bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular karyotype (amplotype) of metastatic colorectal cancer by unbiased arbitrarily primed PCR DNA fingerprinting
Authors: Malkhosyan Sergei , Yasuda Jun , Soto Jose Luis , Sekiya Takao , Yokota Jun , Perucho Manuel
Published: July 2002

Genomic instability characterizes the aneuploid cancer cell. Losses of genetic material are critical in cancer by exposing recessive mutations in tumor suppressor genes. Gains of genetic material also may lead to overexpression of genes contributing to tumor progression either in the presence or absence of mutation. However, the detection of moderate gains (such as tri-tetraploidy) has been a challenge in cancer research. Unbiased DNA fingerprinting by the arbitrarily primed PCR allows the detection of moderate gains (in addition to losses) of DNA sequences of known chromosomal localization. We have generated in this manner a molecular karyotype of metastatic colon cancer. This amplotype shows that sequences from several chromosomes undergo both losses (1, 4, 9, 14, and 18) and gains (6, 7, 12, and 20) in over half of the tumors. Moreover, gains of sequences from chromosomes 8 and 13 occurred in most tumors, indicating the existence in these chromosomes of positive regulators of cell growth or survival that are under strong positive selection during tumor progression. We conclude that overrepresentation of these chromosomal regions is a critical step for metastatic colorectal cancer. Comparative amplotype analysis from primary and metastatic tumors suggest the existence in chromosome 4 of gene(s) whose loss is specifically selected in cells that reach the metastatic stage.
Altering hemoglobin levels changes energy status in maize cells under hypoxia
Authors: Sowa Aleksander W. , Duff Stephen M. G. , Guy Phillip A. , Hill Robert D.
Published: July 2002

Nonsymbiotic hemoglobins are broadly present across the plant kingdom; however, the function of these proteins is unknown. Cultured maize cells have been transformed to constitutively express a barley hemoglobin gene in either the sense (HB
+
) or antisense (HB
−
) orientation. Hemoglobin protein in the transformed cell lines correspondingly was higher or lower than in wild-type cells under normal atmospheric conditions. Limiting oxygen availability, by placing the cells in a nitrogen atmosphere for 12 hr, had little effect on the energy status of cells constitutively expressing hemoglobin, but had a pronounced effect on both wild-type and HB
−
cells, where ATP levels declined by 27% and 61%, respectively. Total adenylates in these cells were approximately 35% lower than in HB
+
cells. Energy charge was relatively unaffected by the treatment in HB
+
and wild-type cells, but was reduced from 0.91 to 0.73 in HB
−
cells, suggesting that the latter were incapable of maintaining their energy status under the low oxygen regime. Treatment of the cells grown in an air atmosphere with antimycin A gave essentially the same results. It is suggested that nonsymbiotic hemoglobins act in plants to maintain the energy status of cells in low oxygen environments and that they accomplish this effect by promoting glycolytic flux through NADH oxidation, resulting in increased substrate-level phosphorylation. Hypoxic acclimation of plants is an example of this effect in nature. Nonsymbiotic hemoglobins are likely ancestors of an early form of hemoglobin that sequestered oxygen in low oxygen environments, providing a source of oxygen to oxidize NADH to provide ATP for cell growth and development.
Activation of iron regulatory protein-1 by oxidative stress
in vitro
Authors: Pantopoulos Kostas , Hentze Matthias W.
Published: July 2002

Iron regulatory protein-1 (IRP-1), a central cytoplasmic regulator of cellular iron metabolism, is rapidly activated by oxidative stress to bind to mRNA iron-responsive elements. We have reconstituted the response of IRP-1 to extracellular H
2
O
2
in a system derived from murine B6 fibroblasts permeabilized with streptolysin-O. This procedure allows separation of the cytosol from the remainder of the cells (cell pellet). IRP-1 in the cytosolic fraction fails to be directly activated by addition of H
2
O
2
. IRP-1 activation requires the presence of a nonsoluble, possibly membrane-associated component in the cell pellet. The streptolysin-O-based
in vitro
system faithfully recapitulates characteristic hallmarks of IRP-1 activation by H
2
O
2
in intact cells. We show that the H
2
O
2
-mediated activation of IRP-1 is temperature dependent and sensitive to treatment with calf intestinal alkaline phosphatase (CIAP). Although IRP-1 activation is unaffected by addition of excess ATP or GTP to this
in vitro
system, it is negatively affected by the nonhydrolyzable nucleotide analogs adenylyl-imidodiphosphate and guanylyl-imidophosphate and completely blocked by ATP-γS and GTP-γS. The
in vitro
reconstitution of this oxidative stress-induced pathway has opened a different avenue for the biochemical dissection of the regulation of mammalian iron metabolism by oxidative stress. Our data show that H
2
O
2
must be sensed to stimulate a pathway to activate IRP-1.
Unblocking of chain-terminated primer by HIV-1 reverse transcriptase through a nucleotide-dependent mechanism
Authors: Meyer Peter R. , Matsuura Suzanne E. , So Antero G. , Scott Walter A.
Published: July 2002

