bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb
Authors: Ezhevsky Sergei A. , Nagahara Hikaru , Vocero-Akbani Adita M. , Gius David R. , Wei Michael C. , Dowdy Steven F.
Published: July 2002

In cycling cells, the retinoblastoma protein (pRb) is un- and/or hypo-phosphorylated in early G
1
and becomes hyper-phosphorylated in late G
1
. The role of hypo-phosphorylation and identity of the relevant kinase(s) remains unknown. We show here that hypo-phosphorylated pRb associates with E2F
in vivo
and is therefore active. Increasing the intracellular concentration of the Cdk4/6 specific inhibitor p15
INK4b
by transforming growth factor β treatment of keratinocytes results in G
1
arrest and loss of hypo-phosphorylated pRb with an increase in unphosphorylated pRb. Conversely, p15
INK4b
-independent transforming growth factor β-mediated G
1
arrest of hepatocellular carcinoma cells results in loss of Cdk2 kinase activity with continued Cdk6 kinase activity and pRb remains only hypo-phosphorylated. Introduction of the Cdk4/6 inhibitor p16
INK4a
protein into cells by fusion to a protein transduction domain also prevents pRb hypo-phosphorylation with an increase in unphosphorylated pRb. We conclude that cyclin D:Cdk4/6 complexes hypo-phosphorylate pRb in early G
1
allowing continued E2F binding.
Inhibition of phospholipase D by lysophosphatidylethanolamine, a lipid-derived senescence retardant
Authors: Ryu Stephen B. , Karlsson Björn H. , Özgen Mustafa , Palta Jiwan P.
Published: July 2002

Phospholipid signaling mediated by lipid-derived second messengers or biologically active lipids is still new and is not well established in plants. We recently have found that lysophosphatidylethanolamine (LPE), a naturally occurring lipid, retards senescence of leaves, flowers, and postharvest fruits. Phospholipase D (PLD) has been suggested as a key enzyme in mediating the degradation of membrane phospholipids during the early stages of plant senescence. Here we report that LPE inhibited the activity of partially purified cabbage PLD in a cell-free system in a highly specific manner. Inhibition of PLD by LPE was dose-dependent and increased with the length and unsaturation of the LPE acyl chain whereas individual molecular components of LPE such as ethanolamine and free fatty acid had no effect on PLD activity. Enzyme-kinetic analysis suggested noncompetitive inhibition of PLD by LPE. In comparison, the related lysophospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, and lysophosphotidylserine had no significant effect on PLD activity whereas PLD was stimulated by lysophosphatidic acid and inhibited by lysophosphatidylinositol. Membrane-associated and soluble PLD, extracted from cabbage and castor bean leaf tissues, also was inhibited by LPE. Consistent with acyl-specific inhibition of PLD by LPE, senescence of cranberry fruits as measured by ethylene production was more effectively inhibited according to the increasing acyl chain length and unsaturation of LPE. There are no known specific inhibitors of PLD in plants and animals. We demonstrate specific inhibitory regulation of PLD by a lysophospholipid.
The C9 methyl group of retinal interacts with glycine-121 in rhodopsin
Authors: Han May , Groesbeek Michel , Sakmar Thomas P. , Smith Steven O.
Published: July 2002

The visual pigment rhodopsin is a prototypical G protein-coupled receptor. These receptors have seven transmembrane helices and are activated by specific receptor–ligand interactions. Rhodopsin is unusual in that its retinal prosthetic group serves as an antagonist in the dark in the 11-
cis
conformation but is rapidly converted to an agonist on photochemical
cis
to
trans
isomerization. Receptor–ligand interactions in rhodopsin were studied in the light and dark by regenerating site-directed opsin mutants with synthetic retinal analogues. A progressive decrease in light-dependent transducin activity was observed when a mutant opsin with a replacement of Gly
121
was regenerated with 11-
cis
-retinal analogues bearing progressively larger R groups (methyl, ethyl, propyl) at the C9 position of the polyene chain. A progressive decrease in light activity was also observed as a function of increasing size of the residue at position 121 for both the 11-
cis
-9-ethyl- and the 11-
cis
-9-propylretinal pigments. In contrast, a striking increase of receptor activity in the dark—i.e., without chromophore isomerization—was observed when the molecular volume at either position 121 of opsin or C9 of retinal was increased. The ability of bulky replacements at either position to hinder ligand incorporation and to activate rhodopsin in the dark suggests a direct interaction between these two sites. A molecular model of the retinal-binding site of rhodopsin is proposed that illustrates the specific interaction between Gly
121
and the C9 methyl group of 11-
cis
-retinal. Steric interactions in this region of rhodopsin are consistent with the proposal that movement of transmembrane helices 3 and 6 is concomitant with receptor activation.
Allergic airway sensitization induces T cell activation but not airway hyperresponsiveness in B cell-deficient mice
Authors: Hamelmann E. , Vella A. T. , Oshiba A. , Kappler J. W. , Marrack P. , Gelfand E. W.
Published: July 2002

