membrane antibody-mediated glomerulonephritis were induced in transfected and nontransfected or sham-transfected controls. Studies of glomerular functions (filtration and protein excretion) andex vivoglomerular leukotriene B4biosynthesis at 3 hr, and up to 4 days, after induction of nephritis revealed preservation or normalization of these parameters in transfected kidneys that expressed human 15-LO mRNA and mature protein, but not in contralateral control kidneys or sham-transfected animals. The results providein vivo-derived data supporting a direct anti-inflammatory role for 15-LO during immune-mediated tissue injury.
Expression of the
Bs2
pepper gene confers resistance to bacterial spot disease in tomato
Authors: Tai Thomas H. , Dahlbeck Douglas , Clark Eszter T. , Gajiwala Paresh , Pasion Romela , Whalen Maureen C. , Stall Robert E. , Staskawicz Brian J.
Published: July 2002

The
Bs2
resistance gene of pepper specifically recognizes and confers resistance to strains of
Xanthomonas campestris
pv.
vesicatoria
that contain the corresponding bacterial avirulence gene,
avrBs2
. The involvement of
avrBs2
in pathogen fitness and its prevalence in many
X. campestris
pathovars suggests that the
Bs2
gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the
Bs2
locus was isolated and the gene was identified by coexpression with
avrBs2
in an
Agrobacterium
-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site–leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species,
Nicotiana benthamiana
, and was designated as
Bs2
. Functional expression of
Bs2
in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species.
Genomic structure and ecdysone regulation of the prophenoloxidase 1 gene in the malaria vector
Anopheles gambiae
Authors: Ahmed A. , Martín D. , Manetti A. G. O. , Han S.-J. , Lee W.-J. , Mathiopoulos K. D. , Müller H.-M. , Kafatos F. C. , Raikhel A. , Brey P. T.
Published: July 2002

Prophenoloxidase, a melanin-synthesizing enzyme, is considered to be an important arthropod immune protein. In mosquitoes, prophenoloxidase has been shown to be involved in refractory mechanisms against malaria parasites. In our study we used
Anopheles gambiae
, the most important human malaria vector, to characterize the first arthropod prophenoloxidase gene at the genomic level. The complete nucleotide sequence, including the immediate 5′ flanking sequence (−855 bp) of the prophenoloxidase 1 gene, was determined. The gene spans 10 kb and is composed of five exons and four introns coding for a 2.5-kb mRNA. In the 5′ flanking sequence, we found several putative regulatory motifs, two of which were identified as ecdysteroid regulatory elements. Electrophoretic mobility gel-shift assays and supershift assays demonstrated that the
Aedes aegypti
ecdysone receptor/
Ultraspiracle
nuclear receptor complex, and, seemingly, the endogenous
Anopheles gambiae
nuclear receptor complex, was able to bind one of the ecdysteroid response elements. Furthermore, 20-hydroxyecdysone stimulation was shown to up-regulate the transcription of the prophenoloxidase 1 gene in an
A. gambiae
cell line.
A plant virus-encoded protein facilitates long-distance movement of heterologous viral RNA
Authors: Ryabov Eugene V. , Robinson David J. , Taliansky Michael E.
Published: July 2002

Transport of plant viruses from cell to cell typically involves one
or more viral proteins that supply specific cell-to-cell movement
functions. Long-distance transport of viruses through the vascular
system is a less well understood process with requirements different
from those of cell-to-cell movement. Usually viral coat protein (CP) is
required for long-distance movement, but groundnut rosette umbravirus
(GRV) does not code for a CP. However, this virus moves efficiently
from cell to cell and long distance. We demonstrate here that the
protein encoded by
ORF3
of GRV can functionally replace
the CP of tobacco mosaic virus (TMV) for long-distance movement. In
spite of low levels of virus RNA accumulation in infected cells,
chimeric TMV with a replacement of the
CP
gene by GRV
ORF3
was able to move rapidly through the phloem. Moreover,
this chimeric virus complemented long-distance movement of another
CP-deficient TMV derivative expressing the gene encoding the green
fluorescent protein. Thus, the GRV
ORF3
-encoded protein
represents a class of trans-acting long-distance movement factors that
can facilitate trafficking of an unrelated viral RNA.
Origin and evolution of the 1918 “Spanish” influenza virus hemagglutinin gene
Authors: Reid Ann H. , Fanning Thomas G. , Hultin Johan V. , Taubenberger Jeffery K.
Published: July 2002

