bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of inhibitory factor κBα mRNA in the central nervous system after peripheral lipopolysaccharide administration: Anin situhybridization histochemistry study in the rat
Authors: Quan Ning , Whiteside Michael , Kim Leopold , Herkenham Miles
Published: July 2002

In this study we investigate the mRNA expression of inhibitory factor κBα (IκBα) in cells of the rat brain induced by an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). IκB controls the activity of nuclear factor κB, which regulates the transcription of many immune signal molecules. The detection of IκB induction, therefore, would reveal the extent and the cellular location of brain-derived immune molecules in response to peripheral immune challenges. Low levels of IκBα mRNA were found in the large blood vessels and in circumventricular organs (CVOs) of saline-injected control animals. After an i.p. LPS injection (2.5 mg/kg), dramatic induction of IκBα mRNA occurred in four spatio-temporal patterns. Induced signals were first detected at 0.5 hr in the lumen of large blood vessels and in blood vessels of the choroid plexus and CVOs. Second, at 1–2 hr, labeling dramatically increased in the CVOs and choroid plexus and spread to small vascular and glial cells throughout the entire brain; these responses peaked at 2 hr and declined thereafter. Third, cells of the meninges became activated at 2 hr and persisted until 12 hr after the LPS injection. Finally, only at 12 hr, induced signals were present in ventricular ependyma. Thus, IκBα mRNA is induced in brain after peripheral LPS injection, beginning in cells lining the blood side of the blood–brain barrier and progressing to cells inside brain. The spatiotemporal patterns suggest that cells of the blood–brain barrier synthesize immune signal molecules to activate cells inside the central nervous system in response to peripheral LPS. The cerebrospinal fluid appears to be a conduit for these signal molecules.
A new approach for containment of microorganisms: Dual control of streptavidin expression by antisense RNA and the T7 transcription system
Authors: Szafranski Przemyslaw , Mello Charlene M. , Sano Takeshi , Smith Cassandra L. , Kaplan David L. , Cantor Charles R.
Published: July 2002

The use of microorganisms in the open environment would be of less concern if they were endowed with programmed self-destruction mechanisms. Here, we propose a new genetic design to increase the effectiveness of cell suicide systems. It ensures very tight control of the derepression of cell death by the combination of the bacteriophage T7 RNA polymerase-lysozyme system and an inducible synthesis of antisense RNA and the
Escherichia coli
LacI repressor. Functionality of this regulatory concept was tested by applying it to containment of Gram-negative bacteria, based on the conditional expression of the lethal
Streptomyces avidinii
streptavidin gene. Toxicity of streptavidin is derived from its exceptionally high binding affinity for an essential prosthetic group,
d
-biotin. The entire construct was designed to allow the soil bacterium
Pseudomonas putida
to survive only in the presence of aromatic hydrocarbons and their derivatives which it can degrade. Under favorable growth conditions, clones escaping killing appeared at frequencies of only 10
−7
–10
−8
per cell per generation. The general requirement for biotin through the living world should make streptavidin-based conditional lethal designs applicable to a broad range of containment strategies.
Rapid, sensitive structure analysis of oligosaccharides
Authors: Zhao Yingming , Kent Stephen B. H. , Chait Brian T.
Published: July 2002

We have developed an efficient method for the derivatization of oligosaccharides, wherein the oligosaccharide is efficiently ligated to a basic aminooxyacetyl peptide by oxime formation. The resulting glycopeptide yields much higher sensitivity in matrix-assisted laser desorption/ionization mass spectrometry than does the underivatized oligosaccharide. Digestion of the glycopeptide by a exoglycosidase array and subsequent mass spectrometric assay of the digestion products provide a sensitive and rapid way to elucidate the structure of the oligosaccharide. In addition to oligosaccharide sequencing, the ligation reaction between an oligosaccharide and an aminooxyacetyl peptide also provides a potentially very convenient and efficient way for the synthesis of glycopeptides or glycoproteins.
bax
-deficiency promotes drug resistance and oncogenic transformation by attenuating p53-dependent apoptosis
Authors: McCurrach Mila E. , Connor Thomas M. F. , Knudson C. Michael , Korsmeyer Stanley J. , Lowe Scott W.
Published: July 2002