B cells play an important role in the allergic response by producing allergen-specific Igs as well as by serving as antigen-presenting cells. We studied the involvement of B cells in the development of responses in a murine model of allergic airway sensitization. Normal and B cell-deficient (μMt−/−) B10.BR mice were sensitized via the airways to ovalbumin; Ig production, cytokine elaboration from local lymph node cells, development of airway hyperresponsiveness, and histological changes in the airways were evaluated. Both strains of mice had increased production of T helper 2-like cytokines and developed an accumulation of eosinophils in the bronchial tissue after airway sensitization. However, only wild-type mice produced allergen-specific antibodies and exhibited altered airway function. B cell-deficient mice reconstituted with anti-ovalbumin IgE during the course of sensitization developed increases in airway responsiveness. These results indicated that neither B cells nor IgE were necessary for the induction of a T helper 2-type cytokine response or eosinophil infiltration of the airways after allergic sensitization but that IgE was required as a second signal for the development of airway hyperresponsiveness in this model of airway sensitization.
T cell receptor restriction of diabetogenic autoimmune NOD T cells
Authors: Simone E. , Daniel D. , Schloot N. , Gottlieb P. , Babu S. , Kawasaki E. , Wegmann D. , Eisenbarth G. S.
Published: July 2002

Restricted use of T cell receptor (TCR) gene segments is characteristic of several induced autoimmune disease models. TCR sequences have previously been unavailable for pathogenic T cells which react with a defined autoantigen in a spontaneous autoimmune disease. The majority of T cell clones, derived from islets of NOD mice which spontaneously develop type I diabetes, react with insulin peptide B-(9–23). We have sequenced the α and β chains of TCRs from these B-(9–23)-reactive T cell clones. No TCR β chain restriction was found. In contrast, the clones (10 of 13) used Vα13 coupled with one of two homologous Jα segments (Jα45 or Jα34 in 8 of 13 clones). Furthermore, 9 of 10 of the Vα13 segments are a novel NOD sequence that we have tentatively termed Vα13.3. This dramatic α chain restriction, similar to the β chain restriction of other autoimmune models, provides a target for diagnostics and immunomodulatory therapy.
Cyclooxygenase-2 expression in human colon cancer cells increases metastatic potential
Authors: Tsujii Masahiko , Kawano Sunao , DuBois Raymond N.
Published: July 2002

Recent epidemiologic studies have shown a 40–50% reduction in mortality from colorectal cancer in individuals who take nonsteroidal antiinflammatory drugs on a regular basis compared with those not taking these agents. One property shared by all of these drugs is their ability to inhibit cyclooxygenase (COX), a key enzyme in the conversion of arachidonic acid to prostaglandins. Two isoforms of COX have been characterized, COX-1 and COX-2. COX-2 is expressed at high levels in intestinal tumors in humans and rodents. Human colon cancer cells (Caco-2) were permanently transfected with a COX-2 expression vector or the identical vector lacking the COX-2 insert. The Caco-2 cells, which constitutively expressed COX-2, acquired increased invasiveness compared with the parental Caco-2 cells or the vector transfected control cells. Biochemical changes associated with this phenotypic change included activation of metalloproteinase-2 and increased RNA levels for the membrane-type metalloproteinase. Increased invasiveness and prostaglandin production were reversed by treatment with sulindac sulfide, a known COX inhibitor. These studies demonstrate that constitutive expression of COX-2 can lead to phenotypic changes that alter the metastatic potential of colorectal cancer cells.
Genotypes at the GluR6 kainate receptor locus are associated with variation in the age of onset of Huntington disease
Authors: Rubinsztein David C. , Leggo Jayne , Chiano Mathias , Dodge Alan , Norbury Gail , Rosser Elisabeth , Craufurd David
Published: July 2002

Huntington disease (HD) is associated with abnormal expansions of a CAG repeat close to the 5′ end of the
IT15
gene. We have assembled a set of 293 HD subjects whose ages of onset were known and sized their HD CAG repeats. These repeats accounted for 69% of the variance of age of onset when we used the most parsimonious model, which relates the logarithm of age of onset to a function of CAG repeat number. Since other familial factors have been proposed to influence the age of onset of HD, we have examined a number of candidate loci. The CAG repeat number on normal chromosomes, the Δ2642 polymorphism in the HD gene, and apolipoprotein E genotypes did not affect the age of onset of HD. Although mitochondrial energy production defects in HD have led to suggestions that variants in the mitochondrial genome may be associated with clinical variability in HD, this suggestion was not supported by our preliminary experiments that examined the
Dde
I mitochondrial restriction fragment length polymorphism at position 10,394. Excitotoxicity has been a favored mechanism to explain the cell death in HD, particularly since intrastriatal injection of excitatory amino acids in animals creates HD-like pathology. Accordingly, we investigated the GluR6 kainate receptor. Of the variance in the age of onset of HD that was not accounted for by the CAG repeats, 13% could be attributed to GluR6 genotype variation. These data implicate GluR6-mediated excitotoxicity in the pathogenesis of HD and highlight the potential importance of this process in other polyglutamine repeat expansion diseases.
Mice devoid of γ-aminobutyrate type A receptor β3 subunit have epilepsy, cleft palate, and hypersensitive behavior
Authors: Homanics Gregg E. , DeLorey Timothy M. , Firestone Leonard L. , Quinlan Joseph J. , Handforth Adrian , Harrison Neil L. , Krasowski Matthew D. , Rick Caroline E. M. , Korpi Esa R. , Mäkelä Riikka , Brilliant Murray H. , Hagiwara Nobuko , Ferguson Carolyn , Snyder Kimberly , Olsen Richard W.
Published: July 2002

