bluuebunny/crossref_metadata_2025 huggingface dataset:
The CXC chemokine stromal cell-derived factor 1 is not responsible for CD8+T cell suppression of syncytia-inducing strains of HIV-1
Authors: Lacey Simon F. , McDanal Charlene B. , Horuk Richard , Greenberg Michael L.
Published: July 2002

Primary CD8+T cells from HIV+asymptomatics can suppress virus production from CD4+T cells acutely infected with either non-syncytia-inducing (NSI) or syncytia-inducing (SI) HIV-1 isolates. NSI strains of HIV-1 predominantly use the CCR5 chemokine receptor as a fusion cofactor, whereas fusion of T cell line-adapted SI isolates is mediated by another chemokine receptor, CXCR4. The CCR5 ligands RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein 1α (MIP-1α), and MIP-1β are HIV-1 suppressive factors secreted by CD8+cells that inhibit NSI viruses. Recently, the CXC chemokine stromal cell-derived factor 1 (SDF-1) was identified as a ligand for CXCR4 and shown to inhibit SI strains. We speculated that SDF-1 might be an effector molecule for CD8+suppression of SI isolates and assessed several SDF-1 preparations for inhibition of HIV-1LAI-mediated cell–cell fusion, and examined levels of SDF-1 transcripts in CD8+T cells. SDF-1 fusion inhibitory activity correlated with the N terminus, and the α and β forms of SDF-1 exhibited equivalent fusion blocking activity. SDF-1 preparations having the N terminus described by Bleulet al.(Bleul, C.C., Fuhlbrigge, R.C., Casasnovas, J.M., Aiuti, A. & Springer, T.A. (1996)J. Exp. Med.184, 1101–1109) readily blocked HIV-1LAI-mediated fusion, whereas forms containing two or three additional N-terminal amino acids lacked this activity despite their ability to bind and/or signal through CXCR4. Though SDF-1 is constitutively expressed in most tissues, CD8 T cells contained extremely low levels of SDF-1 mRNA transcripts (<1 transcript/5,000 cells), and these levels did not correlate with virus suppressive activity. We conclude that suppression of SI strains of HIV-1 by CD8+T cells is unlikely to involve SDF-1.
Lipoprotein lipase controls fatty acid entry into adipose tissue, but fat mass is preserved by endogenous synthesis in mice deficient in adipose tissue lipoprotein lipase
Authors: Weinstock Peter H. , Levak-Frank Sanja , Hudgins Lisa C. , Radner Herbert , Friedman Jeffrey M. , Zechner Rudolf , Breslow Jan L.
Published: July 2002

Lipoprotein lipase (LPL) is the rate-limiting enzyme for the import of triglyceride-derived fatty acids by muscle, for utilization, and adipose tissue (AT), for storage. Relative ratios of LPL expression in these two tissues have therefore been suggested to determine body mass composition as well as play a role in the initiation and/or development of obesity. To test this, LPL knockout mice were mated to transgenics expressing LPL under the control of a muscle-specific promoter (MCK) to generate induced mutants with either relative (L2-MCK) or absolute AT LPL deficiency (L0-MCK). L0-MCK mice had normal weight gain and body mass composition. However, AT chemical composition indicated that LPL deficiency was compensated for by large increases in endogenous AT fatty acid synthesis. Histological analysis confirmed that such up-regulation of
de novo
fatty acid synthesis in L0-MCK mice could produce normal amounts of AT as early as 20 h after birth. To assess the role of AT LPL during times of profound weight gain, L0-MCK and L2-MCK genotypes were compared on the obese
ob/ob
background.
ob/ob
mice rendered deficient in AT LPL (L0-MCK-
ob/ob
) also demonstrated increased endogenous fatty acid synthesis but had diminished weight and fat mass. These findings reveal marked alterations in AT metabolism that occur during LPL deficiency and provide strong evidence for a role of AT LPL in one type of genetic obesity.
Cloning of a trypanosomatid gene coding for an ornithine decarboxylase that is metabolically unstable even though it lacks the C-terminal degradation domain
Authors: Svensson Fredrik , Ceriani Carolina , Wallström Eva Lövkvist , Kockum Inger , Algranati Israel D. , Heby Olle , Persson Lo
Published: July 2002

Mammalian ornithine decarboxylase (ODC) is among the most labile of cellular proteins, with a half-life of usually less than an hour. Like other short-lived proteins ODC is degraded by the 26S proteasome. Its degradation is not triggered by ubiquitination, but is stimulated by the binding of an inducible protein, antizyme. Truncations and mutations in the C terminus of mammalian ODC have been shown to prevent the rapid turnover of the enzyme, demonstrating the presence of a degradation signal in this region. Moreover, ODCs from the trypanosomatid parasites
Trypanosoma brucei
and
Leishmania donovani
, which lack this C-terminal domain, are metabolically stable, and recombination of
T. brucei
ODC with the C terminus of mammalian ODC confers a short half-life to the fusion protein when expressed in mammalian cells. In the present study we have cloned and sequenced the ODC gene from the trypanosomatid
Crithidia fasciculata
. To our knowledge, this is the first protozoan shown to have an ODC with a rapid turnover. The sequence analysis revealed a high homology between
C. fasciculata
ODC and
L. donovani
ODC, despite the difference in stability. We demonstrate that
C. fasciculata
ODC has a very rapid turnover even when expressed in mammalian cells. Moreover, ODC from
C. fasciculata
is shown to lack the C-terminal degradation domain of mammalian ODC. Our findings indicate that
C. fasciculata
ODC contains unique signals, targeting the enzyme for rapid degradation not only in the parasite but also in mammalian cells.
Molecular characterization of two mammalian bHLH-PAS domain proteins selectively expressed in the central nervous system
Authors: Zhou Yu-Dong , Barnard Mary , Tian Hui , Li Xu , Ring Huijun Z. , Francke Uta , Shelton John , Richardson James , Russell David W. , McKnight Steven L.
Published: July 2002