Inactivation of p53-dependent apoptosis promotes oncogenic transformation, tumor development, and resistance to many cytotoxic anticancer agents. p53 can transcriptionally activate
bax
, a
bcl-2
family member that promotes apoptosis. To determine whether
bax
is required for p53-dependent apoptosis, the effects of
bax
deficiency were examined in primary fibroblasts expressing the
E1A
oncogene, a setting where apoptosis is dependent on endogenous p53. We demonstrate that
bax
can function as an effector of
p53
in chemotherapy-induced apoptosis and contributes to a
p53
pathway to suppress oncogenic transformation. Furthermore, we show that additional p53 effectors participate in these processes. These p53-controlled factors act synergistically with Bax to promote a full apoptotic response, and their action is suppressed by the Bcl-2 and E1B 19K oncoproteins. These studies demonstrate that Bax is a determinant of p53-dependent chemosensitivity and illustrate how p53 can promote apoptosis by coordinating the activities of multiple effectors.
Identification of ERF-1 as a member of the AP2 transcription factor family
Authors: McPherson Lisa A. , Baichwal Vijay R. , Weigel Ronald J.
Published: July 2002

The ERF-1 transcription factor was previously shown to be involved in the regulation of estrogen receptor (ER) gene transcription in hormonally responsive breast and endometrial carcinomas. In this study we sought to identify the gene for ERF-1. ERF-1 activates ER gene transcription by binding to the imperfect palindrome CCCTGCGGGG within the promoter of the ER gene. ERF-1 protein was purified from the ER-positive breast carcinoma cell line, MCF7, utilizing ion exchange and DNA affinity chromatography. Peptide sequence analysis was used to isolate a 2.7 kb cDNA clone from an MCF7 cDNA library. This cDNA encodes a protein of 48 kDa previously identified as the AP2γ transcription factor. By gel-shift analysis,
in vitro
synthesized ERF-1 comigrates with MCF7 native ERF-1 complex and demonstrates identical sequence binding specificity as native ERF-1. In addition, AP2 polyclonal antisera supershifts both
in vitro
synthesized and native ERF-1 complexes. These results show that ERF-1 is a member of the AP2 family of developmentally regulated transcription factors. Given the central role of ER expression in breast carcinoma biology, ERF-1 is likely to regulate expression of a set of genes characteristic of the hormonally-responsive breast cancer phenotype.
Particle components of dark matter
Authors: Ellis John
Published: July 2002

Particle candidates for astrophysical dark matter are reviewed,
with particular emphasis on the lightest supersymmetric particle and
the axion. The former is now constrained by accelerator experiments to
have a mass above about 40 GeV, and ongoing searches at accelerators,
in space, and in underground experiments have a good chance to detect
it. A reevaluation of the constraint on the axion from supernova 1987a
leaves open an interesting window where it may be detected if it
constitutes the galactic halo.
P/NP, and the quantum field computer
Authors: Freedman Michael H.
Published: July 2002

The central problem in computer science is the conjecture that two complexity classes,P(polynomial time) andNP(nondeterministic polynomial time—roughly those decision problems for which a proposed solution can be checked in polynomial time), are distinct in the standard Turing model of computation:P≠NP. As a generality, we propose that each physical theory supports computational models whose power is limited by the physical theory. It is well known that classical physics supports a multitude of implementation of the Turing machine. Non-Abelian topological quantum field theories exhibit the mathematical features necessary to support a model capable of solving all ⧣Pproblems, a computationally intractable class, in polynomial time. Specifically, Witten [Witten, E. (1989)Commun. Math. Phys.121, 351–391] has identified expectation values in a certainSU(2)-field theory with values of the Jones polynomial [Jones, V. (1985)Bull. Am. Math. Soc.12, 103–111] that are ⧣P-hard [Jaeger, F., Vertigen, D. & Welsh, D. (1990)Math. Proc. Comb. Philos. Soc.108, 35–53]. This suggests that some physical system whose effective Lagrangian contains a non-Abelian topological term might be manipulated to serve as an analog computer capable of solvingNPor even ⧣P-hard problems in polynomial time. Defining such a system and addressing the accuracy issues inherent in preparation and measurement is a major unsolved problem.
Nitric oxide mediatesN-methyl-d-aspartate receptor-induced activation of p21ras
Authors: Yun Hye-Young , Gonzalez-Zulueta Mirella , Dawson Valina L. , Dawson Ted M.
Published: July 2002

