bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of “substrate-trapping” mutants to identify physiological substrates of protein tyrosine phosphatases
Authors: Flint Andrew J. , Tiganis Tony , Barford David , Tonks Nicholas K.
Published: July 2002

The identification of substrates of protein tyrosine phosphatases (PTPs) is an essential step toward a complete understanding of the physiological function of members of this enzyme family. PTPs are defined by a conserved catalytic domain harboring 27 invariant residues. From a mutagenesis study of these invariant residues that was guided by our knowledge of the crystal structure of PTP1B, we have discovered a mutation of the invariant catalytic acid (Asp-181 in PTP1B) that converts an extremely active enzyme into a “substrate trap.” Expression of this D181A mutant of PTP1B in COS and 293 cells results in an enzyme that competes with endogenous PTP1B for substrates and promotes the accumulation of phosphotyrosine primarily on the epidermal growth factor (EGF) receptor as well as on proteins of 120, 80, and 70 kDa. The association between the D181A mutant of PTP1B and these substrates was sufficiently stable to allow isolation of the complex by immunoprecipitation. As predicted for an interaction between the substrate-binding site of PTP1B and its substrates, the complex is disrupted by vanadate and, for the EGF receptor, the interaction absolutely requires receptor autophosphorylation. Furthermore, from immunofluorescence studies, the D181A mutant of PTP1B appeared to retain the endogenous EGF receptor in an intracellular complex. These results suggest that the EGF receptor is a bona fide substrate for PTP1B
in vivo
and that one important function of PTP1B is to prevent the inappropriate, ligand-independent, activation of newly synthesized EGF receptor in the endoplasmic reticulum. This essential catalytic aspartate residue is present in all PTPs and has structurally equivalent counterparts in the dual-specificity phosphatases and the low molecular weight PTPs. Therefore we anticipate that this method may be widely applicable to facilitate the identification of substrates of other members of this enzyme family.
Silent information regulator protein complexes in
Saccharomyces cerevisiae
: A SIR2/SIR4 complex and evidence for a regulatory domain in SIR4 that inhibits its interaction with SIR3
Authors: Moazed Danesh , Kistler Amy , Axelrod Amy , Rine Jasper , Johnson Alexander D.
Published: July 2002

The SIR2, SIR3, and SIR4 silent information regulator proteins are involved in the assembly of silent chromatin domains in the budding yeast
Saccharomyces cerevisiae
. Using a series of biochemical experiments, we have studied protein–protein interactions involving these proteins. We found that yeast extracts contained a SIR2/SIR4 complex that was associated with little or no SIR3. However, truncations of the N-terminal two-thirds of the SIR4 protein allowed it to efficiently associate with SIR3, suggesting that the N-terminal domain of SIR4 inhibited its interaction with SIR3. We propose that the SIR3 and SIR4 proteins interact only during the assembly of the SIR protein complex at the silencer and that an early step in assembly unmasks the SIR4 protein to allow its association with SIR3. To test whether the interactions observed in yeast extracts were direct, we tested these SIR-SIR interactions using bacterially expressed SIR proteins. We observed direct interactions between SIR4 and SIR2, SIR4 and SIR3, SIR2 and SIR3, SIR2 and SIR2, and SIR4 and SIR4, indicating that the associations observed in yeast extracts were direct.
Molecular characterization of human and mouse fatty acid amide hydrolases
Authors: Giang Dan K. , Cravatt Benjamin F.
Published: July 2002

Recently, we reported the isolation, cloning, and expression of a rat enzyme, fatty acid amide hydrolase (FAAH), that degrades bioactive fatty acid amides like oleamide and anandamide to their corresponding acids, thereby serving to terminate the signaling functions of these molecules. Here, we report the molecular characterization of both a mouse and a human FAAH and compare these enzymes to the rat FAAH. The enzymes are well conserved in primary structure, with the mouse and rat FAAHs sharing 91% amino acid identity and the human FAAH sharing 82% and 84% identity with the rat FAAH and mouse FAAH, respectively. In addition, the expressed human and rat FAAHs behave biochemically as membrane proteins of comparable molecular size and show similar, but distinguishable, enzymological properties. The identification of highly homologous FAAH proteins in rat, mouse, and human supports a general role for the fatty acid amides in mammalian biology.
Neuronal (type I) nitric oxide synthase regulates nuclear factorκB activity and immunologic (type II) nitric oxide synthase expression
Authors: Togashi Hitoshi , Sasaki Masayuki , Frohman Elliot , Taira Eichi , Ratan Rajiv R. , Dawson Ted M. , Dawson Valina L.
Published: July 2002

Nitric oxide subserves diverse physiologic roles in the nervous system. NO is produced from at least three different NO synthase (NOS) isoforms: neuronal NOS (nNOS), endothelial NOS, and immunologic NOS (iNOS). We show that nNOS is the predominant isoform constitutively expressed in glia. NO derived from nNOS in glia inhibits the transcription factor nuclear factor κB (NFκB) as NOS inhibitors enhance basal NFκB activation. Pyrrolidine dithiocarbamate (PDTC) is an inhibitor of NFκB in most cells; however, we show that PDTC is also a potent scavenger of NO through formation of mononitrosyl iron complexes with PDTC. In Jurkat cells, a human T-cell lymphoma cell line, tumor necrosis factor-α (TNF-α) induces NFκB activation that is inhibited by PDTC. Contrary to the results in Jurkat cells, PDTC did not inhibit tumor necrosis factor-α-induced NFκB activation in astrocytes; instead PDTC itself induces NFκB activation in astrocytes, and this may be related to scavenging of endogenously produced NO by the PDTC iron complex. In astrocytes PDTC also dramatically induces the NFκB-dependent enzyme, iNOS, supporting the physiologic relevance of endogenous NO regulation of NFκB. NFκB activation in glia from mice lacking nNOS responds more rapidly to PDTC compared with astrocytes from wild-type mice. Our data suggest that nNOS in astrocytes regulates NFκB activity and iNOS expression, and indicate a novel regulatory role for nNOS in tonically suppressing central nervous system, NFκB-regulated genes.
cAMP-mediated inhibition of the epithelial brush border Na
+
/H
+
exchanger, NHE3, requires an associated regulatory protein
Authors: Yun C. H. Chris , Oh Soohyuk , Zizak Mirza , Steplock Deborah , Tsao Su , Tse Chung-Ming , Weinman Edward J. , Donowitz Mark
Published: July 2002