HIV-1 replication is inhibited by the incorporation of chain-terminating nucleotides at the 3′ end of the growing DNA chain. Here we show a nucleotide-dependent reaction catalyzed by HIV-1 reverse transcriptase that can efficiently remove the chain-terminating residue, yielding an extendible primer terminus. Radioactively labeled 3′-terminal residue from the primer can be transferred into a product that is resistant to calf intestinal alkaline phosphatase and sensitive to cleavage by snake venom phosphodiesterase. The products formed from different nucleotide substrates have unique electrophoretic migrations and have been identified as dinucleoside tri- or tetraphosphates. The reaction is inhibited by dNTPs that are complementary to the next position on the template (
K
i
≈ 5 μM), suggesting competition between dinucleoside polyphosphate synthesis and DNA polymerization. Dinucleoside polyphosphate synthesis was inhibited by an HIV-1 specific non-nucleoside inhibitor and was absent in mutant HIV-1 reverse transcriptase deficient in polymerase activity, indicating that this activity requires a functional polymerase active site. We suggest that dinucleoside polyphosphate synthesis occurs by transfer of the 3′ nucleotide from the primer to the pyrophosphate moiety in the nucleoside di- or triphosphate substrate through a mechanism analogous to pyrophosphorolysis. Unlike pyrophosphorolysis, however, the reaction is nucleotide-dependent, is resistant to pyrophosphatase, and produces dinucleoside polyphosphates. Because it occurs at physiological concentrations of ribonucleoside triphosphates, this reaction may determine the
in vivo
activity of many nucleoside antiretroviral drugs.
Concerted biosynthesis of an insect elicitor of plant volatiles
Authors: Paré P. W. , Alborn H. T. , Tumlinson J. H.
Published: July 2002

A variety of agricultural plant species, including corn, respond to insect herbivore damage by releasing large quantities of volatile compounds and, as a result, become highly attractive to parasitic wasps that attack the herbivores. An elicitor of plant volatiles,
N
-(17-hydroxylinolenoyl)-
l
-glutamine, named volicitin and isolated from beet armyworm caterpillars, is a key component in plant recognition of damage from insect herbivory. Chemical analysis of the oral secretion from beet armyworms that have fed on
13
C-labeled corn seedlings established that the fatty acid portion of volicitin is plant derived whereas the 17-hydroxylation reaction and the conjugation with glutamine are carried out by the caterpillar by using glutamine of insect origin. Ironically, these insect-catalyzed chemical modifications to linolenic acid are critical for the biological activity that triggers the release of plant volatiles, which in turn attract natural enemies of the caterpillar.
Improved cervical smear assessment using antibodies against proteins that regulate DNA replication
Authors: Williams Gareth H. , Romanowski Piotr , Morris Lesley , Madine Mark , Mills Anthony D. , Stoeber Kai , Marr Jackie , Laskey Ronald A. , Coleman Nicholas
Published: July 2002

Carcinoma of the cervix is one of the most common malignancies. Papanicolaou (Pap) smear tests have reduced mortality by up to 70%. Nevertheless their interpretation is notoriously difficult with high false-negative rates and frequently fatal consequences. We have addressed this problem by using affinity-purified antibodies against human proteins that regulate DNA replication, namely Cdc6 and Mcm5. These antibodies were applied to sections and smears of normal and diseased uterine cervix by using immunoperoxidase or immunofluorescence to detect abnormal precursor malignant cells. Antibodies against Cdc6 and Mcm5 stain abnormal cells in cervical smears and sections with remarkably high specificity and sensitivity. Proliferation markers Ki-67 and proliferating cell nuclear antigen are much less effective. The majority of abnormal precursor malignant cells are stained in both low-grade and high-grade squamous intraepithelial lesions. Immunostaining of cervical smears can be combined with the conventional Pap stain so that all the morphological information from the conventional method is conserved. Thus antibodies against proteins that regulate DNA replication can reduce the high false-negative rate of the Pap smear test and may facilitate mass automated screening.
Phospholipase Cβ4 is specifically involved in climbing fiber synapse elimination in the developing cerebellum
Authors: Kano Masanobu , Hashimoto Kouichi , Watanabe Masahiko , Kurihara Hideo , Offermanns Stefan , Jiang Huiping , Wu Yanping , Jun Kisun , Shin Hee-Sup , Inoue Yoshiro , Simon Melvin I. , Wu Dianqing
Published: July 2002

Elimination of excess climbing fiber (CF)–Purkinje cell synapses during cerebellar development involves a signaling pathway that includes type 1 metabotropic glutamate receptor, Gαq, and the γ isoform of protein kinase C. To identify phospholipase C (PLC) isoforms involved in this process, we generated mice deficient in PLCβ4, one of two major isoforms expressed in Purkinje cells. PLCβ4 mutant mice are viable but exhibit locomotor ataxia. Their cerebellar histology, parallel fiber synapse formation, and basic electrophysiology appear normal. However, developmental elimination of multiple CF innervation clearly is impaired in the rostral portion of the cerebellar vermis, in which PLCβ4 mRNA is predominantly expressed. By contrast, CF synapse elimination is normal in the caudal cerebellum, in which low levels of PLCβ4 mRNA but reciprocally high levels of PLCβ3 mRNA are found. These results indicate that PLCβ4 transduces signals that are required for CF synapse elimination in the rostral cerebellum.
A glycosidase antibody elicited against a chair-like transition state analog by
in vitro
immunization
Authors: Yu Jaehoon , Choi So Young , Moon Kyung-Duk , Chung Hyun-Ho , Youn Hyun Joo , Jeong Sunjoo , Park Hokoon , Schultz Peter G.
Published: July 2002

Antibodies were generated against the positively charged chair-like glycosidase inhibitor nojirimycin by
in vitro
immunization. A number of catalytic antibodies were isolated, one of which catalyzes the hydrolysis of
p
-nitrophenyl β-
d
-glucopyranoside 3 with a rate enhancement (
k
cat
/
k
uncat
) of 10
5
M over the HOAC-catalyzed reaction. The antibody discriminates modifications in the pyranoside ring of substrate 3 at the C2, C4, and the anomeric positions. The pH dependence of the reaction and chemical modification studies suggest the presence of an active-site Asp or Glu residue that may function as a general acid. This study further defines those requirements necessary to generate antibodies that efficiently cleave glycosidic bonds.
Verbal novelty detection within the human hippocampus proper
Authors: Grunwald Thomas , Lehnertz Klaus , Heinze Hans J. , Helmstaedter Christoph , Elger Christian E.
Published: July 2002