Here we describe two mammalian transcription factors selectively expressed in the central nervous system. Both proteins, neuronal PAS domain protein (NPAS) 1 and NPAS2, are members of the basic helix–loop–helix-PAS family of transcription factors. cDNAs encoding mouse and human forms of NPAS1 and NPAS2 have been isolated and sequenced. RNA blotting assays demonstrated the selective presence of NPAS1 and NPAS2 mRNAs in brain and spinal cord tissues of adult mice. NPAS1 mRNA was first detected at embryonic day 15 of mouse development, shortly after early organogenesis of the brain. NPAS2 mRNA was first detected during early postnatal development of the mouse brain.
In situ
hybridization assays using brain tissue of postnatal mice revealed an exclusively neuronal pattern of expression for NPAS1 and NPAS2 mRNAs. The human
NPAS1
gene was mapped to chromosome 19q13.2–q13.3, and the mouse
Npas1
gene to chromosome 7 at 2 centimorgans. Similarly, the human
NPAS2
gene was assigned to chromosome 2p11.2–2q13, and the mouse
Npas2
gene to chromosome 1 at 21–22 centimorgans. The chromosomal regions to which human
NPAS1
and
NPAS2
map are syntenic with those containing the mouse
Npas1
and
Npas2
genes, indicating that the mouse and human genes are true homologs.
Gilbert syndrome and glucose-6-phosphate dehydrogenase deficiency: A dose-dependent genetic interaction crucial to neonatal hyperbilirubinemia
Authors: Kaplan Michael , Renbaum Paul , Levy-Lahad Ephrat , Hammerman Cathy , Lahad Amnon , Beutler Ernest
Published: July 2002

Severe jaundice leading to kernicterus or death in the newborn is the most devastating consequence of glucose-6-phosphate dehydrogenase (EC
1.1.1.49
; G-6-PD) deficiency. We asked whether the TA repeat promoter polymorphism in the gene for uridinediphosphoglucuronate glucuronosyltransferase 1 (EC
2.4.1.17
; UDPGT1), associated with benign jaundice in adults (Gilbert syndrome), increases the incidence of neonatal hyperbilirubinemia in G-6-PD deficiency. DNA from term neonates was analyzed for UDPGT1 polymorphism (normal homozygotes, heterozygotes, variant homozygotes), and for G-6-PD Mediterranean deficiency. The variant UDPGT1 promoter allele frequency was similar in G-6-PD-deficient and normal neonates. Thirty (22.9%) G-6-PD deficient neonates developed serum total bilirubin ≥ 257 μmol/liter, vs. 22 (9.2%) normals (
P
= 0.0005). Of those with the normal homozygous UDPGT1 genotype, the incidence of hyperbilirubinemia was similar in G-6-PD-deficients and controls (9.7% and 9.9%). In contrast, in the G-6-PD-deficient neonates, those with the heterozygous or homozygous variant UDPGT1 genotype had a higher incidence of hyperbilirubinemia than corresponding controls (heterozygotes: 31.6% vs. 6.7%,
P
< 0.0001; variant homozygotes: 50% vs. 14.7%,
P
= 0.02). Among G-6-PD-deficient infants the incidence of hyperbilirubinemia was greater in those with the heterozygous (31.6%,
P
= 0.006) or variant homozygous (50%,
P
= 0.003) UDPGT1 genotype than in normal homozygotes. In contrast, among those normal for G-6-PD, the UDPGT1 polymorphism had no significant effect (heterozygotes: 6.7%; variant homozygotes: 14.7%). Thus, neither G-6-PD deficiency nor the variant UDPGT1 promoter, alone, increased the incidence of hyperbilirubinemia, but both in combination did. This gene interaction may serve as a paradigm of the interaction of benign genetic polymorphisms in the causation of disease.
An interaction between DNA ligase I and proliferating cell nuclear antigen: Implications for Okazaki fragment synthesis and joining
Authors: Levin David S. , Bai Wei , Yao Nina , O’Donnell Michael , Tomkinson Alan E.
Published: July 2002

Although three human genes encoding DNA ligases have been isolated, the molecular mechanisms by which these gene products specifically participate in different DNA transactions are not well understood. In this study, fractionation of a HeLa nuclear extract by DNA ligase I affinity chromatography resulted in the specific retention of a replication protein, proliferating cell nuclear antigen (PCNA), by the affinity resin. Subsequent experiments demonstrated that DNA ligase I and PCNA interact directly via the amino-terminal 118 aa of DNA ligase I, the same region of DNA ligase I that is required for localization of this enzyme at replication foci during S phase. PCNA, which forms a sliding clamp around duplex DNA, interacts with DNA pol δ and enables this enzyme to synthesize DNA processively. An interaction between DNA ligase I and PCNA that is topologically linked to DNA was detected. However, DNA ligase I inhibited PCNA-dependent DNA synthesis by DNA pol δ. These observations suggest that a ternary complex of DNA ligase I, PCNA and DNA pol δ does not form on a gapped DNA template. Consistent with this idea, the cell cycle inhibitor p21, which also interacts with PCNA and inhibits processive DNA synthesis by DNA pol δ, disrupts the DNA ligase I–PCNA complex. Thus, we propose that after Okazaki fragment DNA synthesis is completed by a PCNA–DNA pol δ complex, DNA pol δ is released, allowing DNA ligase I to bind to PCNA at the nick between adjacent Okazaki fragments and catalyze phosphodiester bond formation.
The phosphoprotein DARPP-32 mediates cAMP-dependent potentiation of striatal
N
-methyl-
d
-aspartate responses
Authors: Blank Thomas , Nijholt Ingrid , Teichert Ulrich , Kügler Heidemarie , Behrsing Holger , Fienberg Allen , Greengard Paul , Spiess Joachim
Published: July 2002