NHE3 is the Na
+
/H
+
exchanger located on the intestinal and renal brush border membrane, where it functions in transepithelial Na
+
absorption. The brush border Na
+
absorptive process is acutely inhibited by activation of cAMP-dependent protein kinase, but the molecular mechanism of this inhibitory effect is poorly understood. We have identified two regulatory proteins, E3KARP and NHERF, that interact with NHE3 to enable cAMP to inhibit NHE3. The two regulatory proteins are structurally related, sharing ≈50% identity in amino acid sequences. It has been previously shown that when NHE3 is transfected into PS120 fibroblasts or Caco-2 cells, cAMP failed to inhibit NHE3 activity. Northern blot analysis showed that both PS120 and Caco-2 cells lacked the expression of both E3KARP and NHERF. In contrast, other cell lines in which cAMP inhibits NHE3, including OK, CHO, and LLC-PK
1
cells, expressed NHERF-related regulatory proteins. To determine their functions in cAMP-dependent inhibition of NHE3, E3KARP and NHERF were transfected into PS120/NHE3 fibroblasts. Transfection in PS120/NHE3 fibroblasts with either NHERF or E3KARP reconstituted cAMP-induced inhibition of NHE3, resulting in 25–30% inhibition in these cells.
AIM1
, a novel non-lens member of the βγ-crystallin superfamily, is associated with the control of tumorigenicity in human malignant melanoma
Authors: Ray Michael E. , Wistow Graeme , Su Yan A. , Meltzer Paul S. , Trent Jeffrey M.
Published: July 2002

AIM1
is a novel gene whose expression is associated with the experimental reversal of tumorigenicity of human malignant melanoma. The predicted protein product of the major 4.1-kb transcript shows striking similarity to the βγ-crystallin superfamily. All known members of this superfamily contain two or four characteristic motifs arranged as one or two symmetrical domains.
AIM1
, in contrast, contains 12 βγ motifs, suggesting a 6-domain structure resembling a trimer of β- or γ-crystallin subunits. The structure of the
AIM1
gene shows remarkable similarity to β-crystallin genes, with homologous introns delineating equivalent protein structural units. AIM1 is the first mammalian member of the βγ superfamily with a primarily non-lens role. Other parts of the predicted AIM1 protein sequence have weak similarity with filament or actin-binding proteins. AIM1 is a good candidate for the putative suppressor of malignant melanoma on chromosome 6, possibly exerting its effects through interactions with the cytoskeleton.
RecQ DNA helicase is a suppressor of illegitimate recombination in
Escherichia coli
Authors: Hanada Katsuhiro , Ukita Toshiyuki , Kohno Yuko , Saito Kazue , Kato Jun-ichi , Ikeda Hideo
Published: July 2002

Bloom syndrome and Werner syndrome are genetic disorders in which an increased rate of chromosomal abnormality is observed. The genes responsible for these diseases,
BLM
and
WRN
, have been cloned and identified as homologs of the
Escherichia coli recQ
genes. We studied the effect of
recQ
mutations on illegitimate recombination, which is an aberrant biological event related to the chromosomal abnormality in humans, and found that a variety of
recQ
mutations increased spontaneous illegitimate recombination by 20- to 300-fold and increased UV light-induced illegitimate recombination by 10- to 100-fold. Most λ
bio
or λ
pro
transducing phages are formed by the recombination events at several hot spots, which are enhanced by the
recQ
mutation. The analysis of nucleotide sequences at the recombination junction in the transducing phages indicates that recombination at the hot spot sites as well as the non-hot spot sites takes place between short homologous sequences. Enhancement of the recombination in the
recQ
mutants also occurs in the
recA
,
recBC sbcBC
, or
recBC sbcA
backgrounds, indicating that these recombination events are mediated by none of the known recombination pathways, RecBC, RecF, and RecE. We therefore concluded that the RecQ function suppresses illegitimate recombination that depends on short homologous regions. We discuss a model, based on the 3′-to-5′ helicase activity of RecQ, to explain the role of this protein as a suppressor of illegitimate recombination.
In vitrorestoration of T cell responses in tuberculosis and augmentation of monocyte effector function againstMycobacterium tuberculosisby natural inhibitors of transforming growth factor β
Authors: Hirsch Christina S. , Ellner Jerrold J. , Blinkhorn Richard , Toossi Zahra
Published: July 2002