Animal studies and neuropsychological tests of patients with temporal lobe epilepsy have demonstrated the importance of human medial temporal lobes for memory formation. In addition, more recent studies have shown that the human hippocampal region is also involved in novelty detection. However, the exact contribution of the hippocampus proper to these processes is still unknown. To examine further its role we compared event-related potentials recorded within the medial temporal lobes in 29 temporal lobe epilepsy patients with and 21 without hippocampal sclerosis. While in patients with extrahippocampal lesions but without hippocampal sclerosis event-related potentials to first presentations and repetitions of words were reduced on the side of the epileptogenic focus, in patients with hippocampal sclerosis only those to first presentations but not to repetitions were affected. Because sclerosis of the hippocampus proper selectively reduced event-related potentials to new but not old verbal stimuli, it can be concluded that the human hippocampus proper contributes to verbal novelty detection.
Differential regulation of IκB kinase α and β by two upstream kinases, NF-κB-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1
Authors: Nakano Hiroyasu , Shindo Masahisa , Sakon Sachiko , Nishinaka Shigeyuki , Mihara Motoyuki , Yagita Hideo , Okumura Ko
Published: July 2002

NF-κB is activated by various stimuli including inflammatory cytokines and stresses. A key step in the activation of NF-κB is the phosphorylation of its inhibitors, IκBs, by an IκB kinase (IKK) complex. Recently, two closely related kinases, designated IKKα and IKKβ, have been identified to be the components of the IKK complex that phosphorylate critical serine residues of IκBs for degradation. A previously identified NF-κB-inducing kinase (NIK), which mediates NF-κB activation by TNFα and IL-1, has been demonstrated to activate IKKα. Previous studies showed that mitogen-activated protein kinase/ERK kinase kinase-1 (MEKK1), which constitutes the c-Jun N-terminal kinase/stress-activated protein kinase pathway, also activates NF-κB by an undefined mechanism. Here, we show that overexpression of MEKK1 preferentially stimulates the kinase activity of IKKβ, which resulted in phosphorylation of IκBs. Moreover, a catalytically inactive mutant of IKKβ blocked the MEKK1-induced NF-κB activation. By contrast, overexpression of NIK stimulates kinase activities of both IKKα and IKKβ comparably, suggesting a qualitative difference between NIK- and MEKK1-mediated NF-κB activation pathways. Collectively, these results indicate that NIK and MEKK1 independently activate the IKK complex and that the kinase activities of IKKα and IKKβ are differentially regulated by two upstream kinases, NIK and MEKK1, which are responsive to distinct stimuli.
The tetravalent guanylhydrazone CNI-1493 blocks the toxic effects of interleukin-2 without diminishing antitumor efficacy
Authors: Kemeny M. Margaret , Botchkina Galina I. , Ochani Mahander , Bianchi Marina , Urmacher Carlos , Tracey Kevin J.
Published: July 2002

The use of interleukin 2 (IL-2) as an antineoplastic agent has been limited by the serious toxicities that accompany the doses necessary for a tumor response. Elevation of nitric oxide (NO) and tumor necrosis factor (TNF) both have been implicated in IL-2 toxicities. CNI-1493, a tetravalent guanylhydrazone, is an inhibitor of macrophage activation including the synthesis of TNF and other cytokines. Doses of CNI-1493 as low as 1 mg/kg/day conferred complete protection against fatal toxicity of IL-2 with IL-2 doses tenfold higher than the safely tolerated level in Sprague–Dawley rats. Moreover, typical pathologic changes in the lungs, kidneys, and the liver caused by IL-2 infusion were blocked by cotreatment with CNI-1493. When animals bearing established hepatomas were given IL-2 and CNI-1493 combination therapy, 10 of 10 hepatomas regressed from 1 cm3to <1 mm3. Intracytoplasmic TNF levels were increased in normal tissues from IL-2 treated animals, and treatment with CNI-1493 maintained TNF at control levels. The degree of apoptosis measured by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining of tumors following IL-2 therapy was not reduced compared with IL-2 cotreated with CNI-1493. In contrast, apoptosis in the liver and lung parenchyma following IL-2 therapy was blocked completely by cotreatment with CNI-1493. Taken together, these data showed that low and infrequent doses of CNI-1493 markedly protected animals from IL-2 systemic toxicities whereas not affecting tumor response to IL-2 therapy. With the protection afforded by CNI-1493 treatment, IL-2 therapy dose levels could be increased to provide significant antitumor effects in animals with established hepatomas.
Snt309p modulates interactions of Prp19p with its associated components to stabilize the Prp19p-associated complex essential for pre-mRNA splicing
Authors: Chen Hau-Ren , Tsao Twee Y. , Chen Chun-Hong , Tsai Wei-Yü , Her Lu-Shiun , Hsu Milton Ming-Tao , Cheng Soo-Chen
Published: July 2002