N-methyl-d-aspartate (NMDA) glutamate receptor-mediated increases in intracellular calcium are thought to play a critical role in synaptic plasticity. The mechanisms by which changes in cytoplasmic calcium transmit the glutamate signal to the nucleus, which is ultimately important for long-lasting neuronal responses, are poorly understood. We show that NMDA receptor stimulation leads to activation of p21ras(Ras) through generation of nitric oxide (NO) via neuronal NO synthase. The competitive NO synthase inhibitor,l-nitroarginine methyl ester, prevents Ras activation elicited by NMDA and this effect is competitively reversed by the NO synthase substrate,l-arginine. NMDA receptor stimulation fails to activate Ras in neuronal cultures from mice lacking neuronal NO synthase. NMDA-induced Ras activation occurs through a cGMP-independent pathway as 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a potent and selective inhibitor of guanylyl cyclase, has no effect on NMDA receptor-induced activation of Ras, and the cell-permeable cGMP analog, 8Br-cGMP, does not activate Ras. Furthermore, NO directly activates immunoprecipitated Ras from neurons. NMDA also elicits tyrosine phosphorylation of extracellular signal-regulated kinases, a downstream effector pathway of Ras, through a NO/non-cGMP dependent mechanism, thus supporting the physiologic relevance of endogenous NO regulation of Ras. These results suggest that Ras is a physiologic target of endogenously produced NO and indicates a signaling pathway for NMDA receptor activation that may be important for long-lasting neuronal responses.
Optimal selectin-mediated rolling of leukocytes during inflammationin vivorequires intercellular adhesion molecule-1 expression
Authors: Steeber Douglas A. , Campbell Matthew A. , Basit Abdul , Ley Klaus , Tedder Thomas F.
Published: July 2002

Leukocyte interactions with vascular endothelium during inflammation occur through discrete steps involving selectin-mediated leukocyte rolling and subsequent firm adhesion mediated by members of the integrin and Ig families of adhesion molecules. To identify functional synergy between selectin and Ig family members, mice deficient in both L-selectin and intercellular adhesion molecule 1 (ICAM-1) were generated. Leukocyte rolling velocities in cremaster muscle venules were increased significantly in ICAM-1-deficient mice during both trauma- and tumor necrosis factor α-induced inflammation, but rolling leukocyte flux was not reduced. Elimination of ICAM-1 expression in L-selectin-deficient mice resulted in a sharp reduction in the flux of rolling leukocytes during tumor necrosis factor α-induced inflammation. The observed differences in leukocyte rolling behavior demonstrated that ICAM-1 expression was required for optimal P- and L-selectin-mediated rolling. Increased leukocyte rolling velocities presumably translated into decreased tissue emigration because circulating neutrophil, monocyte, and lymphocyte numbers were increased markedly in L-selectin/ICAM-1-deficient mice. Furthermore, neutrophil emigration during acute peritonitis was reduced by 80% in the double-deficient mice compared with either L-selectin or ICAM-1-deficient mice. Thus, members of the selectin and Ig families function synergistically to mediate optimal leukocyte rollingin vivo, which is essential for the generation of effective inflammatory responses.
Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells
Authors: Pereira Daniel S. , Dorrell Craig , Ito Caryn Y. , Gan Olga I. , Murdoch Barbara , Rao Veena N. , Zou Jian-Ping , Reddy E. Shyam P. , Dick John E.
Published: July 2002

Many chimeric oncogenes have been identified by virtue of the association between chromosomal translocation and specific human leukemias. However, the biological mechanism by which these oncogenes disrupt the developmental program of normal human hematopoietic cells during the initiation of the leukemogenic process is poorly understood due to the absence of an appropriate experimental system to study their function. Here, we report that retroviral transduction of TLS-ERG, a myeloid leukemia-associated fusion gene, to human cord blood cells results in altered myeloid and arrested erythroid differentiation and a dramatic increase in the proliferative and self-renewal capacity of transduced myeloid progenitors. Thus, TLS-ERG expression alone induced a leukemogenic program that exhibited similarities to the human disease associated with this translocation. These results provide an experimental examination of the early stages of the human leukemogenic process induced by a single oncogene and establish a paradigm to functionally assay putative leukemogenic genes in normal human hematopoietic cells.
Altering the biochemical state of individual cultured cells and organelles with ultramicroelectrodes
Authors: Lundqvist J. Anders , Sahlin Frida , Åberg Maria A. I. , Strömberg Anette , Eriksson Peter S. , Orwar Owe
Published: July 2002