The signal transduction pathway underlying the cAMP-dependent modulation of rat striatal
N
-methyl-
d
-aspartate (NMDA) responses was investigated by using the two-electrode voltage-clamp technique. In oocytes injected with rat striatal poly(A)
+
mRNA, activation of cAMP-dependent protein kinase (PKA) by forskolin potentiated NMDA responses. Inhibition of protein phosphatase 1 (PP1) and/or protein phosphatase 2A (PP2A) by the specific inhibitor calyculin A occluded the PKA-mediated potentiation of striatal NMDA responses, suggesting that the PKA effect was mediated by inhibition of a protein phosphatase. Coinjection of oocytes with striatal mRNA and antisense oligodeoxynucleotides directed against the protein phosphatase inhibitor DARPP-32 dramatically reduced the PKA enhancement of NMDA responses. NMDA responses recorded from oocytes injected with rat hippocampal poly(A)
+
mRNA were not affected by stimulation of PKA. When oocytes were coinjected with rat hippocampal poly(A)
+
mRNA plus complementary RNA coding for DARPP-32, NMDA responses were potentiated after stimulation of PKA. The results provide evidence that DARPP-32, which is enriched in the striatum, may participate in the signaling between the two major afferent striatal pathways, the glutamatergic and the dopaminergic projections, by the cAMP-dependent regulation of striatal NMDA currents.
Why do mitochondria synthesize fatty acids? Evidence for involvement in lipoic acid production
Authors: Wada Hajime , Shintani David , Ohlrogge John
Published: July 2002

The function of acyl carrier protein (ACP) in mitochondria isolated from pea leaves has been investigated. When pea leaf mitochondria were labeled with [2-
14
C]malonic acid
in vitro
, radioactivity was incorporated into fatty acids, and, simultaneously, ACP was acylated. [1-
14
C]Acetate was much less effective as a precursor for fatty acid synthesis, suggesting that mitochondria do not possess acetyl-CoA carboxylase. The incorporation of radioactivity from [2-
14
C]malonate into fatty acids and the labeling of ACP were inhibited by cerulenin and required ATP and Mg
2+
. These findings indicate that plant mitochondria contain not only ACP, but all enzymes required for
de novo
fatty acid synthesis. Over 30% of the radioactive products from pea mitochondria labeled with [2-
14
C]malonate were recovered in H protein, which is a subunit of glycine decarboxylase and contains lipoic acid as an essential constituent. In similar experiments, the H protein of
Neurospora
mitochondria was also labeled by [2-
14
C]malonate. The labeling of pea H protein was inhibited by addition of cerulenin into the assay medium. Together, these findings indicate that ACP is involved in the
de novo
synthesis of fatty acids in plant mitochondria and that a major function of this pathway is production of lipoic acid precursors.
A general purpose RNA-cleaving DNA enzyme
Authors: Santoro Stephen W. , Joyce Gerald F.
Published: July 2002

An
in vitro
selection procedure was used to develop a DNA enzyme that can be made to cleave almost any targeted RNA substrate under simulated physiological conditions. The enzyme is comprised of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of seven to eight deoxynucleotides each. The RNA substrate is bound through Watson–Crick base pairing and is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. Despite its small size, the DNA enzyme has a catalytic efficiency (
k
cat
/
K
m
) of ≈10
9
M
−1
⋅min
−1
under multiple turnover conditions, exceeding that of any other known nucleic acid enzyme. Its activity is dependent on the presence of Mg
2+
ion. By changing the sequence of the substrate-recognition domains, the DNA enzyme can be made to target different RNA substrates. In this study, for example, it was directed to cleave synthetic RNAs corresponding to the start codon region of HIV-1
gag
/
pol
,
env
,
vpr
,
tat
, and
nef
mRNAs.
Calpain activity promotes the sealing of severed giant axons
Authors: Godell Christopher M. , Smyers Mark E. , Eddleman Christopher S. , Ballinger Martis L. , Fishman Harvey M. , Bittner George D.
Published: July 2002

A barrier (seal) must form at the cut ends of a severed axon if a neuron is to survive and eventually regenerate. Following severance of crayfish medial giant axons in physiological saline, vesicles accumulate at the cut end and form a barrier (seal) to ion and dye diffusion. In contrast, squid giant axons do not seal, even though injury-induced vesicles form after axonal transection and accumulate at cut axonal ends. Neither axon seals in Ca
2+
-free salines. The addition of calpain to the bath saline induces the sealing of squid giant axons, whereas the addition of inhibitors of calpain activity inhibits the sealing of crayfish medial giant axons. These complementary effects involving calpain in two different axons suggest that endogenous calpain activity promotes plasmalemmal repair by vesicles or other membranes which form a plug or a continuous membrane barrier to seal cut axonal ends.
Elicitor-stimulated ion fluxes and O
2
−
from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley
Authors: Jabs Thorsten , Tschöpe Markus , Colling Christiane , Hahlbrock Klaus , Scheel Dierk
Published: July 2002