We examined the capacity of the naturally occurring inhibitors of transforming growth factor β (TGF-β), decorin and latency associated peptide (LAP), to reverse depressed T cell functions in peripheral blood mononuclear cells (PBMCs) from patients with pulmonary tuberculosis (TB)in vitroand to counteract the suppressive properties of TGF-β on mycobacterial replication in blood monocytes (MN)in vitro. T cell blastogenesis in response to purified protein derivative (PPD) in PBMCs of TB patients that were cocultured with decorin or LAP reached levels comparable to those observed in healthy tuberculin-responsive control subjects. Decorin and LAP were as effective as neutralizing antibody to TGF-β in correcting depressed T cell proliferation. Coculture of PBMCs from healthy PPD reactive individuals with neutralizing antibody to TGF-β, decorin, or LAP did not affect T cell blastogenesis. Levels of interferon-γ in cultures of PPD-stimulated PBMCs from patients with TB increased by more than 2-fold in the presence of maximal concentrations of either of the inhibitors of TGF-β, whereas TGF-β immunoreactivity declined to background levels. Coculture with optimal concentrations of decorin or LAP also led to reductions in mycobacterial growth in MN infected withMycobacterium tuberculosis(MTB)in vitroby 51% and 62%, respectively, when compared with cells left untreated. In parallel, levels of immunoreactive TGF-β in MTB-infected MN cultures containing decorin or LAP decreased to background levels. These data indicate that the naturally occurring inhibitors of TGF-β, decorin and LAP, efficiently abrogate the suppressive effects of TGF-β in PBMCs of TB patients and in MN infected with MTBin vitro. Therefore, these agents may be considered as adjuncts to antituberculous chemotherapy, and may be particularly useful in treatment of TB that is unresponsive to conventional chemotherapy.
The regulator of early gliogenesis
glial cells missing
is a transcription factor with a novel type of DNA-binding domain
Authors: Schreiber Jörg , Sock Elisabeth , Wegner Michael
Published: July 2002

Absence or presence of
glial cells missing
(GCM) in cells of the developing nervous system of
Drosophila
decides over their future fate as neurons or glia with only those cells turning into glia that express GCM. To understand how GCM exerts its function we performed a detailed structure–function analysis. Using fusions between the DNA binding domain of the yeast GAL4 protein and GCM, we detected a transactivation function within the C-terminal part of GCM. In addition to this transactivation domain we mapped a sequence-specific DNA-binding domain within the N-terminal part of the GCM protein in close proximity to a bipartite nuclear localization signal. Binding site selection assays determined the motif 5′-AT(G/A)CGGGT-3′ as the preferred binding site for GCM. Both the lack of homology to known proteins and the novel DNA binding specificity indicate that GCM contained a new type of DNA-binding domain. In transiently transfected cells, GCM also activated transcription from promoters consisting of the newly identified GCM-binding site and a TATA box. Thus, GCM is a novel type of transcription factor involved in early gliogenesis.
Mutations in the
MDR
3 gene cause progressive familial intrahepatic cholestasis
Authors: de Vree J. Marleen L. , Jacquemin Emmanuel , Sturm Ekkehard , Cresteil Danièle , Bosma Piter J. , Aten Jan , Deleuze Jean-François , Desrochers Marc , Burdelski Martin , Bernard Olivier , Elferink Ronald P. J. Oude , Hadchouel Michelle
Published: July 2002

Class III multidrug resistance (MDR) P-glycoproteins (P-gp), mdr2 in mice and MDR3 in man, mediate the translocation of phosphatidylcholine across the canalicular membrane of the hepatocyte. Mice with a disrupted
mdr2
gene completely lack biliary phospholipid excretion and develop progressive liver disease, characterized histologically by portal inflammation, proliferation of the bile duct epithelium, and fibrosis. This disease phenotype is very similar to a subtype of progressive familial intrahepatic cholestasis, hallmarked by a high serum γ-glutamyltransferase (γ-GT) activity. We report immunohistochemistry for MDR3 P-gp, reverse transcription-coupled PCR sequence analysis, and genomic DNA analysis of
MDR3
from two progressive familial intrahepatic cholestasis patients with high serum γ-GT. Canalicular staining for MDR3 P-gp was negative in liver tissue of both patients. Reverse transcription-coupled PCR sequencing of the first patient’s sequence demonstrated a homozygous 7-bp deletion, starting at codon 132, which results in a frameshift and introduces a stop codon 29 codons downstream. The second patient is homozygous for a nonsense mutation in codon 957 (C → T) that introduces a stop codon (TGA). Our results demonstrate that mutations in the human
MDR3
gene lead to progressive familial intrahepatic cholestasis with high serum γ-GT. The histopathological picture in these patients is very similar to that in the corresponding
mdr2
(−/−) mouse, in which mdr2 P-gp deficiency induces complete absence of phospholipid in bile.
Diapause-specific gene expression in pupae of the flesh flySarcophaga crassipalpis
Authors: Flannagan Ronald D. , Tammariello Steven P. , Joplin Karl H. , Cikra-Ireland Rebecca A. , Yocum George D. , Denlinger David L.
Published: July 2002

Several cDNAs isolated from brains of diapausing pupae of the flesh fly,Sarcophaga crassipalpis, show expression patterns unique to diapause. To isolate such cDNAs a diapause pupal brain cDNA library was screened by using an elimination hybridization technique, and cDNAs that did not hybridize with cDNA probes constructed from the RNA of nondiapausing pupae were selected for further screening. The 95 clones that did not hybridize in the initial library screen were selected for further characterization. These clones were then screened against diapause and nondiapause pupal poly(A)+Northern blots. The secondary screen identified 4 diapause-up-regulated clones, 7 diapause-down-regulated clones, 8 clones expressed equally in both diapause and nondiapause, and 75 clones without detectable expression. The diapause-up-regulated and down-regulated clones were further characterized by partial DNA sequencing and identity searches by using GenBank. Identities between our cloned cDNAs and other genes included those linked to cell cycle progression, stress responses, and DNA repair processes. The results suggest that insect diapause is not merely a shutdown of gene expression but is a unique, developmental pathway characterized by the expression of a novel set of genes.
Interactions with multiple peptide ligands determine the fate of developing thymocytes
Authors: Williams Owen , Tarazona Raquel , Wack Andreas , Harker Nicola , Roderick Kathleen , Kioussis Dimitris
Published: July 2002