γ-Aminobutyric acid type A receptors (GABA
A
-Rs) mediate the bulk of rapid inhibitory synaptic transmission in the central nervous system. The β3 subunit is an essential component of the GABA
A
-R in many brain regions, especially during development, and is implicated in several pathophysiologic processes. We examined mice harboring a β3 gene inactivated by gene targeting. GABA
A
-R density is approximately halved in brain of β3-deficient mice, and GABA
A
-R function is severely impaired. Most β3-deficient mice die as neonates; some neonatal mortality, but not all, is accompanied by cleft palate. β3-deficient mice that survive are runted until weaning but achieve normal body size by adulthood, although with reduced life span. These mice are fertile but mothers fail to nurture offspring. Brain morphology is grossly normal, but a number of behaviors are abnormal, consistent with the widespread location of the β3 subunit. The mice are very hyperactive and hyperresponsive to human contact and other sensory stimuli, and often run continuously in tight circles. When held by the tail, they hold all paws in like a ball, which is frequently a sign of neurological impairment. They have difficulty swimming, walking on grids, and fall off platforms and rotarods, although they do not have a jerky gait. β3-deficient mice display frequent myoclonus and occasional epileptic seizures, documented by electroencephalographic recording. Hyperactivity, lack of coordination, and seizures are consistent with reduced presynaptic inhibition in spinal cord and impaired inhibition in higher cortical centers and/or pleiotropic developmental defects.
On the actions that one nerve cell can have on another: Distinguishing “drivers” from “modulators”
Authors: Sherman S. Murray , Guillery R. W.
Published: July 2002

When one nerve cell acts on another, its postsynaptic effect can vary greatly. In sensory systems, inputs from “drivers” can be differentiated from those of “modulators.” The driver can be identified as the transmitter of receptive field properties; the modulator can be identified as altering the probability of certain aspects of that transmission. Where receptive fields are not available, the distinction is more difficult and currently is undefined. We use the visual pathways, particularly the thalamic geniculate relay for which much relevant evidence is available, to explore ways in which drivers can be distinguished from modulators. The extent to which the distinction may apply first to other parts of the thalamus and then, possibly, to other parts of the brain is considered. We suggest the following distinctions: Cross-correlograms from driver inputs have sharper peaks than those from modulators; there are likely to be few drivers but many modulators for any one cell; and drivers are likely to act only through ionotropic receptors having a fast postsynaptic effect whereas modulators also are likely to activate metabotropic receptors having a slow and prolonged postsynaptic effect.
Differential roles of Ca
2+
/calmodulin-dependent kinases in posttetanic potentiation at input selective glutamatergic pathways
Authors: Wang Daliang , Maler Leonard
Published: July 2002

The electrosensory lateral line lobe (ELL) of the electric fish
Apteronotus leptorhynchus
is a layered medullary region receiving electroreceptor input that terminates on basal dendrites of interneurons and projection (pyramidal) cells. The molecular layer of the ELL contains two distinct glutamatergic feedback pathways that terminate on the proximal (ventral molecular layer, VML) and distal (dorsal molecular layer) apical dendrites of pyramidal cells. Western blot analysis with an antibody directed against mammalian Ca
2+
/calmodulin-dependent kinase 2, α subunit (CaMK2α) recognized a protein of identical size in the brain of
A. leptorhynchus
. Immunohistochemistry demonstrated that CaMK2 α expression in the ELL was restricted to fibers and terminals in the VML. Posttetanic potentiation (PTP) could be readily elicited in pyramidal cells by stimulation of either VML or DML in brain slices of the ELL. PTP in the VML was blocked by extracellular application of a CaMK2 antagonist (KN62) while intracellular application of KN62 or a CaMK2 inhibitory peptide had no effect, consistent with the presynaptic localization of CaMK2 α in VML. PTP in the dorsal molecular layer was not affected by extracellular application of KN62. Anti-Hebbian plasticity has also been demonstrated in the VML, but was not affected by KN62. These results demonstrate that, while PTP can occur independent of CaMK2, it is, in some synapses, dependent on this kinase.
Su(UR)ES: A gene suppressing DNA underreplication in intercalary and pericentric heterochromatin ofDrosophila melanogasterpolytene chromosomes
Authors: Belyaeva Elena S. , Zhimulev Igor F. , Volkova Elena I. , Alekseyenko Artyom A. , Moshkin Yury M. , Koryakov Dmitry E.
Published: July 2002

A genetic locus suppressing DNA underreplication in intercalary heterochromatin (IH) and pericentric heterochromatin (PH) of the polytene chromosomes ofDrosophila melanogastersalivary glands, has been described. Found in theIn(1)scV2strain, the mutation, designated asSu(UR)ES, was located on chromosome 3L at position 34.8 and cytologically mapped to region 68A3-B4. A cytological phenotype was observed in the salivary gland chromosomes of larvae homozygous and hemizygous forSu(UR)ES: (i) in the IH regions, that normally are incompletely polytenized and so they often break to form “weak points,” underreplication is suppressed, breaks and ectopic contacts disappear; (ii) the degree of polytenization in PH grows higher. That is why the regions in chromosome arm basements, normally β-heterochromatic, acquire a distinct banding pattern, i.e., become euchromatic by morphological criteria; (iii) an additional bulk of polytenized material arises between the arms of chromosome 3 to form a fragment with a typical banding pattern. Chromosome 2 PH reveals additional α-heterochromatin.Su(UR)ESdoes not affect the viability, fertility, or morphological characters of the imago, and has semidominant expression in the heterozygote and distinct maternal effect. The results obtained provide evidence that the processes leading to DNA underreplication in IH and PH are affected by the same genetic mechanism.
Structural characterization and immunochemical detection of a fluorophore derived from 4-hydroxy-2-nonenal and lysine
Authors: Tsai Lin , Szweda Pamela A. , Vinogradova Olga , Szweda Luke I.
Published: July 2002