Fungal elicitor stimulates a multicomponent defense response in cultured parsley cells (
Petroselinum crispum
). Early elements of this receptor-mediated response are ion fluxes across the plasma membrane and the production of reactive oxygen species (ROS), sequentially followed by defense gene activation and phytoalexin accumulation. Omission of Ca
2+
from the culture medium or inhibition of elicitor-stimulated ion fluxes by ion channel blockers prevented the latter three reactions, all of which were triggered in the absence of elicitor by amphotericin B-induced ion fluxes. Inhibition of elicitor-stimulated ROS production using diphenylene iodonium blocked defense gene activation and phytoalexin accumulation. O
2
−
but not H
2
O
2
stimulated phytoalexin accumulation, without inducing proton fluxes. These results demonstrate a causal relationship between early and late reactions of parsley cells to the elicitor and indicate a sequence of signaling events from receptor-mediated activation of ion channels via ROS production and defense gene activation to phytoalexin synthesis. Within this sequence, O
2
−
rather than H
2
O
2
appears to trigger the subsequent reactions.
Prokaryotes: The unseen majority
Authors: Whitman William B. , Coleman David C. , Wiebe William J.
Published: July 2002

The number of prokaryotes and the total amount of their cellular
carbon on earth are estimated to be 4–6 × 10
30
cells
and 350–550 Pg of C (1 Pg = 10
15
g), respectively.
Thus, the total amount of prokaryotic carbon is 60–100% of the
estimated total carbon in plants, and inclusion of prokaryotic carbon
in global models will almost double estimates of the amount of carbon
stored in living organisms. In addition, the earth’s prokaryotes
contain 85–130 Pg of N and 9–14 Pg of P, or about 10-fold more of
these nutrients than do plants, and represent the largest pool of these
nutrients in living organisms. Most of the earth’s prokaryotes occur
in the open ocean, in soil, and in oceanic and terrestrial subsurfaces,
where the numbers of cells are 1.2 × 10
29
, 2.6
× 10
29
, 3.5 × 10
30
, and 0.25–2.5
× 10
30
, respectively. The numbers of heterotrophic
prokaryotes in the upper 200 m of the open ocean, the ocean below
200 m, and soil are consistent with average turnover times of
6–25 days, 0.8 yr, and 2.5 yr, respectively. Although subject to a
great deal of uncertainty, the estimate for the average turnover time
of prokaryotes in the subsurface is on the order of 1–2 ×
10
3
yr. The cellular production rate for all prokaryotes on
earth is estimated at 1.7 × 10
30
cells/yr and is
highest in the open ocean. The large population size and rapid growth
of prokaryotes provides an enormous capacity for genetic diversity.
Additional evidence for an eight-transmembrane-domain topology for
Caenorhabditis elegans
and human presenilins
Authors: Li Xiajun , Greenwald Iva
Published: July 2002

Presenilins have been implicated in the genesis of Alzheimer’s disease and in facilitating LIN-12/Notch activity during development. All presenilins have multiple hydrophobic regions that could theoretically span a membrane, and a description of the membrane topology is a crucial step toward deducing the mechanism of presenilin function. Previously, we proposed an eight-transmembrane-domain model for presenilin, based on studies of the
Caenorhabditis elegans
SEL-12 presenilin. Here, we describe experiments that support the view that two of the hydrophobic regions of SEL-12 function as the seventh and eighth transmembrane domains. Furthermore, we have shown that human presenilin 1 behaves like SEL-12 presenilin when analyzed by our methods. Our results provide additional experimental support for the eight-transmembrane-domain model of presenilin topology.
Mutations in the Δ7-sterol reductase gene in patients with the Smith–Lemli–Opitz syndrome
Authors: Fitzky Barbara U. , Witsch-Baumgartner Martina , Erdel Martin , Lee Joon No , Paik Young-Ki , Glossmann Hartmut , Utermann Gerd , Moebius Fabian F.
Published: July 2002

The Smith–Lemli–Opitz syndrome (SLOS) is an inborn disorder of sterol metabolism with characteristic congenital malformations and dysmorphias. All patients suffer from mental retardation. Here we identify the SLOS gene as a Δ7-sterol reductase (
DHCR7
, EC
1.3.1.21
) required for the
de novo
biosynthesis of cholesterol. The human and murine genes were characterized and assigned to syntenic regions on chromosomes 11q13 and 7F5 by fluorescense
in situ
hybridization. Among the mutations found in patients with the SLOS, are missense (P51S, T93M, L99P, L157P, A247V, V326L, R352W, C380S, R404C, and G410S), nonsense (W151X), and splice site (IVS8–1G>C) mutations as well as an out of frame deletion (720–735 del). The missense mutations L99P, V326L, R352W, R404C, and G410S reduced heterologous protein expression by >90%. Our results strongly suggest that defects in the
DHCR7
gene cause the SLOS.
An entropy criterion to detect minimally frustrated intermediates in native proteins
Authors: Compiani Mario , Fariselli Piero , Martelli Pier Luigi , Casadio Rita
Published: July 2002

The analysis of the information flow in a feed-forward neural network suggests that the output of the network can be used to compute a structural entropy for the sequence-to-secondary structure mapping. On this basis, we formulate a minimum entropy criterion for the identification of minimally frustrated traits with helical conformation that correspond to initiation sites of protein folding. The entropy of protein segments can be viewed as a nucleation propensity that is useful to characterize putative regions where folding is likely to be initiated with the formation of stretches of α-helices under the predominant influence of local interactions. Our procedure is successfully tested in the search for initiation sites of protein folding for which independent experimental and computational evidence exists. Our results lend support to the view that folding is a hierarchical event in which, in harmony with the minimal frustration principle, the final conformation preserves structural modules formed in the early stages of the process.
An immune cell-selective interleukin 4 agonist
Authors: Shanafelt Armen B. , Forte Carla P. , Kasper James J. , Sanchez-Pescador Lisa , Wetzel Monte , Gundel Robert , Greve Jeffrey M.
Published: July 2002