Thymocytes are positively or negatively selected depending on interactions between their T cell receptors (TCR) and peptides presented by major histocompatibility complex molecules. We have previously shown that apoptosis of thymocytes from an αβ TCR-transgenic mouse (F5), induced by antigenic peptide, can be inhibited specifically by an antagonist peptide variant in anin vitroculture model. We have now extended these experiments by demonstrating that the antagonist peptide can inhibit natural negative selection of maturing thymocytes, induced by endogenously expressed antigen, in fetal thymic organ cultures (FTOC). This inhibition resulted in the rescue and maturation of thymocytes that would otherwise have been deleted. Mature T cells generated in these cultures were able to respond to antigen by producing limited quantities of interferon-γ, but unlike T cells from control FTOC, they required exogenous interleukin-2 to generate cytolytic effector cells. Interestingly, the antagonist peptide also accelerated the development of F5 thymocytes in the absence of the negatively selecting ligand. These data suggest that the developmental fate of a thymocyte may be determined by the recognition of multiple distinct peptide ligands during thymic selection. Alterations in the profiles of selecting peptides presented in the thymus would thus have profound effects on the size and autoreactive potential of the T cell repertoire generated.
Expression of the peroxisome proliferator-activated receptor γ (PPARγ) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein
Authors: Ricote Mercedes , Huang Jannet , Fajas Luis , Li Andrew , Welch John , Najib Jamila , Witztum Joseph L. , Auwerx Johan , Palinski Wulf , Glass Christopher K.
Published: July 2002

The peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent transcription factor that has been demonstrated to regulate fat cell development and glucose homeostasis. PPARγ is also expressed in a subset of macrophages and negatively regulates the expression of several proinflammatory genes in response to natural and synthetic ligands. We here demonstrate that PPARγ is expressed in macrophage foam cells of human atherosclerotic lesions, in a pattern that is highly correlated with that of oxidation-specific epitopes. Oxidized low density lipoprotein (oxLDL) and macrophage colony-stimulating factor, which are known to be present in atherosclerotic lesions, stimulated PPARγ expression in primary macrophages and monocytic cell lines. PPARγ mRNA expression was also induced in primary macrophages and THP-1 monocytic leukemia cells by the phorbol ester 12-
O
-tetradecanoylphorbol 13-acetate (TPA). Inhibition of protein kinase C blocked the induction of PPARγ expression by TPA, but not by oxLDL, suggesting that more than one signaling pathway regulates PPARγ expression in macrophages. TPA induced the expression of PPARγ in RAW 264.7 macrophages by increasing transcription from the PPARγ1 and PPARγ3 promoters. In concert, these observations provide insights into the regulation of PPARγ expression in activated macrophages and raise the possibility that PPARγ ligands may influence the progression of atherosclerosis.
Inhibition in verbal working memory revealed by brain activation
Authors: Jonides John , Smith Edward E. , Marshuetz Christy , Koeppe Robert A. , Reuter-Lorenz Patricia A.
Published: July 2002

There are many occasions in which humans and other animals must inhibit the production of some behavior or inhibit the processing of some internal representation. Success in inhibitory processing under normal circumstances can be revealed by the fact that certain brain pathologies render inhibitory processing ineffective. These pathologies often have been associated with damage to frontal cortex, including lateral and inferior aspects. We provide behavioral evidence of a verbal working memory task that, by hypothesis, engaged inhibitory processing, and we show (by using positron emission tomograpny) that the inhibitory processing is associated with a lateral portion of the left prefrontal cortex. The task in which subjects engaged was item-recognition: Four target letters were presented for storage followed, after a brief interval, by a probe letter that could match a target letter or not. On some trials, when the probe did not match a target letter and therefore required a “no” response, the probe had matched a target letter of the previous trial, so on these trials a “yes” response was prepotent and had to be inhibited, by hypothesis. Compared with a condition in which no prepotent response was created, this condition yielded brain activation in left inferior frontal gyrus, in the region of Brodmann’s area 45.
K-Ar ages of Pleistocene lava dams in the Grand Canyon in Arizona
Authors: Dalrymple G. Brent , Hamblin W. K.
Published: July 2002

At least 13 times during the Pleistocene Epoch lava flowed into the inner gorge of the Grand Canyon and formed lava dams, as high as 600 m, that temporarily blocked the flow of the Colorado River. K-Ar ages on these lava dams indicate that the seven youngest formed within a short period of time between about 0.6 and 0.4 mega-annum (Ma). The physiography of the lava dam remnants within the canyon shows that each dam was destroyed by erosion, the Colorado River rapidly reaching its pre-existing grade level, before the next dam was emplaced by new eruptions. The total time for emplacement and destruction for an individual lava dam was probably as little as 0.01–0.02 million years. The K-Ar ages of the two oldest dams, the Lava Butte dam (0.577 ± 0.054 Ma) and the Prospect dam (0.684 ± 0.051 Ma) are somewhat younger than the physiography of their remnants suggest.
A cis-acting element that directs the activity of the murine methylation modifier locusSsm1
Authors: Engler Peter , Doglio Lynn T. , Bozek Grazyna , Storb Ursula
Published: July 2002