Aging and the progression of certain degenerative diseases are accompanied by increases in intracellular fluorescent material, termed “lipofuscin” and ceroid, respectively. These pigments are observed within granules composed, in part, of damaged protein and lipid. Modification of various biomolecules by aldehyde products of lipid peroxidation is believed to contribute to lipofuscin and ceroid formation. However, little direct evidence currently exists because the structures responsible for the fluorescent, cross-linked nature of this material are not well characterized. In this study, we have identified a fluorescent product formed in the reaction of
N
α
-acetyllysine and 4-hydroxy-2-nonenal (HNE), a major product of lipid peroxidation and the most reactive of these compounds under physiological conditions [Esterbauer, H., Shaur, R. J. & Zollner, H. (1991)
Free Radical Biol. Med.
11, 81–128]. This fluorescent compound, characterized as a 2-hydroxy-3-imino-1,2-dihydropyrrol derivative, appears to form upon oxidative cyclization of the nonfluorescent 2:1 lysine-HNE Michael adduct-Schiff base cross-link. Polyclonal antibody was raised to the
N
α
-acetyllysine-HNE fluorophore and found to be highly specific to the chromophore structure of the compound. This antibody has been used to conclusively demonstrate that the lysine-HNE derivative of this fluorophore forms on protein upon exposure to HNE. The results of this study therefore provide the basis for future investigations on the contribution(s) of HNE-derived fluorophore formation to lipofuscin and ceroid accumulation.
New enzyme lineages by subdomain shuffling
Authors: Hopfner Karl-Peter , Kopetzki Erhard , Kreße Georg-Burkhard , Bode Wolfram , Huber Robert , Engh Richard A.
Published: July 2002

Protein functions have evolved in part via domain recombination events. Such events, for example, recombine structurally independent functional domains and shuffle targeting, regulatory, and/or catalytic functions. Domain recombination, however, can generate new functions, as implied by the observation of catalytic sites at interfaces of distinct folding domains. If useful to an evolving organism, such initially rudimentary functions would likely acquire greater efficiency and diversity, whereas the initially distinct folding domains would likely develop into single functional domains. This represents the probable evolution of the S1 serine protease family, whose two homologous β-barrel subdomains assemble to form the binding sites and the catalytic machinery. Among S1 family members, the contact interface and catalytic residues are highly conserved whereas surrounding surfaces are highly variable. This observation suggests a new strategy to engineer viable proteins with novel properties, by swapping folding subdomains chosen from among protein family members. Such hybrid proteins would retain properties conserved throughout the family, including folding stability as single domain proteins, while providing new surfaces amenable to directed evolution or engineering of specific new properties. We show here that recombining the N-terminal subdomain from coagulation factor X with the C-terminal subdomain from trypsin creates a potent enzyme (fXYa) with novel properties, in particular a broad substrate specificity. As shown by the 2.15-Å crystal structure, plasticity at the hydrophobic subdomain interface maintains activity, while surface loops are displaced compared with the parent subdomains. fXYa thus represents a new serine proteinase lineage with hybrid fX, trypsin, and novel properties.
Mechanism of capsid maturation in a double-stranded DNA virus
Authors: Tuma Roman , Prevelige Peter E. , Thomas George J.
Published: July 2002

Folding mechanisms of proteins incorporated within supramolecular assemblies, including viruses, are little understood and may differ fundamentally from folding mechanisms of small globular proteins. We describe a novel Raman dynamic probe of hydrogen-isotope exchange to investigate directly these protein folding/assembly pathways. The method is applied to subunit folding in assembly intermediates of the double-stranded DNA bacteriophage P22. The icosahedral procapsid-to-capsid maturation (shell expansion) of P22 is shown to be accompanied by a large increase in exchange protection of peptide β-strands. The molecular mechanism of shell expansion involves unfolding of metastable tertiary structure to form more stable quaternary contacts and is governed by a surprisingly high activation energy. The results demonstrate that coat subunit folding and capsid expansion are strongly coupled processes. Subunit structure in the procapsid represents a late intermediate along the folding/assembly pathway to the mature capsid. Coupling of folding and assembly is proposed as a general pathway for the construction of supramolecular complexes.
Expression of homeobox genes shows chelicerate arthropods retain their deutocerebral segment
Authors: Telford Maximilian J. , Thomas Richard H.
Published: July 2002

Expression patterns of six homeobox containing genes in a model chelicerate, the oribatid mite
Archegozetes longisetosus
, were examined to establish homology of chelicerate and insect head segments and to investigate claims that the chelicerate deutocerebral segment has been reduced or lost.
engrailed
(
en
) expression, which has been used to demonstrate the presence of segments in insects, fails to demonstrate a reduced deutocerebral segment. Expression patterns of the chelicerate homologs of the
Drosophila
genes
Antennapedia
(
Antp
),
Sex combs reduced
(
Scr
),
Deformed
(
Dfd
),
proboscipedia
(
pb
), and
orthodenticle
(
otd
) confirm direct correspondence of head segments. The chelicerate deutocerebral segment has not been reduced or lost. We make further inferences concerning the evolution of heads and Hox genes in arthropods.
Loss of cell adhesion in
Xenopus laevis
embryos mediated by the cytoplasmic domain of XLerk, an erythropoietin-producing hepatocellular ligand
Authors: Jones Teri L. , Chong Lisa D. , Kim Jaebong , Xu Ren-He , Kung Hsiang-Fu , Daar Ira O.
Published: July 2002