Interleukin 4 (IL-4) is a pleiotropic cytokine. Of the cell types responsive to IL-4, T cells express one IL-4 receptor (IL-4R) type, IL-4Rα/IL-2Rγ (class I IL-4R), whereas endothelial cells express another type, IL-4Rα/IL-13Rα (class II IL-4R). It was hypothesized that IL-4 variants could be generated that would be selective for cell types expressing the different IL-4Rs. A series of IL-4 muteins were generated that were substituted in the region of IL-4 implicated in interactions with IL-2Rγ. These muteins were evaluated in T cell and endothelial cell assays. One of these muteins, containing the mutation Arg-121 to Glu (IL-4/R121E), exhibited complete biological selectivity for T cells, B cells, and monocytes, but showed no activity on endothelial cells. Receptor binding studies indicated that IL-4/R121E retained physical interaction with IL-2Rγ but not IL-13Rα; consistent with this observation, IL-4/R121E was an antagonist of IL-4-induced activity on endothelial cells. IL-4/R121E exhibits a spectrum of activitiesin vitrothat suggest utility in the treatment of certain autoimmune diseases.
Amitochondriate amoebae and the evolution of DNA-dependent RNA polymerase II
Authors: Stiller John W. , Duffield Ellen C. S. , Hall Benjamin D.
Published: July 2002

Unlike parasitic protist groups that are defined by the absence of mitochondria, the Pelobiontida is composed mostly of free-living species. Because of the presence of ultrastructural and cellular features that set them apart from all other eukaryotic organisms, it has been suggested that pelobionts are primitively amitochondriate and may represent the earliest-evolved lineage of extant protists. Analyses of rRNA genes, however, have suggested that the group arose well after the diversification of the earliest-evolved protists. Here we report the sequence of the gene encoding the largest subunit of DNA-dependent RNA polymerase II (
RPB
1) from the pelobiont
Mastigamoeba invertens
. Sequences within RPB1 encompass several of the conserved catalytic domains that are common to eubacterial, archaeal, and eukaryotic nuclear-encoded RNA polymerases. In RNA polymerase II, these domains catalyze the transcription of all nuclear pre-mRNAs, as well as the majority of small nuclear RNAs. In contrast with rDNA-based trees, phylogenetic analyses of
RPB
1 sequences indicate that
Mastigamoeba
represents an early branch of eukaryotic evolution. Unlike sequences from parasitic amitochondriate protists that were included in our study, there is no indication that
Mastigamoeba RPB
1 is attracted to the base of the eukaryotic tree artifactually. In addition, the presence of introns and a heptapeptide C-terminal repeat in the
Mastigamoeba
RPB1 sequence, features that are typically associated with more recently derived eukaryotic groups, raise provocative questions regarding models of protist evolution that depend almost exclusively on rDNA sequence analyses.
Overexpression of leptin receptors in pancreatic islets of Zucker diabetic fatty rats restores GLUT-2, glucokinase, and glucose-stimulated insulin secretion
Authors: Wang May-Yun , Koyama Kazunori , Shimabukuro Michio , Mangelsdorf David , Newgard Christopher B. , Unger Roger H.
Published: July 2002

The high-
K
m
glucose transporter, GLUT-2, and the high-
K
m
hexokinase of β cells, glucokinase (GK), are required for glucose-stimulated insulin secretion (GSIS). GLUT-2 expression in β cells of Zucker diabetic fatty (ZDF) rats is profoundly reduced at the onset of β-cell dysfunction of diabetes. Because ZDF rats are homozygous for a mutation in their leptin receptor (OB-R) gene and are therefore leptin-insensitive, we expressed the wild-type OB-R gene in diabetic islets by infusing a recombinant adenovirus (AdCMV-OB-Rb) to determine whether this reversed the abnormalities. Leptin induced a rise in phosphorylated STAT3, indicating that the transferred wild-type OB-R was functional. GLUT-2 protein rose 17-fold in AdCMV-OB-Rb-treated ZDF islets without leptin, and leptin caused no further rise. GK protein rose 7-fold without and 12-fold with leptin. Preproinsulin mRNA increased 64% without leptin and rose no further with leptin, but leptin was required to restore GSIS. Clofibrate and 9-
cis
-retinoic acid, the partner ligands for binding to peroxisome proliferator-activator receptor α (PPARα) and retinoid X receptor, up-regulated GLUT-2 expression in islets of normal rats, but not in ZDF rats, in which PPARα is very low. Because the fat content of islets of diabetic ZDF rats remains high unless they are treated with leptin, it appears that restoration of GSIS requires normalization of intracellular nutrient homeostasis, whereas up-regulation of GLUT-2 and GK is leptin-independent, requiring only high expression of OB-Rb.
Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis
Authors: Cases Sylvaine , Smith Steven J. , Zheng Yao-Wu , Myers Heather M. , Lear Steven R. , Sande Eric , Novak Sabine , Collins Colin , Welch Carrie B. , Lusis Aldons J. , Erickson Sandra K. , Farese Robert V.
Published: July 2002