TheSNT309gene was identified via a mutation that causes lethality of cells in combination with aprp19mutation. We showed previously that Snt309p is a component of the Prp19p-associated complex and that Snt309p, like Prp19p, is associated with the spliceosome immediately after or concomitantly with dissociation of U4 from the spliceosome. We show here that extracts prepared from theSNT309-deleted strain (ΔSNT309) were defective in splicing but could be complemented by addition of the purified Prp19p-associated complex. Isolation of the Prp19p-associated complex from ΔSNT309 extracts indicated that the complex was destabilized in the absence of Snt309p and dissociated on affinity chromatography, suggesting a role of Snt309p in stabilization of the Prp19p-associated complex. Addition of the affinity-purified Prp19p–Snt309p binary complex to ΔSNT309 extracts could reconstitute the Prp19p-associated complex. Genetic analysis further suggests that Snt309p plays a role in modulating interactions of Prp19p with other associated components to facilitate formation of the Prp19p-associated complex. A model for how Snt309p modulates such interactions is proposed.
Activation of flavin-containing oxidases underlies light-induced production of H
2
O
2
in mammalian cells
Authors: Hockberger Philip E. , Skimina Timothy A. , Centonze Victoria E. , Lavin Colleen , Chu Su , Dadras Soheil , Reddy Janardan K. , White John G.
Published: July 2002

Violet-blue light is toxic to mammalian cells, and this toxicity has been linked with cellular production of H
2
O
2
. In this report, we show that violet-blue light, as well as UVA, stimulated H
2
O
2
production in cultured mouse, monkey, and human cells. We found that H
2
O
2
originated in peroxisomes and mitochondria, and it was enhanced in cells overexpressing flavin-containing oxidases. These results support the hypothesis that photoreduction of flavoproteins underlies light-induced production of H
2
O
2
in cells. Because H
2
O
2
and its metabolite, hydroxyl radicals, can cause cellular damage, these reactive oxygen species may contribute to pathologies associated with exposure to UVA, violet, and blue light. They may also contribute to phototoxicity often encountered during light microscopy. Because multiphoton excitation imaging with 1,047-nm wavelength prevented light-induced H
2
O
2
production in cells, possibly by minimizing photoreduction of flavoproteins, this technique may be useful for decreasing phototoxicity during fluorescence microscopy.
Efficient export of the glucose transporter Hxt1p from the endoplasmic reticulum requires Gsf2p
Authors: Sherwood Peter W. , Carlson Marian
Published: July 2002

Mutations in the
GSF2
gene cause glucose starvation phenotypes in
Saccharomyces cerevisiae
. We have isolated the
HXT1
gene, which encodes a low-affinity, high-capacity glucose transporter, as a multicopy suppressor of a
gsf2
mutation. We show that
gsf2
mutants accumulate Hxt1p in the endoplasmic reticulum (ER) and that Gsf2p is a 46-kDa integral membrane protein localized to the ER.
gsf2
mutants also display a galactose growth defect and abnormal localization of the galactose transporter Gal2p but are not defective in function or localization of the high-affinity glucose transporter Hxt2p. These findings suggest that Gsf2p functions in the ER to promote the secretion of certain hexose transporters.
Osteopontin-deficient mice are resistant to ovariectomy-induced bone resorption
Authors: Yoshitake Hiroyuki , Rittling Susan R. , Denhardt David T. , Noda Masaki
Published: July 2002

Osteopontin is one of the major noncollagenous bone matrix proteins produced by osteoblasts and osteoclasts, bone cells that are uniquely responsible for the remodeling of mineralized tissues. Osteoclasts express the αvβ3 integrin, which is one of the receptors for osteopontin. Recent knockout studies revealed that noncollagenous bone matrix proteins are functionally important in regulation of bone metabolism. However, the significance of the presence of osteopontin in
in vivo
has not been known. We report here that osteopontin knockout mice are resistant to ovariectomy-induced bone resorption compared with wild-type mice. Microcomputed tomography analysis indicated about 60% reduction in bone volume by ovariectomy in wild-type mice, whereas the osteopontin-deficient mice exhibited only about 10% reduction in trabecular bone volume after ovariectomy. Reduction in uterine weight was observed similarly in both wild-type and osteopontin-deficient mice, indicating the specificity of the effect of osteopontin deficiency on bone metabolism. We propose that osteopontin is essential for postmenopausal osteoporosis in women. Strategies to counteract osteopontin’s action may prove effective in suppressing osteoporosis.
Crystal structure of ERA: A GTPase-dependent cell cycle regulator containing an RNA binding motif
Authors: Chen Xin , Court Donald L. , Ji Xinhua
Published: July 2002

ERA forms a unique family of GTPase. It is widely conserved and essential in bacteria. ERA functions in cell cycle control by coupling cell division with growth rate. ERA homologues also are found in eukaryotes. Here we report the crystal structure of ERA from
Escherichia coli
. The structure has been determined at 2.4-Å resolution. It reveals a two-domain arrangement of the molecule: an N-terminal domain that resembles p21 Ras and a C-terminal domain that is unique. Structure-based topological search of the C domain fails to reveal any meaningful match, although sequence analysis suggests that it contains a KH domain. KH domains are RNA binding motifs that usually occur in tandem repeats and exhibit low sequence similarity except for the well-conserved segment VIGxxGxxIK. We have identified a
βα
α
β
fold that contains the VIGxxGxxIK sequence and is shared by the C domain of ERA and the KH domain. We propose that this
βα
α
β
fold is the RNA binding motif, the minimum structural requirement for RNA binding. ERA dimerizes in crystal. The dimer formation involves a significantly distorted switch II region, which may shed light on how ERA protein regulates downstream events.
Oreopithecus
was a bipedal ape after all: Evidence from the iliac cancellous architecture
Authors: Rook Lorenzo , Bondioli Luca , Köhler Meike , Moyà-Solà Salvador , Macchiarelli Roberto
Published: July 2002

Textural properties and functional morphology of the hip bone cancellous network of
Oreopithecus bambolii
, a 9- to 7-million-year-old Late Miocene hominoid from Italy, provide insights into the postural and locomotor behavior of this fossil ape. Digital image processing of calibrated hip bone radiographs reveals the occurrence of trabecular features, which, in humans and fossil hominids, are related to vertical support of the body weight, i.e., to bipedality.
Deletion of Ku86 causes early onset of senescence in mice
Authors: Vogel Hannes , Lim Dae-Sik , Karsenty Gerard , Finegold Milton , Hasty Paul
Published: July 2002