The erythropoietin-producing hepatocellular (Eph) family of ligands and receptors has been implicated in the control of axon guidance and the segmental restriction of cells during embryonic development. In this report, we show that ectopic expression of XLerk, a
Xenopus
homologue of the murine Lerk-2 (ephrin-B1) transmembrane ligand, causes dissociation of
Xenopus
embryonic blastomeres by the mid-blastula transition. Moreover, a mutant that lacks the extracellular receptor binding domain can induce this phenotype. The carboxyl-terminal 19 amino acids of the cytoplasmic domain of XLerk are necessary but not sufficient to induce cellular dissociation. Basic fibroblast growth factor, but not activin, can rescue both the loss of cell adhesion and mesoderm induction in ectodermal explants expressing XLerk. Collectively, these results show that the cytoplasmic domain of XLerk has a signaling function that is important for cell adhesion, and fibroblast growth factor signaling modulates this function.
Abnormal regulation of the leptin gene in the pathogenesis of obesity
Authors: Ioffe Ella , Moon Byoung , Connolly Eileen , Friedman Jeffrey M.
Published: July 2002

A subset of obese humans has relatively low plasma levels of leptin. This finding has suggested that in some cases abnormal regulation of the leptin gene in adipose tissue is etiologic in the pathogenesis of the obese state. The possibility that a relative decrease in leptin production can lead to obesity was tested by mating animals carrying a weakly expressed adipocyte specific aP2-human leptin transgene to C57BL/6J
ob/ob
mice (which do not express leptin). The transgene does not contain the regulatory elements of the leptin gene and is analogous to a circumstance in which the
cis
elements and/or
trans
factors regulating leptin RNA production are abnormal. The
ob/ob
mice carrying the transgene had a plasma leptin level of 1.78 ng/ml, which is ≈one-half that found in normal, nontransgenic mice (3.72 ng/ml,
P
< 0.01). The
ob/ob
animals expressing the leptin transgene were markedly obese though not as obese as
ob/ob
mice without the transgene. The infertility as well as several of the endocrine abnormalities generally evident in
ob/ob
mice were normalized in the
ob/ob
transgenic mice. However, the
ob/ob
transgenic mice had an abnormal response when placed at an ambient temperature of 4°C, suggesting that different thresholds exist for the different biologic effects of leptin. Leptin treatment of the
ob/ob
transgenic mice resulted in marked weight loss with efficacy similar to that seen after treatment of wild-type mice. In aggregate these data suggest that dysregulation of leptin gene can result in obesity with relatively normal levels of leptin and that this form of obesity is responsive to leptin treatment.
A perceptual memory for low-contrast visual signals
Authors: Tanaka Yasuto , Sagi Dov
Published: July 2002

Detection of a visual signal can be facilitated by simultaneous presentation of a similar subthreshold signal. Here we show that the facilitatory effect of a subthreshold signal can persist for more than 16 s. Presenting a near-threshold Gabor signal (prime) produced a phase-independent increase in contrast sensitivity (40%) to similar successive signals (target) for a period of up to 16 s. This effect was obtained only when both prime and target were presented to the same eye. We further show that the memory trace is inactivated by presenting high-contrast signals before the target. These results suggest that activated neurons in the primary visual cortex retain a near-threshold memory trace that persists until reactivated.
HIV-1 Tat protein mimicry of chemokines
Authors: Albini Adriana , Ferrini Silvano , Benelli Roberto , Sforzini Sabrina , Giunciuglio Daniela , Aluigi Maria Grazia , Proudfoot Amanda E. I. , Alouani Sami , Wells Timothy N. C. , Mariani Giuliano , Rabin Ronald L. , Farber Joshua M. , Noonan Douglas M.
Published: July 2002

The HIV-1 Tat protein is a potent chemoattractant for monocytes. We observed that Tat shows conserved amino acids corresponding to critical sequences of the chemokines, a family of molecules known for their potent ability to attract monocytes. Synthetic Tat and a peptide (CysL24–51) encompassing the “chemokine-like” region of Tat induced a rapid and transient Ca2+influx in monocytes and macrophages, analogous to β-chemokines. Both monocyte migration and Ca2+mobilization were pertussis toxin sensitive and cholera toxin insensitive. Cross-desensitization studies indicated that Tat shares receptors with MCP-1, MCP-3, and eotaxin. Tat was able to displace binding of β-chemokines from the β-chemokine receptors CCR2 and CCR3, but not CCR1, CCR4, and CCR5. Direct receptor binding experiments with the CysL24–51peptide confirmed binding to cells transfected with CCR2 and CCR3. HIV-1 Tat appears to mimic β-chemokine features, which may serve to locally recruit chemokine receptor-expressing monocytes/macrophages toward HIV producing cells and facilitate activation and infection.
Adenoviral-mediated gene transfer in lymphocytes
Authors: Leon Ronald P. , Hedlund Tammy , Meech Sandra J. , Li Shaobing , Schaack Jerome , Hunger Stephen P. , Duke Richard C. , DeGregori James
Published: July 2002