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC
2.3.1.20
) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.
Ca
2+
/calmodulin-dependent kinase II mediates simultaneous enhancement of gap-junctional conductance and glutamatergic transmission
Authors: Pereda Alberto E. , Bell Theodore D. , Chang Bill H. , Czernik Andrew J. , Nairn Angus C. , Soderling Thomas R. , Faber Donald S.
Published: July 2002

While chemical synapses are very plastic and modifiable by defined activity patterns, gap junctions, which mediate electrical transmission, have been classically perceived as passive intercellular channels. Excitatory transmission between auditory afferents and the goldfish Mauthner cell is mediated by coexisting gap junctions and glutamatergic synapses. Although an increased intracellular Ca
2+
concentration is expected to reduce gap junctional conductance, both components of the synaptic response were instead enhanced by postsynaptic increases in Ca
2+
concentration, produced by patterned synaptic activity or intradendritic Ca
2+
injections. The synaptically induced potentiations were blocked by intradendritic injection of KN-93, a Ca
2+
/calmodulin-dependent kinase (CaM-K) inhibitor, or CaM-KIINtide, a potent and specific peptide inhibitor of CaM-KII, whereas the responses were potentiated by injection of an activated form of CaM-KII. The striking similarities of the mechanisms reported here with those proposed for long-term potentiation of mammalian glutamatergic synapses suggest that gap junctions are also similarly regulated and indicate a primary role for CaM-KII in shaping and regulating interneuronal communication, regardless of its modality.
Promoting detachment of neutrophils adherent to murine postcapillary venules to control inflammation: Effect of lipocortin 1
Authors: Lim Lina H. K. , Solito Egle , Russo-Marie Françoise , Flower Roderick J. , Perretti Mauro
Published: July 2002

In this study we investigated, using intravital microscopy, how neutrophil extravasation across mouse mesenteric postcapillary venules is inhibited by the glucocorticoid-regulated protein lipocortin (LC; also termed annexin) 1. Intraperitoneal injection of 1 mg of zymosan into mice induced neutrophil rolling on the activated mesenteric endothelium followed by adhesion (maximal at 2 hr: 5–6 cells per 100-μm of vessel length) and emigration (maximal at 4 hr: 8–10 cells per high-powered field). Treatment of mice with human recombinant LC1 (2 mg/kg s.c.) or its mimetic peptide Ac2–26 (13 mg/kg s.c.) did not modify cell rolling but markedly reduced (≥50%) the degree of neutrophil adhesion and emigration (P< 0.05). Intravenous treatment with peptide Ac2–26 (13 mg/kg) or recombinant human LC1 (0.7–2 mg/kg) promoted detachment of neutrophils adherent to the endothelium 2 hr after zymosan administration, with adherent cells detaching within 4.12 ± 0.75 min and 2.36 ± 0.31 min, respectively (n= 20–25 cells). Recruitment of newly adherent cells to the endothelium was unaffected. The structurally related protein LC5 was inactive in this assay, whereas a chimeric molecule constructed from the N terminus of LC1 (49 aa) attached to the core region of LC5 produced cell detachment with kinetics similar to LC1. Removal of adherent neutrophils from activated postcapillary endothelium is a novel pharmacological action, and it is at this site where LC1 and its mimetics operate to down-regulate this aspect of the host inflammatory response.
Memory and long-term potentiation (LTP) dissociated: Normal spatial memory despite CA1 LTP elimination with
Kv1.4
antisense
Authors: Meiri Noam , Sun Miao-Kun , Segal Zachary , Alkon Daniel L.
Published: July 2002

Long-term potentiation (LTP) in the hippocampal slice preparation has been proposed as an
in vitro
model for long-term memory. However, correlation of LTP with memory in living animals has been difficult to demonstrate. Furthermore, in the last few years evidence has accumulated that dissociate the two. Because potassium channels might determine the weight of synapses in networks, we studied the role of Kv1.4, a presynaptic A-type voltage-dependent K
+
channel, in both memory and LTP. Reverse transcription–PCR and Western blot analysis with specific antibodies showed that antisense oligodeoxyribonucleotide to Kv1.4 microinjected intraventricularly into rat brains obstructed hippocampal Kv1.4 mRNA, “knocking down” the protein in the hippocampus. This antisense knockdown had no effect on rat spatial maze learning, memory, or exploratory behavior, but eliminated both early- and late-phase LTP and reduced paired-pulse facilitation (a presynaptic effect) in CA1 pyramidal neurons without affecting dentate gyrus LTP. This presynaptic Kv1.4 knockdown together with previous postsynaptic Kv1.1 knockdown demonstrates that CA1 LTP is neither necessary nor sufficient for rat spatial memory.
Overexpression of Mdm2 in mice reveals a p53-independent role for Mdm2 in tumorigenesis
Authors: Jones Stephen N. , Hancock Amy R. , Vogel Hannes , Donehower Lawrence A. , Bradley Allan
Published: July 2002

The
Mdm2
proto-oncogene is amplified to high copy numbers in human sarcomas and is overexpressed in a wide variety of other human cancers. Because Mdm2 protein forms a complex with the p53 tumor suppressor protein and down-regulates p53 function, the oncogenic potential of Mdm2 is presumed to be p53-dependent. To model these conditions in mice, we have used the entire
Mdm2
gene, under transcriptional control of its native promoter region, as a transgene to create mice that overexpress Mdm2. The transgenic mice are predisposed to spontaneous tumor formation, and the incidence of sarcomas observed in the Mdm2-transgenic mice in the presence or absence of functional p53 demonstrates that, in addition to Mdm2-mediated inactivation of p53, there exists a p53-independent role for Mdm2 in tumorigenesis.
Generation and reproductive phenotypes of mice lacking estrogen receptor β
Authors: Krege John H. , Hodgin Jeffrey B. , Couse John F. , Enmark Eva , Warner Margaret , Mahler Joel F. , Sar Madhabananda , Korach Kenneth S. , Gustafsson Jan-Åke , Smithies Oliver
Published: July 2002