The “Spanish” influenza pandemic killed over 20 million
people in 1918 and 1919, making it the worst infectious pandemic in
history. Here, we report the complete sequence of the hemagglutinin
(HA) gene of the 1918 virus. Influenza RNA for the analysis was
isolated from a formalin-fixed, paraffin-embedded lung tissue sample
prepared during the autopsy of a victim of the influenza pandemic in
1918. Influenza RNA was also isolated from lung tissue samples from two
additional victims of the lethal 1918 influenza: one formalin-fixed,
paraffin-embedded sample and one frozen sample obtained by
in
situ
biopsy of the lung of a victim buried in permafrost since
1918. The complete coding sequence of the A/South Carolina/1/18
HA gene was obtained. The HA1 domain sequence was confirmed by using
the two additional isolates (A/New York/1/18 and A/Brevig
Mission/1/18). The sequences show little variation. Phylogenetic
analyses suggest that the 1918 virus HA gene, although more closely
related to avian strains than any other mammalian sequence, is
mammalian and may have been adapting in humans before 1918.
Elimination of zinc from synaptic vesicles in the intact mouse brain by disruption of the
ZnT
3
gene
Authors: Cole Toby B. , Wenzel H. Jürgen , Kafer Kathy E. , Schwartzkroin Philip A. , Palmiter Richard D.
Published: July 2002

The mammalian protein ZnT3 resides on synaptic vesicle membranes of
zinc-containing neurons, suggesting its possible role in vesicular zinc
transport. We show here that histochemically reactive zinc,
corresponding to the zinc found within synaptic vesicles, was
undetectable in the brains of mice with targeted disruption of the
ZnT3
gene. Total zinc levels in the hippocampus and
cortex of these mice were reduced by about 20%. The ultrastructure of
mossy fiber boutons, which normally store the highest levels of
vesicular zinc, was unaffected. Mice with one normal
ZnT3
allele had reduced levels of ZnT3 protein on
synaptic vesicle membranes and had intermediate amounts of vesicular
zinc. These results demonstrate that ZnT3 is required for transport of
zinc into synaptic vesicles and suggest that vesicular zinc
concentration is determined by the abundance of ZnT3.
TheSalmonellainvasin SipB induces macrophage apoptosis by binding to caspase-1
Authors: Hersh David , Monack Denise M. , Smith Mark R. , Ghori Nafisa , Falkow Stanley , Zychlinsky Arturo
Published: July 2002

Recently,Salmonellaspp. were shown to induce apoptosis in infected macrophages. The mechanism responsible for this process is unknown. In this report, we establish that the Inv-Spa type III secretion apparatus target invasin SipB is necessary and sufficient for the induction of apoptosis. Purified SipB microinjected into macrophages led to cell death. Binding studies show that SipB associates with the proapoptotic protease caspase-1. This interaction results in the activation of caspase-1, as seen in its proteolytic maturation and the processing of its substrate interleukin-1β. Caspase-1 activity is essential for the cytotoxicity. Functional inhibition of caspase-1 activity by acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone blocks macrophage cytotoxicity, and macrophages lacking caspase-1 are not susceptible toSalmonella-induced apoptosis. Taken together, the data demonstrate that SipB functions as an analog of theShigellainvasin IpaB.
Molecular evidence of cryptic speciation in planktonic foraminifers and their relation to oceanic provinces
Authors: de Vargas Colomban , Norris Richard , Zaninetti Louisette , Gibb Stuart W. , Pawlowski Jan
Published: July 2002

The fossil record of planktonic foraminifers is a key source of data on the biodiversity and evolution of marine plankton. One of the most distinctive foraminiferal taxa,
Orbulina universa
, widely used as a stratigraphic and paleoclimatic index, has always been regarded as a single species. Here we present a phylogenetic analysis of
Orbulina
small subunit rDNA sequences from 25 pelagic stations covering 100° latitude in the Atlantic Ocean. The genetic data reveal the presence of three cryptic species, whose distribution is clearly correlated to hydrographic provinces, and particularly to sea-surface total chlorophyll a concentration. Our results, together with previous studies, suggest that a considerable part of the diversity among planktonic foraminifers has been overlooked in morphological taxonomies. Our data also support the idea that planktonic foraminifers, even if adapted to particular hydrographic conditions, are high-dispersal organisms whose speciation may be similar to that of other high-dispersal taxa in which reproductive mechanisms and behavior, rather than just geographic barriers to dispersal, play key roles in species formation and maintenance.
Strand asymmetry and codon usage bias in the chloroplast genome of
Euglena gracilis
Authors: Morton Brian R.
Published: July 2002