Although adenovirus can infect a wide range of cell types, lymphocytes are not generally susceptible to adenovirus infection, in part because of the absence of the expression of the cellular receptor for the adenoviral fiber protein. The cellular receptor for adenovirus and coxsackievirus (CAR) recently was cloned and shown to mediate adenoviral entry by interaction with the viral fiber protein. We show that the ectopic expression of CAR in various lymphocyte cell lines, which are almost completely resistant to adenovirus infection, is sufficient to facilitate the efficient transduction of these cells by recombinant adenoviruses. Furthermore, this property of CAR does not require its cytoplasmic domain, consistent with the idea that CAR primarily serves as a high affinity binding site for the adenoviral fiber protein, and that viral entry is mediated by interaction of the viral penton base proteins with cellular integrins. As a demonstration of their functional utility, we used CAR-expressing lymphocytes transduced with an adenovirus expressing Fas ligand to efficiently kill Fas receptor-expressing tumor cells. The ability to efficiently manipulate gene expression in lymphocyte cells by using adenovirus vectors should facilitate the functional characterization of pathways affecting lymphocyte physiology.
Polyandrous females discriminate against previous mates
Authors: Zeh Jeanne A. , Newcomer Scott D. , Zeh David W.
Published: July 2002

In most animal species, particularly those in which females engage in polyandry, mate choice is a sequential process in which a female must choose to mate or not to mate with each male encountered. Although a number of theoretical and empirical investigations have examined the effects of sequential mate choice on the operation of sexual selection, how females respond to solicitation by previous mates has received little attention. Here, we report the results of a study carried out on the polyandrous pseudoscorpion,
Cordylochernes scorpioides
, that assessed the sexual receptivity of once-mated females presented after a lapse of 1.5 hr or 48 hr with either their first mate or a different male. Females exhibited a high level of receptivity to new males, irrespective of intermating interval. By contrast, time between matings exerted a strong effect on female receptivity to previous mates. After a lapse of 48 hr, females did not differ significantly in their receptivity toward previous mates and different males, whereas at 1.5 hr after first mating, females were almost invariably unreceptive to males from whom they had previously accepted sperm. This result could not be attributed to male size or mating experience or to male sexual receptivity. Indeed, males were as willing to transfer sperm to a previous mate as they were to a new female. This difference between males and females in their propensity to remate with the same individual may reflect a conflict between the sexes, with males seeking to minimize postcopulatory sexual selection and females actively keeping open the opportunity for sperm competition and female choice of sperm by discriminating against previous mates.
Uncoupling signal transducers from oncogenic
MET
mutants abrogates cell transformation and inhibits invasive growth
Authors: Bardelli Alberto , Longati Paola , Gramaglia Daniela , Basilico Cristina , Tamagnone Luca , Giordano Silvia , Ballinari Dario , Michieli Paolo , Comoglio Paolo M.
Published: July 2002

The assumption that genes encoding tyrosine kinase receptors could play a role in human cancers has been confirmed by the identification of oncogenic mutations in the kinase domain of
RET
and
KIT
. Recently, homologous residues were found mutated in
MET
, in papillary renal carcinomas (PRCs). The link coupling these genetic lesions to cellular transformation is still unclear.
MET
PRC
mutations result in increased kinase activity and—in some instances, i.e., M1250T substitution—in changes in substrate specificity. A direct correlation occurs between the transforming potential of
MET
PRC
mutants and their ability to constitutively associate with signal transducers through two phosphorylated tyrosines (Y
1349
VHVNATY
1356
VNV) located in the receptor tail. Substitution of these “docking tyrosines” with phenylalanines leaves unaffected the altered properties of the kinase but abrogates transformation and invasiveness
in vitro
. Uncoupling the receptor from signal transducers with a tyrosine-phosphorylated peptide derivative (Y
p
VNV) inhibits invasive growth induced by
MET
PRC
mutants. These data indicate that constitutive receptor coupling to downstream signal transducers is a key mechanism in neoplastic transformation driven by mutated
MET
and suggest a therapeutic strategy to target neoplastic diseases associated with this oncogene.
Roles for mannitol and mannitol dehydrogenase in active oxygen-mediated plant defense
Authors: Jennings Dianne B. , Ehrenshaft Marilyn , Pharr D. Mason , Williamson John D.
Published: July 2002

Reactive oxygen species (ROS) are both signal molecules and direct participants in plant defense against pathogens. Many fungi synthesize mannitol, a potent quencher of ROS, and there is growing evidence that at least some phytopathogenic fungi use mannitol to suppress ROS-mediated plant defenses. Here we show induction of mannitol production and secretion in the phytopathogenic fungusAlternaria alternatain the presence of host-plant extracts. Conversely, we show that the catabolic enzyme mannitol dehydrogenase is induced in a non-mannitol-producing plant in response to both fungal infection and specific inducers of plant defense responses. This provides a mechanism whereby the plant can counteract fungal suppression of ROS-mediated defenses by catabolizing mannitol of fungal origin.
Information about movement direction obtained from synchronous activity of motor cortical neurons
Authors: Hatsopoulos Nicholas G. , Ojakangas Catherine L. , Paninski Liam , Donoghue John P.
Published: July 2002

Although neuronal synchronization has been shown to exist in primary motor cortex (MI), very little is known about its possible contribution to coding of movement. By using cross-correlation techniques from multi-neuron recordings in MI, we observed that activity of neurons commonly synchronized around the time of movement initiation. For some cell pairs, synchrony varied with direction in a manner not readily predicted by the firing of either neuron. Information theoretic analysis demonstrated quantitatively that synchrony provides information about movement direction beyond that expected by simple rate changes. Thus, MI neurons are not simply independent encoders of movement parameters but rather engage in mutual interactions that could potentially provide an additional coding dimension in cortex.
Evidence for distinct mechanisms of transition state stabilization of GTPases by fluoride
Authors: Vincent Sylvie , Brouns Madeleine , Hart Matthew J. , Settleman Jeffrey
Published: July 2002