Silencing of chromosomal domains has been described in diverse systems such as position effect variegation in insects, silencing near yeast telomeres, and mammalian X chromosome inactivation. In mammals, silencing is associated with methylation at CpG dinucleotides, but little is known about how methylation patterns are established or altered during development. We previously described a strain-specific modifier locus,Ssm1, that controls the methylation of a complex transgene. In this study we address the questions of the nature ofSsm1’s targets and whether its effect extends into adjacent sequences. By examining the inheritance of methylation patterns in a series of mice harboring deletion derivatives of the original transgene, we have identified a discrete segment, derived from thegptgene ofEscherichia coli, that is a major determinant forSsm1-mediated methylation. Methylation analysis of sequences adjacent to a transgenic target indicates that the influence of this modifier extends into the surrounding chromosome in a strain-dependent fashion. Implications for the mechanism ofSsm1action are discussed.
Cortical areas supporting category learning identified using functional MRI
Authors: Reber P. J. , Stark C. E. L. , Squire L. R.
Published: July 2002

Functional MRI was used to identify cortical areas involved in category learning by prototype abstraction. Participants studied 40 dot patterns that were distortions of an underlying prototype and then, while functional MRI data were collected, made yes-no category judgments about new dot patterns. The dot patterns alternated between ones mostly requiring a “yes” response and ones mostly requiring a “no” response. Activity in four cortical areas correlated with the category judgment task. A sizeable posterior occipital cortical area (BA 17/18) exhibited significantly less activity during processing of the categorical patterns than during processing of noncategorical patterns. Significant increases in activity during processing the categorical patterns were observed in left and right anterior frontal cortex (BA 10) and right inferior lateral frontal cortex (BA 44/47). Decreases in activation of visual cortex when categorical patterns were being evaluated suggest that these patterns could be processed in a more rapid or less effortful manner after the prototype had been learned. Increases in prefrontal activity associated with processing categorical patterns could be related to any of several processes involved in retrieving information about the learned exemplars.
Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes
Authors: Ivanov Sergey V. , Kuzmin Igor , Wei Ming-Hui , Pack Svetlana , Geil Laura , Johnson Bruce E. , Stanbridge Eric J. , Lerman Michael I.
Published: July 2002

To discover genes involved in von Hippel-Lindau (VHL)-mediated carcinogenesis, we used renal cell carcinoma cell lines stably transfected with wild-type VHL-expressing transgenes. Large-scale RNA differential display technology applied to these cell lines identified several differentially expressed genes, including an alpha carbonic anhydrase gene, termed
CA12
. The deduced protein sequence was classified as a one-pass transmembrane CA possessing an apparently intact catalytic domain in the extracellular CA module. Reintroduced wild-type VHL strongly inhibited the overexpression of the
CA12
gene in the parental renal cell carcinoma cell lines. Similar results were obtained with
CA9
, encoding another transmembrane CA with an intact catalytic domain. Although both domains of the VHL protein contribute to regulation of
CA12
expression, the elongin binding domain alone could effectively regulate
CA9
expression. We mapped
CA12
and
CA9
loci to chromosome bands 15q22 and 17q21.2 respectively, regions prone to amplification in some human cancers. Additional experiments are needed to define the role of CA IX and CA XII enzymes in the regulation of pH in the extracellular microenvironment and its potential impact on cancer cell growth.
Changes in cell-cycle kinetics during the development and evolution of primate neocortex
Authors: Kornack David R. , Rakic Pasko
Published: July 2002

The evolutionary expansion of neocortical size in mammals is particularly prominent in anthropoid primates (i.e., monkeys, apes, and humans) and reflects an increased number of cortical cells, yet the developmental basis for this increase remains undefined. Cortical cell production depends on the length of the cell-division cycle of progenitor cells during neurogenesis, which previously has been measured only in smaller-brained rodents. To investigate whether cortical expansion in primates reflects modification of cell-cycle kinetics, we determined cell-cycle length during neurogenesis in the proliferative cerebral ventricular zone of fetal rhesus monkeys, by using cumulative S-phase labeling with bromodeoxyuridine. Cell-cycle durations in monkeys were as much as 5 times longer than those reported in rodents. Nonetheless, substantially more total rounds of cell division elapsed during the prolonged neurogenetic period of the monkey cortex, providing a basis for increased cell production. Moreover, unlike the progressive slowing that occurs during cortical development in rodents, cell division accelerated during neurogenesis of the enlarged cortical layers in monkeys. These findings suggest that evolutionary modification of the duration and number of progenitor cell divisions contributed to both the expansion and laminar elaboration of the primate neocortex.
Neural components of topographical representation
Authors: Aguirre Geoffrey K. , Zarahn Eric , D’Esposito Mark
Published: July 2002

Studies of patients with focal brain damage suggest that
topographical representation is subserved by dissociable neural
subcomponents. This article offers a condensed review of the literature
of “topographical disorientation” and describes several
functional MRI studies designed to test hypotheses generated by that
review. Three hypotheses are considered: (
i
) The
parahippocampal cortex is critically involved in the acquisition of
exocentric spatial information in humans; (
ii
)
separable, posterior, dorsal, and ventral cortical regions subserve the
perception and long term representation of position and identity,
respectively, of landmarks; and (
iii
) there is a
distinct area of the ventral occipitotemporal cortex that responds
maximally to building stimuli and may play a role in the perception of
salient landmarks. We conclude with a discussion of the inferential
limitations of neuroimaging and lesion studies. It is proposed that
combining these two approaches allows for inferences regarding the
computational involvement of a neuroanatomical substrate in a given
cognitive process although neither method can strictly support this
conclusion alone.
A neural system for human visual working memory
Authors: Ungerleider Leslie G. , Courtney Susan M. , Haxby James V.
Published: July 2002