It is shown that the two strands of the chloroplast genome from
Euglena gracilis
are asymmetric with regards to nucleotide composition. This asymmetry switches at both the origin of replication and a location that is halfway around the circular genome from the origin. In both halves of the genome the leading strand is G+T-rich, having a bias toward G over C and T over A, and the lagging strand is A+C-rich. This asymmetry is probably the result of a difference in mutation dynamics between the leading and lagging strands. In addition to composition asymmetry, the two strands differ with regards to coding content. In both halves of the genome the vast majority of genes are coded by the leading strand. These two aspects of strand asymmetry are then applied to a statistical test for selection on codon usage. The results indicate that selection on codon usage is limited to genes on the leading strand; no gene on the A+C-rich lagging strand shows evidence for selection, suggesting that highly expressed genes are coded predominantly on the strand of DNA that is the leading strand during replication. On the basis of these observations it is proposed that the coding strand bias is generated by selection to code highly expressed genes on the leading strand to coordinate the direction of replication and transcription, thereby increasing the potential rate of both reactions.
Vascular endothelial growth factor: Direct neuroprotective effect in
in vitro
ischemia
Authors: Jin Kun Lin , Mao Xiao Ou , Greenberg David A.
Published: July 2002

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenic peptide with recently identified neurotrophic effects. Because some neurotrophic factors can protect neurons from hypoxic or ischemic injury, we investigated the possibility that VEGF has similar neuroprotective properties. In HN33, an immortalized hippocampal neuronal cell line, VEGF reduced cell death associated with an
in vitro
model of cerebral ischemia: at a maximally effective concentration of 50 ng/ml, VEGF approximately doubled the number of cells surviving after 24 h of hypoxia and glucose deprivation. To investigate the mechanism of neuroprotection by VEGF, the expression of known target receptors for VEGF was measured by Western blotting, which showed that HN33 cells expressed VEGFR-2 receptors and neuropilin-1, but not VEGFR-1 receptors. The neuropilin-1 ligand placenta growth factor-2 failed to reproduce the protective effect of VEGF, pointing to VEGFR-2 as the site of VEGF's neuroprotective action. Two phosphatidylinositol 3′-kinase inhibitors, wortmannin and LY294002, reversed the neuroprotective effect of VEGF, implicating the phosphatidylinositol 3′-kinase/Akt signal transduction system in VEGF-mediated neuroprotection. VEGF also protected primary cultures of rat cerebral cortical neurons from hypoxia and glucose deprivation. We conclude that in addition to its known role as an angiogenic factor, VEGF may exert a direct neuroprotective effect in hypoxic-ischemic injury.
A method for detecting abasic sites in living cells: Age-dependent changes in base excision repair
Authors: Atamna Hani , Cheung Ivana , Ames Bruce N.
Published: July 2002

Apurinic/apyrimidinic (AP) sites are common DNA lesions that arise from spontaneous depurination or by base excision repair (BER) of modified bases. A biotin-containing aldehyde-reactive probe (ARP) [Kubo, K., Ide, H., Wallace, S. S. & Kow, Y. W. (1992)
Biochemistry
31, 3703–3708] is used to measure AP sites in living cells. ARP penetrates the plasma membrane of cells and reacts with AP sites in DNA to form a stable ARP–DNA adduct. The DNA is isolated and treated with avidin-horseradish peroxidase (HRP), forming a DNA–HRP complex at each biotin residue, which is rapidly separated from free avidin–HRP by selective precipitation with a DNA precipitating dye (DAPER). The number of AP sites is estimated by HRP activity toward chromogenic substrate in an ELISA assay. The assay integrates the AP sites formed by the different glycosylases of BER during a 1-h incubation and eliminates artifactual depurination or loss of AP sites during DNA isolation. The assay was applied to living cells and nuclei. The number of AP sites after a 1-h incubation in old IMR90 cells was about two to three times higher than that in young cells, and the number in human leukocytes from old donors was about seven times that in young donors. The repair of AP sites was slower in senescent compared with young IMR90 cells. An age-dependent decline is shown in the activity of the glycosylase that removes methylated bases in IMR90 cells and in human leukocytes. The decline in excision of methylated bases from DNA suggests an age-dependent decline in 3-methyladenine DNA glycosylase, a BER enzyme responsible for removing alkylated bases.
Heritability of fitness in a wild mammal population
Authors: Kruuk Loeske E. B. , Clutton-Brock Tim H. , Slate Jon , Pemberton Josephine M. , Brotherstone Sue , Guinness Fiona E.
Published: July 2002