Estrogens influence the differentiation and maintenance of reproductive tissues and affect lipid metabolism and bone remodeling. Two estrogen receptors (ERs) have been identified to date, ERα and ERβ. We previously generated and studied knockout mice lacking estrogen receptor α and reported severe reproductive and behavioral phenotypes including complete infertility of both male and female mice and absence of breast tissue development. Here we describe the generation of mice lacking estrogen receptor β (ERβ −/−) by insertion of a neomycin resistance gene into exon 3 of the coding gene by using homologous recombination in embryonic stem cells. Mice lacking this receptor develop normally and are indistinguishable grossly and histologically as young adults from their littermates. RNA analysis and immunocytochemistry show that tissues from ERβ −/− mice lack normal ERβ RNA and protein. Breeding experiments with young, sexually mature females show that they are fertile and exhibit normal sexual behavior, but have fewer and smaller litters than wild-type mice. Superovulation experiments indicate that this reduction in fertility is the result of reduced ovarian efficiency. The mutant females have normal breast development and lactate normally. Young, sexually mature male mice show no overt abnormalities and reproduce normally. Older mutant males display signs of prostate and bladder hyperplasia. Our results indicate that ERβ is essential for normal ovulation efficiency but is not essential for female or male sexual differentiation, fertility, or lactation. Future experiments are required to determine the role of ERβ in bone and cardiovascular homeostasis.
On the role of selective attention in visual perception
Authors: Luck Steven J. , Ford Michelle A.
Published: July 2002

What is the role of selective attention in visual perception? Before answering this question, it is necessary to differentiate between attentional mechanisms that influence the identification of a stimulus from those that operate after perception is complete. Cognitive neuroscience techniques are particularly well suited to making this distinction because they allow different attentional mechanisms to be isolated in terms of timing and/or neuroanatomy. The present article describes the use of these techniques in differentiating between perceptual and postperceptual attentional mechanisms and then proposes a specific role of attention in visual perception. Specifically, attention is proposed to resolve ambiguities in neural coding that arise when multiple objects are processed simultaneously. Evidence for this hypothesis is provided by two experiments showing that attention—as measured electrophysiologically—is allocated to visual search targets only under conditions that would be expected to lead to ambiguous neural coding.
Isolation and expression of a rat brain cDNA encoding glutamate carboxypeptidase II
Authors: Luthi-Carter Ruth , Berger Urs V. , Barczak Amy K. , Enna Matthew , Coyle Joseph T.
Published: July 2002

N-acetylated alpha-linked acidic dipeptidase (NAALADase) hydrolyzes acidic peptides, such as the abundant neuropeptideN-acetyl-α-l-aspartyl-l-glutamate (NAAG), thereby generating glutamate. Previous cDNA cloning efforts have identified a candidate rat brain NAALADase partial cDNA, and Northern analyses have identified a family of related RNA species that are found only in brain and other NAALADase-expressing cells. In this report, we describe the cloning of a set of rat brain cDNAs that describe a full-length NAALADase mRNA. Transient transfection of a full-length cDNA into the PC3 cell line confers NAAG-hydrolyzing activity that is sensitive to the NAALADase inhibitors quisqualic acid and 2-(phosphonomethyl)glutaric acid. Northern hybridization detects the expression of three similar brain RNAs approximately 3,900, 3,000, and 2,800 nucleotides in length.In situhybridization histochemistry shows that NAALADase-related mRNAs have an uneven regional distribution in rat brain and are expressed predominantly by astrocytes as demonstrated by their colocalization with the astrocyte-specific marker glial fibrillary acidic protein.
Reelin is preferentially expressed in neurons synthesizing γ-aminobutyric acid in cortex and hippocampus of adult rats
Authors: Pesold C. , Impagnatiello F. , Pisu M. G. , Uzunov D. P. , Costa E. , Guidotti A. , Caruncho H. J.
Published: July 2002

During embryonic development of brain laminated structures, the protein Reelin, secreted into the extracellular matrix of the cortex and hippocampus by Cajal–Retzius (CR) cells located in the marginal zone, contributes to the regulation of migration and positioning of cortical and hippocampal neurons that do not synthesize Reelin. Soon after birth, the CR cells decrease, and they virtually disappear during the following 3 weeks. Despite their disappearance, we can quantify Reelin mRNA (approximately 200 amol/μg of total RNA) and visualize it by
in situ
hybridization, and we detect the translated product of this mRNA by immunocytochemistry preferentially in γ-aminobutyric acid (GABA)ergic neurons of adult rat cortex and hippocampus. In adult rat cerebellum, Reelin is expressed in glutamatergic neurons (granule cells). The translated product of this mRNA is readily exported from the granule cell somata to the parallel fibers, where it has been detected by electron microscopy in axon terminals located presynaptically to Purkinje cell dendrites.
Cdc6 is regulated by E2F and is essential for DNA replication in mammalian cells
Authors: Yan Zhen , DeGregori James , Shohet Ralph , Leone Gustavo , Stillman Bruce , Nevins Joseph R. , Williams R. Sanders
Published: July 2002