GTPase-activating proteins (GAPs) function by stabilizing the GTPase transition state. This has been most clearly demonstrated by the formation of a high-affinity complex between various GAPs and GDP-bound GTPases in the presence of aluminum tetrafluoride, which can mimic the γ-phosphate of GTP. Herein, we report that p190 RhoGAP forms a high-affinity complex with Rho GTPases in the presence of fluoride ions, suggesting that p190 also functions to stabilize the GTPase transition state. However, this Rho–p190 complex does not require aluminum ions or even guanine nucleotide, indicating a distinct role for fluoride that is not consistent with the γ-phosphate-mimicking hypothesis. These results indicate that it is necessary to reconsider the assumed role of fluoride in stabilizing a variety of other GTPase–GAP interactions where the requirement for aluminum or guanine nucleotide has not yet been addressed.
Overexpression of human copper,zinc-superoxide dismutase (SOD1) prevents postischemic injury
Authors: Wang Penghai , Chen Hua , Qin Honghua , Sankarapandi Sornampillai , Becher Mark W. , Wong Philip C. , Zweier Jay L.
Published: July 2002

Superoxide and superoxide-derived oxidants have been hypothesized to be important mediators of postischemic injury. Whereas copper,zinc-superoxide dismutase, SOD1, efficiently dismutates superoxide, there has been controversy regarding whether increasing intracellular SOD1 expression would protect against or potentiate cellular injury. To determine whether increased SOD1 protects the heart from ischemia and reperfusion, studies were performed in a newly developed transgenic mouse model in which direct measurement of superoxide, contractile function, bioenergetics, and cell death could be performed. Transgenic mice with overexpression of human SOD1 were studied along with matched nontransgenic controls. Immunoblotting and immunohistology demonstrated that total SOD1 expression was increased 10-fold in hearts from transgenic mice compared with nontransgenic controls, with increased expression in both myocytes and endothelial cells. In nontransgenic hearts following 30 min of global ischemia a reperfusion-associated burst of superoxide generation was demonstrated by electron paramagnetic resonance spin trapping. However, in the transgenic hearts with overexpression of SOD1 the burst of superoxide generation was almost totally quenched, and this was accompanied by a 2-fold increase in the recovery of contractile function, a 2.2-fold decrease in infarct size, and a greatly improved recovery of high energy phosphates compared with that in nontransgenic controls. These results demonstrate that superoxide is an important mediator of postischemic injury and that increasing intracellular SOD1 dramatically protects the heart from this injury. Thus, increasing intracellular SOD1 expression may be a highly effective approach to decrease the cellular injury that occurs following reperfusion of ischemic tissues.
How evolution makes proteins fold quickly
Authors: Mirny Leonid A. , Abkevich Victor I. , Shakhnovich Eugene I.
Published: July 2002

Sequences of fast-folding model proteins (48 residues long on a cubic lattice) were generated by an evolution-like selection toward fast folding. We find that fast-folding proteins exhibit a specific folding mechanism in which all transition state conformations share a smaller subset of common contacts (folding nucleus). Acceleration of folding was accompanied by dramatic strengthening of interactions in the folding nucleus whereas average energy of nonnucleus interactions remained largely unchanged. Furthermore, the residues involved in the nucleus are the most conserved ones within families of evolved sequences. Our results imply that for each protein structure there is a small number of conserved positions that are key determinants of fast folding into that structure. This conjecture was tested on two protein superfamilies: the first having the classical monophosphate binding fold (CMBF; 98 families) and the second having type-III repeat fold (47 families). For each superfamily, we discovered a few positions that exhibit very strong and statistically significant “conservatism of conservatism”—amino acids in those positions are conserved within every family whereas the actual types of amino acids varied from family to family. Those amino acids are in spatial contact with each other. The experimental data of Serrano and coworkers [Lopez-Hernandez, E. & Serrano, L. (1996)
Fold. Des. (London)
1, 43–55]. for one of the proteins of the CMBF superfamily (CheY) show that residues identified this way indeed belong to the folding nucleus. Further analysis revealed deep connections between nucleation in CMBF proteins and their function.
Placental failure in mice lacking the homeobox gene
Dlx
3
Authors: Morasso Maria I. , Grinberg Alexander , Robinson Gertraud , Sargent Thomas D. , Mahon Kathleen A.
Published: July 2002

Dlx3 is a homeodomain transcription factor and a member of the vertebrate
Distal-less
family. Targeted deletion of the mouse
Dlx3
gene results in embryonic death between day 9.5 and day 10 because of placental defects that alter the development of the labyrinthine layer.
In situ
hybridization reveals that the
Dlx3
gene is initially expressed in ectoplacental cone cells and chorionic plate, and later in the labyrinthine trophoblast of the chorioallantoic placenta, where major defects are observed in the
Dlx3
−/− embryos. The expression of structural genes, such as
4311
and
PL-1
, which were used as markers to follow the fate of different derivatives of the placenta, was not affected in the
Dlx3
-null embryos. However, by day 10.5 of development, expression of the paired-like homeodomain gene
Esx1
was strongly down-regulated in affected placenta tissue, suggesting that Dlx3 is required for the maintenance of
Esx1
expression, normal placental morphogenesis, and embryonic survival.
Atrial natriuretic peptide and oxytocin induce natriuresis by release of cGMP
Authors: Soares T. J. , Coimbra T. M. , Martins A. R. , Pereira A. G. F. , Carnio E. C. , Branco L. G. S. , Albuquerque-Araujo W. I. C. , de Nucci G. , Favaretto A. L. V. , Gutkowska J. , McCann S. M. , Antunes-Rodrigues J.
Published: July 2002