Working memory is the process of actively maintaining a
representation of information for a brief period of time so that it is
available for use. In monkeys, visual working memory involves the
concerted activity of a distributed neural system, including posterior
areas in visual cortex and anterior areas in prefrontal cortex. Within
visual cortex, ventral stream areas are selectively involved in object
vision, whereas dorsal stream areas are selectively involved in spatial
vision. This domain specificity appears to extend forward into
prefrontal cortex, with ventrolateral areas involved mainly in working
memory for objects and dorsolateral areas involved mainly in working
memory for spatial locations. The organization of this distributed
neural system for working memory in monkeys appears to be conserved in
humans, though some differences between the two species exist. In
humans, as compared with monkeys, areas specialized for object vision
in the ventral stream have a more inferior location in temporal cortex,
whereas areas specialized for spatial vision in the dorsal stream have
a more superior location in parietal cortex. Displacement of both sets
of visual areas away from the posterior perisylvian cortex may be
related to the emergence of language over the course of brain
evolution. Whereas areas specialized for object working memory in
humans and monkeys are similarly located in ventrolateral prefrontal
cortex, those specialized for spatial working memory occupy a more
superior and posterior location within dorsal prefrontal cortex in
humans than in monkeys. As in posterior cortex, this displacement in
frontal cortex also may be related to the emergence of new areas to
serve distinctively human cognitive abilities.
Daidzin and its antidipsotropic analogs inhibit serotonin and dopamine metabolism in isolated mitochondria
Authors: Keung Wing Ming , Vallee Bert L.
Published: July 2002

Daidzin, a major active principle of an ancient Chinese herbal treatment (
Radix puerariae
) for alcohol abuse, selectively suppresses ethanol intake in all rodent models tested. It also inhibits mitochondrial aldehyde dehydrogenase (ALDH-2). Studies on ethanol intake suppression and ALDH-2 inhibition by structural analogs of daidzin established a link between these two activities and suggested that daidzin may suppress ethanol intake by inhibiting ALDH-2. ALDH-2 is a principal enzyme involved in serotonin (5-HT) and dopamine (DA) metabolism. Thus, daidzin may act by inhibiting 5-HT and DA metabolism. To evaluate this possibility, we have studied the effect of daidzin and its analogs on 5-HT and DA metabolism in isolated hamster and rat liver mitochondria. Daidzin potently inhibits the formation of 5-hydroxyindole-3-acetic acid (5-HIAA) and 3,4-dihydroxyphenylacetic acid (DOPAC) from their respective amines in isolated mitochondria. Inhibition is concentration-dependent and is accompanied by a concomitant accumulation of 5-hydroxyindole-3-acetaldehyde and 3,4-dihydroxyphenylacetaldehyde. Daidzin analogs that suppress hamster ethanol intake also inhibit 5-HIAA and DOPAC formation. Comparing their effects on mitochondria-catalyzed 5-HIAA or DOPAC formation and hamster ethanol intake reveals a positive correlation—the stronger the inhibition on 5-HIAA or DOPAC formation, the greater the ethanol intake suppression. Daidzin and its active analogs, at concentrations that significantly inhibit 5-HIAA formation, have little or no effect on mitochondria-catalyzed 5-HT depletion. It appears that the antidipsotropic action of daidzin is not mediated by 5-HT (or DA) but rather by its reactive intermediates 5-hydroxyindole-3-acetaldehyde and, presumably, 3,4-dihydroxyphenylacetaldehyde as well, which accumulates in the presence of daidzin.
A class of hybrid polar inducers of transformed cell differentiation inhibits histone deacetylases
Authors: Richon Victoria M. , Emiliani Stephane , Verdin Eric , Webb Yael , Breslow Ronald , Rifkind Richard A. , Marks Paul A.
Published: July 2002

Hybrid polar compounds (HPCs) have been synthesized that induce terminal differentiation and/or apoptosis in various transformed cells. We have previously reported on the development of the second-generation HPCs suberoylanilide hydroxamic acid (SAHA) and
m
-carboxycinnamic acid bishydroxamide (CBHA) that are 2,000-fold more potent inducers on a molar basis than the prototype HPC hexamethylene bisacetamide (HMBA). Herein we report that CBHA and SAHA inhibit histone deacetylase 1 (HDAC1) and histone deacetylase 3 (HDAC3) activity
in vitro
. Treatment of cells in culture with SAHA results in a marked hyperacetylation of histone H4, but culture with HMBA does not. Murine erythroleukemia cells developed for resistance to SAHA are cross-resistant to trichostatin A, a known deacetylase inhibitor and differentiation inducer, but are not cross-resistant to HMBA. These studies show that the second-generation HPCs, unlike HMBA, are potent inhibitors of HDAC activity. In this sense, HMBA and the second-generation HPCs appear to induce differentiation by different pathways.
Mariner
transposition and transformation of the yellow fever mosquito,
Aedes aegypti
Authors: Coates Craig J. , Jasinskiene Nijole , Miyashiro Linda , James Anthony A.
Published: July 2002