Classical population genetics theory predicts that selection should deplete heritable genetic variance for fitness. We show here that, consistent with this prediction, there was a negative correlation between the heritability of a trait and its association with fitness in a wild population of red deer (
Cervus elaphus
) and there was no evidence of significant heritability of total fitness. However, the decline in heritability was caused, at least in part, by increased levels of residual variance in longevity and, hence, in total fitness: in this population, longevity is known to be heavily influenced by environmental factors. Other life history traits that were not associated with longevity, such as average annual breeding success, had higher heritabilities. Coefficients of additive genetic variance differed markedly between traits, but highly skewed measures, such as male breeding success, generally had greater coefficients of variance than morphometric traits. Finally, there were significant maternal effects in a range of traits, particularly for females.
Adenoviral gene transfer of SERCA2a improves left-ventricular function in aortic-banded rats in transition to heart failure
Authors: Miyamoto Michael I. , del Monte Federica , Schmidt Ulrich , DiSalvo Thomas S. , Kang Zhao Bin , Matsui Takashi , Guerrero J. Luis , Gwathmey Judith K. , Rosenzweig Anthony , Hajjar Roger J.
Published: July 2002

In human and experimental models of heart failure, sarcoplasmic reticulum Ca
2+
ATPase (SERCA2a) activity is decreased, resulting in abnormal calcium handling. The disturbances in calcium metabolism have been shown to contribute significantly to the contractile dysfunction observed in heart failure. We investigated whether increasing SERCA2a expression can improve ventricular function in an animal model of heart failure obtained by creating ascending aortic constriction in rats. After 19–23 wk of banding during the transition from compensated hypertrophy to heart failure (documented by >25% decrease in fractional shortening), rats were randomized to receive either an adenovirus carrying the SERCA2a gene (Ad.SERCA2a,
n
= 13) or β-galactosidase (Ad.βgal,
n
= 14) by using a catheter-based technique. The failing hearts infected with Ad.βgal were characterized by a significant decrease in SERCA2a expression and a decrease in SERCA2a activity compared with nonfailing sham-operated rats (
n
= 11). In addition, these failing hearts had reduced left-ventricular systolic pressure, maximal rate of left-ventricular pressure rise and decline (+
dP/dt
, −
dP/dt
), and rate of isovolumic relaxation (τ). Overexpression of SERCA2a restored both SERCA2a expression and ATPase activity to nonfailing levels. Furthermore, rats infected with Ad.SERCA2a had significant improvement in left-ventricular systolic pressure, +
dP/dt
, −
dP/dt
, and rate of isovolumic relaxation (τ) normalizing them back to levels comparable to sham-operated rats. In this study, we show that in an animal model of heart failure where SERCA2a protein levels and activity are decreased and severe contractile dysfunction is present, overexpression of SERCA2a
in vivo
restores both systolic and diastolic function to normal levels.
Role of Rab GDP dissociation inhibitor α in regulating plasticity of hippocampal neurotransmission
Authors: Ishizaki Hiroyoshi , Miyoshi Jun , Kamiya Haruyuki , Togawa Atsushi , Tanaka Miki , Sasaki Takuya , Endo Katsuaki , Mizoguchi Akira , Ozawa Seiji , Takai Yoshimi
Published: July 2002

Rab GDP dissociation inhibitor α (Rab GDIα) is a regulator of
the Rab small G proteins implicated in neurotransmission, and mutations
of Rab GDIα cause human X-linked mental retardation associated with
epileptic seizures. In Rab GDIα-deficient mice, synaptic potentials
in the CA1 region of the hippocampus displayed larger enhancement
during repetitive stimulation, which was apparently opposite to the
phenotype of Rab3A-deficient mice. Furthermore, the Rab
GDIα-deficient mice showed hypersensitivity to bicuculline, an
inducer of epileptic seizures. These results suggest that Rab GDIα
plays a specialized role in Rab3A recycling to suppress
hyperexcitability via modulation of presynaptic forms of plasticity.