DNA double-strand breaks formed during the assembly of antigen receptors or after exposure to ionizing radiation are repaired by proteins important for nonhomologous end joining that include Ku86, Ku70, DNA-PK
CS
, Xrcc4, and DNA ligase IV. Here we show that
ku86-
mutant mice, compared with control littermates, prematurely exhibited age-specific changes characteristic of senescence that include osteopenia, atrophic skin, hepatocellular degeneration, hepatocellular inclusions, hepatic hyperplastic foci, and age-specific mortality. Cancer and likely sepsis (indicated by reactive immune responses) partly contributed to age-specific mortality for both cohorts, and both conditions occurred earlier in
ku86
−/−
mice. These data indicate that Ku86-dependent chromosomal metabolism is important for determining the onset of age-specific changes characteristic of senescence in mice.
Conversion of mammalian 3α-hydroxysteroid dehydrogenase to 20α-hydroxysteroid dehydrogenase using loop chimeras: Changing specificity from androgens to progestins
Authors: Ma Haiching , Penning Trevor M.
Published: July 2002

Hydroxysteroid dehydrogenases (HSDs) regulate the occupancy and activation of steroid hormone receptors by converting potent steroid hormones into their cognate inactive metabolites. 3α-HSD catalyzes the inactivation of androgens in the prostate by converting 5α-dihydrotestosterone to 3α-androstanediol, where excess 5α-dihydrotestosterone is implicated in prostate disease. By contrast, 20α-HSD catalyzes the inactivation of progestins in the ovary and placenta by converting progesterone to 20α-hydroxyprogesterone, where progesterone is essential for maintaining pregnancy. Mammalian 3α-HSDs and 20α-HSDs belong to the aldo-keto reductase superfamily and share 67% amino acid sequence identity yet show positional and stereospecificity for the formation of secondary alcohols on opposite ends of steroid hormone substrates. The crystal structure of 3α-HSD indicates that the mature steroid binding pocket consists of 10 residues located on five loops, including loop A and the mobile loops B and C. 3α-HSD was converted to 20α-HSD by replacing these loops with those found in 20α-HSD. However, when pocket residues in 3α-HSD were mutated to those found in 20α-HSD altered specificity was not achieved. Replacement of loop A created a 17β-HSD activity that was absent in either 3α- or 20α-HSD. Once loops A and C were replaced, the chimera had both 3α- and 20α-HSD activity. When loops A, B, and C were substituted, 3α-HSD was converted to a stereospecific 20α-HSD with a resultant shift in
k
cat
/
K
m
for the desired reaction of 2 × 10
11
. This study represents an example where sex hormone specificity can be changed at the enzyme level.
Distinctive compartmental organization of human primary visual cortex
Authors: Preuss Todd M. , Qi Huixin , Kaas Jon H.
Published: July 2002

In the primary visual area of macaques and other monkeys, layer 4A is a mosaic of separate tissue compartments related to the parvocellular (P) and magnocellular (M) layers of the lateral geniculate nucleus. This mosaic resembles a honeycomb, with thin walls that receive direct P inputs and cores consisting of columns of dendrites and cell bodies ascending from layer 4B, a layer that receives indirect M inputs. To determine whether apes and humans have a macaque-like layer 4A, we examined the primary visual area in humans, chimpanzees, an orangutan, Old World monkeys, and New World monkeys. Apes and humans lacked the dense band of cytochrome oxidase staining in layer 4A that marks the stratum of P-geniculate afferents in monkeys. Furthermore, humans displayed a unique arrangement of presumed M-related cells and dendrites in layer 4A, as revealed with antibodies against nonphosphorylated neurofilaments and microtubule-associated protein 2. Human 4A contained a large amount of M-like tissue distributed in a complex, mesh-like pattern rather than in simple vertical arrays as in other anthropoid primates. Our results suggest that (
i
) the direct P-geniculate projection to layer 4A was reduced early in the evolution of the ape–human group, (
ii
) the M component of layer 4A was subsequently modified (and possibly enhanced) in the human lineage, and (
iii
) the honeycomb model does not adequately characterize human layer 4A. This is the first demonstration of a difference in the cortical architecture of humans and apes, the animals most closely related to humans.
Imino sugars inhibit the formation and secretion of bovine viral diarrhea virus, a pestivirus model of hepatitis C virus: Implications for the development of broad spectrum anti-hepatitis virus agents
Authors: Zitzmann Nicole , Mehta Anand S. , Carrouée Sandra , Butters Terry D. , Platt Frances M. , McCauley John , Blumberg Baruch S. , Dwek Raymond A. , Block Timothy M.
Published: July 2002