Cdc6 has a critical regulatory role in the initiation of DNA replication in yeasts, but its function in mammalian cells has not been characterized. We show here that Cdc6 is expressed selectively in proliferating but not quiescent mammalian cells, both in culture and within tissues of intact animals. During the transition from a growth-arrested to a proliferative state, transcription of mammalian Cdc6 is regulated by E2F proteins, as revealed by a functional analysis of the human Cdc6 promoter and by the ability of exogenously expressed E2F proteins to stimulate the endogenous Cdc6 gene. Immunodepletion of Cdc6 by microinjection of anti-Cdc6 antibody blocks initiation of DNA replication in a human tumor cell line. We conclude that expression of human Cdc6 is regulated in response to mitogenic signals though transcriptional control mechanisms involving E2F proteins, and that Cdc6 is required for initiation of DNA replication in mammalian cells.
Mapping strain exerted on blood vessel walls using deuterium double-quantum-filtered MRI
Authors: Sharf Yehuda , Seo Yoshiteru , Eliav Uzi , Akselrod Solang , Navon Gil
Published: July 2002

A technique is described for displaying distinct tissue layers of large blood vessel walls as well as measuring their mechanical strain. The technique is based on deuterium double-quantum-filtered (DQF) spectroscopic imaging. The effectiveness of the double-quantum filtration in suppressing the signal of bulk water is demonstrated on a phantom consisting of rat tail tendon fibers. Only intrafibrillar water is displayed, excluding all other signals of water molecules that reorient isotropically. One- and two-dimensional spectroscopic imaging of bovine aorta and coronary arteries show the characteristic DQF spectrum of each of the tissue layers. This property is used to obtain separate images of the outer layer, the tunica adventitia, or the intermediate layer, the tunica media, or both. To visualize the effect of elongation, the average residual quadrupole splitting <Δν

q

> is calculated for each pixel. Two-dimensional deuterium quadrupolar splitting images are obtained for a fully relaxed and a 55% elongated sample of bovine coronary artery. These images indicate that the strong effect of strain is associated with water molecules in the tunica adventitia whereas the DQF NMR signal of water in the tunica media is apparently strain-insensitive. After appropriate calibration, these average quadrupolar splitting images can be interpreted as strain maps.
Molecular coevolution within a
Drosophila
clock gene
Authors: Peixoto Alexandre A. , Hennessy J. Michael , Townson Ian , Hasan Gaiti , Rosbash Michael , Costa Rodolfo , Kyriacou Charalambos P.
Published: July 2002

The
period
(
per
) gene in
Drosophila melanogaster
provides an integral component of biological rhythmicity and encodes a protein that includes a repetitive threonine-glycine (Thr-Gly) tract. Similar repeats are found in the
frq
and
wc2
clock genes of
Neurospora crassa
and in the mammalian
per
homologues, but their circadian functions are unknown. In Drosophilids, the length of the Thr-Gly repeat varies widely between species, and sequence comparisons have suggested that the repeat length coevolves with the immediately flanking amino acids. A functional test of the coevolution hypothesis was performed by generating several hybrid
per
transgenes between
Drosophila pseudoobscura
and
D. melanogaster,
whose repetitive regions differ in length by about 150 amino acids. The positions of the chimeric junctions were slightly altered in each transgene. Transformants carrying
per
constructs in which the repeat of one species was juxtaposed next to the flanking region of the other were almost arrhythmic or showed a striking temperature sensitivity of the circadian period. In contrast, transgenes in which the repeat and flanking regions were conspecific gave wild-type levels of circadian rescue. These results support the coevolutionary interpretation of the interspecific sequence changes in this region of the PER molecule and reveal a functional dimension to this process related to the clock’s temperature compensation.
Alloantigen-induced unresponsiveness in cord blood T lymphocytes is associated with defective activation of Ras
Authors: Porcu Pierluigi , Gaddy Jay , Broxmeyer Hal E.
Published: July 2002

Human umbilical cord blood T lymphocytes (CBTL) respond to primary allostimulation but they do not proliferate upon rechallenge with alloantigen. Using PKH-26-labeled cells created a proliferative block that was observed only in CBTL that have divided during primary stimulation (PKH-26dim) but not in unstimulated (PKH-26bright) CBTL. CBTL’s secondary unresponsiveness resembles anergy and can be overcome by treatment with phorbol myristate acetate (PMA) and ionomycin or by high doses (50–100 units/ml) of interleukin 2. Addition of interleukin 2 to the primary cultures does not prevent the induction of secondary unresponsiveness. Defective Ras activation is detected in PKH-26dimCBTL during secondary response to alloantigen or after antibody-mediated T cell receptor stimulation whereas Ras is activated and proliferation is induced in CBTL during primary alloantigenic stimulation. Upon stimulation with PMA plus ionomycin, PMA plus alloantigen, but not alloantigen plus ionomycin, Ras is activated in PKH-26dimCBTL, and the block in proliferation is overcome. Correction of PKH-26dimCBTL’s proliferative defect correlates with PMA-induced Ras activation, suggesting a defect in the signaling pathway leading to Ras. Ras-independent signals, necessary but not sufficient to induce PKH-26dimCBTL proliferation, are provided by alloantigen exposure, as evident by the ability of PMA plus alloantigen but not PMA alone to overcome the proliferative block. Functional signal transduction through CD28 in PKH-26dimCBTL is supported by detectable CD28-mediated PI-3 kinase activation after PKH-26dimCBTL’s exposure to alloantigen or CD28 cross-linking. These results suggest that defective activation of Ras plays a key role in PKH-26dimCBTL’s secondary unresponsiveness and point to a defect along the T cell receptor rather than the CD28 signaling pathway.