The
mariner
transposable element is capable of interplasmid transposition in the embryonic soma of the yellow fever mosquito,
Aedes aegypti
. To determine if this demonstrated mobility could be utilized to genetically transform the mosquito, a modified
mariner
element marked with a wild-type allele of the
Drosophila melanogaster cinnabar
gene was microinjected into embryos of a kynurenine hydroxylase-deficient, white-eyed recipient strain. Three of 69 fertile male founders resulting from the microinjected embryos produced families with colored-eyed progeny individuals, a transformation rate of 4%. The transgene-mediated complementation of eye color was observed to segregate in a Mendelian manner, although one insertion segregates with the recessive allele (female-determining) of the sex-determining locus, and a separate insertion is homozygous lethal. Molecular analysis of selected transformed families demonstrated that a single complete copy of the construct had integrated independently in each case and that it had done so in a transposase-mediated manner. The availability of a
mariner
transformation system greatly enhances our ability to study and manipulate this important vector species.
In vivo
evidence that erythropoietin protects neurons from ischemic damage
Authors: Sakanaka Masahiro , Wen Tong-Chun , Matsuda Seiji , Masuda Seiji , Morishita Emi , Nagao Masaya , Sasaki Ryuzo
Published: July 2002

Erythropoietin (EPO) produced by the kidney and the liver (in fetuses) stimulates erythropoiesis. In the central nervous system, neurons express EPO receptor (EPOR) and astrocytes produce EPO. EPO has been shown to protect primary cultured neurons from
N
-methyl-
d
-aspartate (NMDA) receptor-mediated glutamate toxicity. Here we report
in vivo
evidence that EPO protects neurons against ischemia-induced cell death. Infusion of EPO into the lateral ventricles of gerbils prevented ischemia-induced learning disability and rescued hippocampal CA1 neurons from lethal ischemic damage. The neuroprotective action of exogenous EPO was also confirmed by counting synapses in the hippocampal CA1 region. Infusion of soluble EPOR (an extracellular domain capable of binding with the ligand) into animals given a mild ischemic treatment that did not produce neuronal damage, caused neuronal degeneration and impaired learning ability, whereas infusion of the heat-denatured soluble EPOR was not detrimental, demonstrating that the endogenous brain EPO is crucial for neuronal survival. The presence of EPO in neuron cultures did not repress a NMDA receptor-mediated increase in intracellular Ca
2+
, but rescued the neurons from NO-induced death. Taken together EPO may exert its neuroprotective effect by reducing the NO-mediated formation of free radicals or antagonizing their toxicity.
A cell surface mucin specifically expressed in the midgut of the malaria mosquito
Anopheles gambiae
Authors: Shen Zhicheng , Dimopoulos George , Kafatos Fotis C. , Jacobs-Lorena Marcelo
Published: July 2002

An invertebrate intestinal mucin gene,
AgMuc1
, was isolated from the malaria vector mosquito
Anopheles gambiae
. The predicted 122-residue protein consists of a central core of seven repeating TTTTVAP motifs flanked by hydrophobic N- and C-terminal domains. This structure is similar to that of mucins that coat the protozoan parasite
Trypanosoma cruzi
. Northern blot analysis indicated that the gene is expressed exclusively in the midgut of adult mosquitoes. A length polymorphism and
in situ
hybridization were used to genetically and cytogenetically map
AgMuc1
to division 7A of the right arm of the second chromosome. The subcellular localization of the encoded protein in tissue culture cells was examined by using a baculovirus vector to express AgMuc1 protein tagged with the green fluorescent protein (GFP). The results indicated that this protein is found at the cell surface and that both hydrophobic domains are required for cell surface targeting. We propose that AgMuc1 is an abundant mucin-like protein that lines the surface of the midgut microvilli, potentially protecting the intestinal epithelium from the proteinase-rich environment of the gut lumen. An intriguing possibility is that, as an abundant surface protein, AgMuc1 may also interact with the malaria parasite during its invasion of the mosquito midgut.
Leptin actions on food intake and body temperature are mediated by IL-1
Authors: Luheshi Giamal N. , Gardner Jason D. , Rushforth David A. , Loudon Andrew S. , Rothwell Nancy J.
Published: July 2002

Leptin regulates energy balance through its actions in the brain on appetite and energy expenditure and also shares properties with cytokines such as IL-1. We report here that leptin, injected into rats intracerebroventricularly or peripherally, induces significant dose-dependent increases in core body temperature as well as suppression of appetite. Leptin failed to affect food intake or body temperature in obese (fa/fa) Zucker rats, which posses a defective leptin receptor. Furthermore, injection of leptin increased levels of the proinflammatory cytokine IL-1β in the hypothalamus of normal Sprague–Dawley rats. Central injection of IL-1 receptor antagonist (IL-1ra) inhibited the suppression of food intake caused by central or peripheral injection of leptin (60 and 84%, respectively) and abolished the leptin-induced increase in body temperature in both cases. Mice lacking (gene knockout) the main IL-1 receptor (80 kDa, R1) responsible for IL-1 actions showed no reduction in food intake in response to leptin. These data indicate that leptin actions in the brain depend on IL-1, and we show further that the effect of leptin on fever, but not food intake, is abolished by a cyclooxygenase inhibitor. Thus, we propose that in addition to its role in body weight regulation, leptin may mediate neuroimmune responses via actions in the brain dependent on release of IL-1 and prostaglandins.
Sodium channels and pain
Authors: Waxman S. G. , Dib-Hajj S. , Cummins T. R. , Black J. A.
Published: July 2002