One function of N-linked glycans is to assist in the folding of glycoproteins by mediating interactions of the lectin-like chaperone proteins calnexin and calreticulin with nascent glycoproteins. These interactions can be prevented by inhibitors of the α-glucosidases, such as
N
-butyl-deoxynojirimycin (NB-DNJ) and
N
-nonyl-DNJ (NN-DNJ), and this causes some proteins to be misfolded and retained within the endoplasmic reticulum (ER). We have shown previously that the NN-DNJ-induced misfolding of one of the hepatitis B virus (HBV) envelope glycoproteins prevents the formation and secretion of virus
in vitro
and that this inhibitor alters glycosylation and reduces the viral levels in an animal model of chronic HBV infection. This led us to investigate the effect of glucosidase inhibitors on another ER-budding virus, bovine viral diarrhea virus, a tissue culture surrogate of human hepatitis C virus (HCV). Here we show that in MDBK cells α-glucosidase inhibitors prevented the formation and secretion of infectious bovine viral diarrhea virus. Data also are presented showing that NN-DNJ, compared with NB-DNJ, exhibits a prolonged retention in liver
in vivo
. Because viral secretion is selectively hypersensitive to glucosidase inhibition relative to the secretion of cellular proteins, the possibility that glucosidase inhibitors could be used as broad-based antiviral hepatitis agents is discussed. A single drug against HBV, HCV, and, possibly, HDV, which together chronically infect more than 400 million people worldwide, would be of great therapeutic value.
Spectral bifurcations in dispersive wave turbulence
Authors: Cai David , Majda Andrew J. , McLaughlin David W. , Tabak Esteban G.
Published: July 2002

Dispersive wave turbulence is studied numerically for a class of one-dimensional nonlinear wave equations. Both deterministic and random (white noise in time) forcings are studied. Four distinct stable spectra are observed—the direct and inverse cascades of weak turbulence (WT) theory, thermal equilibrium, and a fourth spectrum (MMT; Majda, McLaughlin, Tabak). Each spectrum can describe long-time behavior, and each can be only metastable (with quite diverse lifetimes)—depending on details of nonlinearity, forcing, and dissipation. Cases of a long-live MMT transient state dcaying to a state with WT spectra,
and vice-versa
, are displayed. In the case of freely decaying turbulence, without forcing, both cascades of weak turbulence are observed. These WT states constitute the clearest and most striking numerical observations of WT spectra to date—over four decades of energy, and three decades of spatial, scales. Numerical experiments that study details of the composition, coexistence, and transition between spectra are then discussed, including: (
i
) for deterministic forcing, sharp distinctions between focusing and defocusing nonlinearities, including the role of long wavelength instabilities, localized coherent structures, and chaotic behavior; (
ii
) the role of energy growth in time to monitor the selection of MMT or WT spectra; (
iii
) a second manifestation of the MMT spectrum as it describes a self-similar evolution of the wave,
without temporal averaging
; (
iv
) coherent structures and the evolution of the direct and inverse cascades; and (
v
) nonlocality (in
k
-space) in the transferral process.
Structure-activity analysis of synthetic autoinducing thiolactone peptides from
Staphylococcus aureus
responsible for virulence
Authors: Mayville Patricia , Ji Guangyong , Beavis Ronald , Yang Hongmei , Goger Michael , Novick Richard P. , Muir Tom W.
Published: July 2002

The synthesis of virulence factors and other extracellular proteins
responsible for pathogenicity in
Staphylococcus aureus
is
under the control of the
agr
locus. A secreted
agr
-encoded peptide, AgrD, processed from the AgrD gene
product, is known to be an effector of self-strain activation and
cross-strain inhibition of the
agr
response. Biochemical
analysis of AgrD peptides isolated from culture supernatants has
suggested that they contain an unusual thiol ester-linked cyclic
structure. In the present work, chemical synthesis is used to confirm
that the mature AgrD peptides contain a thiolactone structure and that
this feature is absolutely necessary for full biological activity. The
AgrD synthetic thiolactone peptides exhibited biological activity
in vivo
in a mouse protection test. Structure-activity
studies have allowed key aspects of the peptide structure involved in
the differential activation and inhibition functions to be identified.
Accordingly, we propose a model for activation and inhibition of the
agr
response in which the former, but not the latter,
involves specific acylation of the
agr
transmembrane
receptor, AgrC.
Overexpression of the
Bacillus thuringiensis
(Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects
Authors: Kota Madhuri , Daniell Henry , Varma Sam , Garczynski Stephen F. , Gould Fred , Moar William J.
Published: July 2002

Evolving levels of resistance in insects to the bioinsecticide
Bacillus thuringiensis
(Bt) can be dramatically reduced through the genetic engineering of chloroplasts in plants. When transgenic tobacco leaves expressing Cry2Aa2 protoxin in chloroplasts were fed to susceptible, Cry1A-resistant (20,000- to 40,000-fold) and Cry2Aa2-resistant (330- to 393-fold) tobacco budworm
Heliothis virescens
, cotton bollworm
Helicoverpa zea
, and the beet armyworm
Spodoptera exigua
, 100% mortality was observed against all insect species and strains. Cry2Aa2 was chosen for this study because of its toxicity to many economically important insect pests, relatively low levels of cross-resistance against Cry1A-resistant insects, and its expression as a protoxin instead of a toxin because of its relatively small size (65 kDa). Southern blot analysis confirmed stable integration of
cry
2Aa2 into all of the chloroplast genomes (5,000–10,000 copies per cell) of transgenic plants. Transformed tobacco leaves expressed Cry2Aa2 protoxin at levels between 2% and 3% of total soluble protein, 20- to 30-fold higher levels than current commercial nuclear transgenic plants. These results suggest that plants expressing high levels of a nonhomologous Bt protein should be able to overcome or at the very least, significantly delay, broad spectrum Bt-resistance development in the field.
Immune surveillance against a solid tumor fails because of immunological ignorance
Authors: Ochsenbein Adrian F. , Klenerman Paul , Karrer Urs , Ludewig Burkhard , Pericin Marcus , Hengartner Hans , Zinkernagel Rolf M.
Published: July 2002