Our hypothesis is that oxytocin (OT) causes natriuresis by activation of renal NO synthase that releases NO followed by cGMP that mediates the natriuresis. To test this hypothesis, an inhibitor of NO synthase,
l
-nitroarginine methyl ester (NAME), was injected into male rats. Blockade of NO release by NAME had no effect on natriuresis induced by atrial natriuretic peptide (ANP). This natriuresis presumably is caused by cGMP because ANP also activates guanylyl cyclase, which synthesizes cGMP from GTP. The 18-fold increase in sodium (Na
+
) excretion induced by OT (1 μg) was accompanied by an increase in urinary cGMP and preceded by 20 min a 20-fold increase in NO
3
−
excretion. NAME almost completely inhibited OT-induced natriuresis and increased NO
3
−
excretion; however, when the dose of OT was increased 10-fold, a dose that markedly increases plasma ANP concentrations, NAME only partly inhibited the natriuresis. We conclude that the natriuretic action of OT is caused by a dual action: generation of NO leading to increased cGMP and at higher doses release of ANP that also releases cGMP. OT-induced natriuresis is caused mainly by decreased tubular Na
+
reabsorption mediated by cGMP. In contrast to ANP that releases cGMP in the renal vessels and the tubules, OT acts on its receptors on NOergic cells demonstrated in the macula densa and proximal tubules to release cGMP that closes Na
+
channels. Both ANP- and OT-induced kaliuresis also appear to be mediated by cGMP. We conclude that cGMP mediates natriuresis and kaliuresis induced by both ANP and OT.
Cell cycle-dependent regulation of RNA polymerase I transcription: The nucleolar transcription factor UBF is inactive in mitosis and early G
1
Authors: Klein Joachim , Grummt Ingrid
Published: July 2002

Transcription of ribosomal RNA genes by RNA polymerase (pol) I oscillates during the cell cycle, being maximal in S and G
2
phase, repressed during mitosis, and gradually recovering during G
1
progression. We have shown that transcription initiation factor (TIF)-IB/SL1 is inactivated during mitosis by cdc2/cyclin B-directed phosphorylation of TAF
I
110. In this study, we have monitored reactivation of transcription after exit from mitosis. We demonstrate that the pol I factor UBF is also inactivated by phosphorylation but recovers with different kinetics than TIF-IB/SL1. Whereas TIF-IB/SL1 activity is rapidly regained on entry into G
1
, UBF is reactivated later in G
1
, concomitant with the onset of pol I transcription. Repression of pol I transcription in mitosis and early G
1
can be reproduced with either extracts from cells synchronized in M or G
1
phase or with purified TIF-IB/SL1 and UBF isolated in the presence of phosphatase inhibitors. The results suggest that two basal transcription factors, e.g., TIF-IB/SL1 and UBF, are inactivated at mitosis and reactivated by dephosphorylation at the exit from mitosis and during G
1
progression, respectively.
Cytochrome
b
562
folding triggered by electron transfer: Approaching the speed limit for formation of a four-helix-bundle protein
Authors: Wittung-Stafshede Pernilla , Lee Jennifer C. , Winkler Jay R. , Gray Harry B.
Published: July 2002

Ferrocytochrome
b
562
[Fe(II)cyt
b
562
] folding can be triggered by photoinduced electron transfer to unfolded Fe(III)cyt
b
562
in 2–3 M guanidine hydrochloride solutions. The folding rates increase with decreasing guanidine hydrochloride; the extrapolated time constant for this folding process in the absence of denaturant (5 μs) is near the predicted value for intrachain diffusion. The relatively smooth energy landscape indicated for Fe(II)cyt
b
562
folding accords with the helical, highly symmetrical structure of the protein.
Arrangement of radial actin bundles in the growth cone ofAplysiabag cell neurons shows the immediate past history of filopodial behavior
Authors: Katoh Kaoru , Hammar Katherine , Smith Peter J. S. , Oldenbourg Rudolf
Published: July 2002

Filopodia that protrude forward from the lamellipodium, located at the leading edge of a neuronal growth cone, are needed to guide the extension of a nerve cell. At the core of each filopodium an actin bundle forms and grows into the lamellipodium. By using kymographs of time-lapse polarized light images we examined the relationship between the behavior of the filopodia, the actin bundles immediately proximal to the filopodia, and the shapes and composition of actin bundles in the whole lamellipodium. We find that the shapes of actin bundles, such as tilt, fork, and fused zones, originate at the leading edge and are surprisingly well preserved during retrograde transport of the actin cytoskeleton in the whole lamellipodium. The number of filaments that make up the radial actin bundles, as displayed by their birefringence retardation, also is preserved during retrograde flow over a distance of 4–8 μm from the leading edge into the lamellipodium. Thus, the disposition of the actin bundles in the lamellipodium frozen at any time point preserves and portrays a history of the past behavior of actin bundles proximal to the filopodia and the behavior of the filopodia themselves. These findings suggest that the arrangement of actin bundles in static image records, such as electron or fluorescence micrographs of fixed and stained specimens, can in fact reveal the sequence of the past history of filopodial behavior and the generation, density, fusion, etc. of the filaments in the actin bundles.