Although it is well established that hyperexcitability and/or
increased baseline sensitivity of primary sensory neurons can lead to
abnormal burst activity associated with pain, the underlying molecular
mechanisms are not fully understood. Early studies demonstrated that,
after injury to their axons, neurons can display changes in
excitability, suggesting increased sodium channel expression, and, in
fact, abnormal sodium channel accumulation has been observed at the
tips of injured axons. We have used an ensemble of molecular,
electrophysiological, and pharmacological techniques to ask: what types
of sodium channels underlie hyperexcitability of primary sensory
neurons after injury? Our studies demonstrate that multiple sodium
channels, with distinct electrophysiological properties, are encoded by
distinct mRNAs within small dorsal root ganglion (DRG) neurons, which
include nociceptive cells. Moreover, several DRG neuron-specific sodium
channels now have been cloned and sequenced. After injury to the axons
of DRG neurons, there is a dramatic change in sodium channel expression
in these cells, with down-regulation of some sodium channel genes and
up-regulation of another, previously silent sodium channel gene. This
plasticity in sodium channel gene expression is accompanied by
electrophysiological changes that poise these cells to fire
spontaneously or at inappropriate high frequencies. Changes in sodium
channel gene expression also are observed in experimental models of
inflammatory pain. Thus, sodium channel expression in DRG neurons is
dynamic, changing significantly after injury. Sodium channels within
primary sensory neurons may play an important role in the
pathophysiology of pain.
Neurotrophins and hyperalgesia
Authors: Shu X.-Q. , Mendell L. M.
Published: July 2002

Nerve growth factor (NGF), a member of the neurotrophin family, is
crucial for survival of nociceptive neurons during development.
Recently, it has been shown to play an important role in nociceptive
function in adults. NGF is up-regulated after inflammatory injury of
the skin. Administration of exogenous NGF either systemically or in the
skin causes thermal hyperalgesia within minutes. Mast cells are
considered important components in the action of NGF, because prior
degranulation abolishes the early NGF-induced component of
hyperalgesia. Substances degranulated by mast cells include serotonin,
histamine, and NGF. Blockade of histamine receptors does not prevent
NGF-induced hyperalgesia. The effects of blocking serotonin receptors
are complex and cannot be interpretable uniquely as NGF losing its
ability to induce hyperalgesia. To determine whether NGF has a direct
effect on dorsal root ganglion neurons, we have begun to investigate
the acute effects of NGF on capsaicin responses of small-diameter
dorsal root ganglion cells in culture. NGF acutely conditions the
response to capsaicin, suggesting that NGF may be important in
sensitizing the response of sensory neurons to heat (a process that is
thought to operate via the capsaicin receptor VR1). We also have found
that ligands for the trkB receptor (brain-derived neurotrophic factor
and neurotrophin-4/5) acutely sensitize nociceptive afferents and
elicit hyperalgesia. Because brain-derived neurotrophic factor is
up-regulated in trkA positive cells after inflammatory injury and is
transported anterogradely, we consider it to be a potentially important
peripheral component involved in neurotrophin-induced hyperalgesia.
SIAH-1 promotes apoptosis and tumor suppression through a network involving the regulation of protein folding, unfolding, and trafficking: Identification of common effectors with p53 and p21
Waf1
Authors: Roperch Jean-Pierre , Lethrone Florence , Prieur Sylvie , Piouffre Laurence , Israeli David , Tuynder Marcel , Nemani Mona , Pasturaud Patricia , Gendron Marie-Claude , Dausset Jean , Oren Moshe , Amson Robert B. , Telerman Adam
Published: July 2002

We have previously described biological model systems for studying tumor suppression in which, by using H-1 parvovirus as a selective agent, cells with a strongly suppressed malignant phenotype (KS or US) were derived from malignant cell lines (K562 or U937). By using cDNA display on the K562/KS cells, 15 cDNAs were now isolated, corresponding to genes differentially regulated in tumor suppression. Of these, TSAP9 corresponds to a TCP-1 chaperonin, TSAP13 to a regulatory proteasome subunit, and TSAP21 to syntaxin 11, a vesicular trafficking molecule. The 15 cDNAs were used as a molecular fingerprint in different tumor-suppression models. We found that a similar pattern of differential regulation is shared by activation of p53, p21
Waf1
, and the human homologue of
Drosophila
seven in absentia, SIAH-1. Because SIAH-1 is differentially expressed in the various models, we characterized it at the protein and functional levels. The 32-kDa, mainly nuclear protein encoded by SIAH-1, can induce apoptosis and promote tumor suppression. These results suggest the existence of a common mechanism of tumor suppression and apoptosis shared by p53, p21
Waf1
, and SIAH-1 and involving regulation of the cellular machinery responsible for protein folding, unfolding, and trafficking.
Transcription in Archaea
Authors: Kyrpides Nikos C. , Ouzounis Christos A.
Published: July 2002

Using the sequences of all the known transcription-associated proteins from Bacteria and Eucarya (a total of 4,147), we have identified their homologous counterparts in the four complete archaeal genomes. Through extensive sequence comparisons, we establish the presence of 280 predicted transcription factors or transcription-associated proteins in the four archaeal genomes, of which 168 have homologs only in Bacteria, 51 have homologs only in Eucarya, and the remaining 61 have homologs in both phylogenetic domains. Although bacterial and eukaryotic transcription have very few factors in common, each exclusively shares a significantly greater number with the Archaea, especially the Bacteria. This last fact contrasts with the obvious close relationship between the archaeal and eukaryotic transcription mechanisms
per se
, and in particular, basic transcription initiation. We interpret these results to mean that the archaeal transcription system has retained more ancestral characteristics than have the transcription mechanisms in either of the other two domains.