Many peripheral solid tumors such as sarcomas and carcinomas express tumor-specific antigens that can serve as targets for immune effector T cells. Nevertheless, overall immune surveillance against such tumors seems relatively inefficient. We studied immune surveillance against a s.c. sarcoma expressing a characterized viral tumor antigen. Surprisingly, the tumor cells were capable of inducing a protective cytotoxic T cell response if transferred as a single-cell suspension. However, if they were transplanted as small tumor pieces, tumors readily grew. Tumor growth correlated strictly with (i) failure of tumor cells to reach the draining lymph nodes and (ii) absence of primed cytotoxic T cells. Cytotoxic T cells were not tolerant or deleted because a tumor antigen-specific cytotoxic T cell response was readily induced in lymphoid tissue by immunization with virus or with tumor cells even in the presence of large tumors. Established tumors were rejected by vaccine-induced effector T cells if effector T cells were maintained by prolonged or repetitive vaccination, but not by single-dose vaccination. Thus, in addition to several other tumor-promoting parameters, some antigenic peripheral sarcomas—and probably carcinomas—may grow not because they anergize or tolerize tumor-specific T cells, but because such tumors are immunologically dealt with as if they were in a so-called immunologically privileged site and are ignored for too long.
Reverse genetic analysis of
Caenorhabditis elegans
presenilins reveals redundant but unequal roles for
sel-
12
and
hop-
1
in Notch-pathway signaling
Authors: Westlund Bethany , Parry Dianne , Clover Ralph , Basson Michael , Johnson Carl D.
Published: July 2002

Mutations in the human presenilin genes
PS1
and
PS2
cause early-onset Alzheimer’s disease. Studies in
Caenorhabditis elegans
and in mice indicate that one function of presenilin genes is to facilitate Notch-pathway signaling. Notably, mutations in the
C. elegans
presenilin gene
sel-12
reduce signaling through an activated version of the Notch receptor LIN-12. To investigate the function of a second
C. elegans
presenilin gene
hop-1
and to examine possible genetic interactions between
hop-1
and
sel-12
, we used a reverse genetic strategy to isolate deletion alleles of both loci. Animals bearing both
hop-1
and
sel-12
deletions displayed new phenotypes not observed in animals bearing either single deletion. These new phenotypes—germ-line proliferation defects, maternal-effect embryonic lethality, and somatic gonad defects—resemble those resulting from a reduction in signaling through the
C. elegans
Notch receptors GLP-1 and LIN-12. Thus SEL-12 and HOP-1 appear to function redundantly in promoting Notch-pathway signaling. Phenotypic analyses of
hop-1
and
sel-12
single and double mutant animals suggest that
sel-12
provides more presenilin function than does
hop-1.
A genetic approach to trace neural circuits
Authors: Horowitz Lisa F. , Montmayeur Jean-Pierre , Echelard Yann , Buck Linda B.
Published: July 2002

Mammalian nervous system function involves billions of neurons which are interconnected in a multitude of neural circuits. Here we describe a genetic approach to chart neural circuits. By using an olfactory-specific promoter, we selectively expressed barley lectin in sensory neurons in the olfactory epithelium and vomeronasal organ of transgenic mice. The lectin was transported through the axons of those neurons to the olfactory bulb, transferred to the bulb neurons with which they synapse, and transported through the axons of bulb neurons to the olfactory cortex. The lectin also was retrogradely transported from the bulb to neuromodulatory brain areas. No evidence could be obtained for adverse effects of the lectin on odorant receptor gene expression, sensory axon targeting in the bulb, or the generation or transmission of signals by olfactory sensory neurons. Transneuronal transfer was detected prenatally in the odor-sensing pathway, but only postnatally in the pheromone-sensing pathway, suggesting that odors, but not pheromones, may be sensedin utero. Our studies demonstrate that a plant lectin can serve as a transneuronal tracer when its expression is genetically targeted to a subset of neurons. This technology can potentially be applied to a variety of vertebrate and invertebrate neural systems and may be particularly valuable for mapping connections formed by small subsets of neurons and for studying the development of connectivity as it occursin utero.
Ectopic expression of the minimal
whiE
polyketide synthase generates a library of aromatic polyketides of diverse sizes and shapes
Authors: Shen Yuemao , Yoon Pall , Yu Tin-Wein , Floss Heinz G. , Hopwood David , Moore Bradley S.
Published: July 2002

The single recombinant expressing the
Streptomyces coelicolor
minimal
whiE
(spore pigment) polyketide synthase (PKS) is uniquely capable of generating a large array of well more than 30 polyketides, many of which, so far, are novel to this recombinant. The characterized polyketides represent a diverse set of molecules that differ in size (chain length) and shape (cyclization pattern). This combinatorial biosynthetic library is, by far, the largest and most complex of its kind described to date and indicates that the minimal
whiE
PKS does not independently control polyketide chain length nor dictate the first cyclization event. Rather, the minimal PKS enzyme complex must rely on the stabilizing effects of additional subunits (i.e., the cyclase
whiE
-ORFVI) to ensure that the chain reaches the full 24 carbons and cyclizes correctly. This dramatic loss of control implies that the growing polyketide chain does not remain enzyme bound, resulting in the spontaneous cyclization of the methyl terminus. Among the six characterized dodecaketides, four different first-ring cyclization regiochemistries are represented, including C7/C12, C8/C13, C10/C15, and C13/C15. The dodecaketide TW93h possesses a unique 2,4-dioxaadamantane ring system and represents a new structural class of polyketides with no related structures isolated from natural or engineered organisms, thus supporting the claim that engineered biosynthesis is capable of producing novel chemotypes.
Factor-specific modulation of CREB-binding protein acetyltransferase activity
Authors: Perissi Valentina , Dasen Jeremy S. , Kurokawa Riki , Wang Zhiyong , Korzus Edward , Rose David W. , Glass Christopher K. , Rosenfeld Michael G.
Published: July 2002

CREB-binding proteins (CBP) and p300 are essential transcriptional coactivators for a large number of regulated DNA-binding transcription factors, including CREB, nuclear receptors, and STATs. CBP and p300 function in part by mediating the assembly of multiprotein complexes that contain additional cofactors such as p300/CBP interacting protein (p/CIP), a member of the p160/SRC family of coactivators, and the p300/CBP associated factor p/CAF. In addition to serving as molecular scaffolds, CBP and p300 each possess intrinsic acetyltransferase activities that are required for their function as coactivators. Here we report that the adenovirus E1A protein inhibits the acetyltransferase activity of CBP on binding to the C/H3 domain, whereas binding of CREB, or a CREB/E1A fusion protein to the KIX domain, fails to inhibit CBP acetyltransferase activity. Surprisingly, p/CIP can either inhibit or stimulate CBP acetyltransferase activity depending on the specific substrate evaluated and the functional domains present in the p/CIP protein. While the CBP interaction domain of p/CIP inhibits acetylation of histones H3, H4, or high mobility group by CBP, it enhances acetylation of other substrates, such as Pit-1. These observations suggest that the acetyltransferase activities of CBP/p300 and p/CAF can be differentially modulated by factors binding to distinct regions of CBP/p300. Because these interactions are likely to result in differential effects on the coactivator functions of CBP/p300 for different classes of transcription factors, regulation of CBP/p300 acetyltransferase activity may represent a mechanism for integration of diverse signaling pathways.
The crystal structure of modified bovine fibrinogen
Authors: Brown Jerry H. , Volkmann Niels , Jun Gyo , Henschen-Edman Agnes H. , Cohen Carolyn
Published: July 2002

Here we report the crystal structure at ≈4-Å resolution of a selectively proteolyzed bovine fibrinogen. This key component in hemostasis is an elongated 340-kDa glycoprotein in the plasma that upon activation by thrombin self-assembles to form the fibrin clot. The crystals are unusual because they are made up of end-to-end bonded molecules that form flexible filaments. We have visualized the entire coiled-coil region of the molecule, which has a planar sigmoidal shape. The primary polymerization receptor pockets at the ends of the molecule face the same way throughout the end-to-end bonded filaments, and based on this conformation, we have developed an improved model of the two-stranded protofibril that is the basic building block in fibrin. Near the middle of the coiled-coil region, the plasmin-sensitive segment is a hinge about which the molecule adopts different conformations. This segment also includes the boundary between the three- and four-stranded portions of the coiled coil, indicating the location on the backbone that anchors the extended flexible Aα arm. We suggest that a flexible branch point in the molecule may help accommodate variability in the structure of the fibrin clot.
Cholesterol-dependent clustering of IL-2Rα and its colocalization with HLA and CD48 on T lymphoma cells suggest their functional association with lipid rafts
Authors: Vereb G. , Matkó J. , Vámosi G. , Ibrahim S. M. , Magyar E. , Varga S. , Szöllősi J. , Jenei A. , Gáspár R. , Waldmann T. A. , Damjanovich S.
Published: July 2002

Immunogold staining and electron microscopy show that IL-2 receptor α-subunits exhibit nonrandom surface distribution on human T lymphoma cells. Analysis of interparticle distances reveals that this clustering on the scale of a few hundred nanometers is independent of the presence of IL-2 and of the expression of the IL-2R β-subunit. Clustering of IL-2Rα is confirmed by confocal microscopy, yielding the same average cluster size, ≈600–800 nm, as electron microscopy. HLA class I and II and CD48 molecules also form clusters of the same size. Disruption of cholesterol-rich lipid rafts with filipin or depletion of membrane cholesterol with methyl-β-cyclodextrin results in the blurring of cluster boundaries and an apparent dispersion of clusters for all four proteins. Interestingly, the transferrin receptor, which is thought to be located outside lipid rafts, exhibits clusters that are only 300 nm in size and are less affected by modifying the membrane cholesterol content. Furthermore, transferrin receptor clusters hardly colocalize with IL-2Rα, HLA, and CD48 molecules (crosscorrelation coefficient is 0.05), whereas IL-2Rα colocalizes with both HLA and CD48 (crosscorrelation coefficient is between 0.37 and 0.46). This coclustering is confirmed by electron microscopy. The submicron clusters of IL-2Rα chains and their coclustering with HLA and CD48, presumably associated with lipid rafts, could underlie the efficiency of signaling in lymphoid cells.
Thyroid hormone receptor-binding protein, an LXXLL motif-containing protein, functions as a general coactivator
Authors: Ko Lan , Cardona Guemalli R. , Chin William W.
Published: July 2002

Nuclear hormone receptors activate gene transcription through ligand-dependent association with coactivators. Specific LXXLL sequence motifs present in these cofactors are sufficient to mediate these ligand-induced interactions. A thyroid hormone receptor (TR)-binding protein (TRBP) was cloned by a Sos-Ras yeast two-hybrid system using TRβ1-ligand binding domain as bait. TRBP contains 2063 amino acid residues, associates with TR through a LXXLL motif, and is ubiquitously expressed in a variety of tissues and cells. TRBP strongly transactivates through TRβ1 and estrogen receptor in a dose-related and ligand-dependent manner, and also exhibits coactivation through AP-1, CRE, and NFκB-response elements, similar to the general coactivator CBP/p300. The C terminus of TRBP binds to CBP/p300 and DRIP130, a component of the DRIP/TRAP/ARC complex, which suggests that TRBP may activate transcription by means of such interactions. Further, the association of TRBP with the DNA-dependent protein kinase (DNA-PK) complex and DNA-independent phosphorylation of TRBP C terminus by DNA-PK point to a potential connection between transcriptional control and chromatin architecture regulation.