We describe an efficient technique for the selective chemical and biological manipulation of the contents of individual cells. This technique is based on the electric-field-induced permeabilization (electroporation) in biological membranes using a low-voltage pulse generator and microelectrodes. A spatially highly focused electric field allows introduction of polar cell-impermeant solutes such as fluorescent dyes, fluorogenic reagents, and DNA into single cells. The high spatial resolution of the technique allows for design of, for example, cellular network constructions in which cells in close contact with each other can be made to possess different biochemical, biophysical, and morphological properties. Fluorescein, and fluo-3 (a calcium-sensitive fluorophore), are electroporated into the soma of cultured single progenitor cells derived from adult rat hippocampus. Fluo-3 also is introduced into individual submicrometer diameter processes of thapsigargin-treated progenitor cells, and a plasmid vector cDNA construct (pRAY 1), expressing the green fluorescent protein, is electroporated into cultured single COS 7 cells. At high electric field strengths, observations of dye-transfer into organelles are proposed.
Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A4orthologs of Antarctic notothenioid fishes
Authors: Fields Peter A. , Somero George N.
Published: July 2002

To elucidate mechanisms of enzymatic adaptation to extreme cold, we determined kinetic properties, thermal stabilities, and deduced amino acid sequences of lactate dehydrogenase A4(A4-LDH) from nine Antarctic (−1.86 to 1°C) and three South American (4 to 10°C) notothenioid teleosts. Higher Michaelis–Menten constants (Km) and catalytic rate constants (kcat) distinguish orthologs of Antarctic from those of South American species, but no relationship exists between adaptation temperature and the rate at which activity is lost because of heat denaturation. In all species, active site residues are conserved fully, and differences inkcatandKmare caused by substitutions elsewhere in the molecule. Within geographic groups, identical kinetic properties are generated by different substitutions. By combining our data with A4-LDH sequences for other vertebrates and information on roles played by localized conformational changes in settingkcat, we conclude that notothenioid A4-LDHs have adapted to cold temperatures by increases in flexibility in small areas of the molecule that affect the mobility of adjacent active-site structures. Using these findings, we propose a model that explains linked temperature-adaptive variation inKmandkcat. Changes in sequence that increase flexibility of regions of the enzyme involved in catalytic conformational changes may reduce energy (enthalpy) barriers to these rate-governing shifts in conformation and, thereby, increasekcat. However, at a common temperature of measurement, the higher configurational entropy of a cold-adapted enzyme may foster conformations that bind ligands poorly, leading to highKmvalues relative to warm-adapted orthologs.
Isolation of yeast mutants defective for localization of vacuolar vital dyes
Authors: Zheng Bing , Wu Jennifer N. , Schober Wendy , Lewis Dorothy E. , Vida Thomas
Published: July 2002

An application of flow cytometric sorting is used for isolation of
Saccharomyces cerevisiae
mutants that mislocalize vacuolar vital dyes. This screen is based on the ability of a lipophilic styryl compound,
N
-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4–64), to label endocytic intermediates from the plasma membrane to the vacuole membrane at 15°C. Cells stained at 15°C for both FM4–64 and carboxydichlorofluorescein diacetate (a vacuolar luminal vital stain), had a pronounced shift in red/green fluorescence from cells stained at 30° or 38°C. Flow cytometric selection based on this characteristic shift allowed the isolation of 16 mutants. These comprised 12 complementation groups, which we have designated
SVL
for
s
tyryl dye
v
acuolar
l
ocalization. These groups were put into three classes. Class I mutants contain very large vacuoles; class II mutants have very fragmented vacuoles; and class III mutants show the strongest svl phenotype with punctate/diffuse FM4–64 staining. Limited genetic overlap was observed with previously isolated mutants, namely
svl2
/
vps41
,
svl6
/
vps16
, and
svl7
/
fab1
. The remaining
svl
mutants appear to represent novel genes, two of which showed temperature-sensitive vacuole staining morphology. Another mutant,
svl8
, displayed defects in uptake and sorting of phosphatidylcholine and phosphatidylethanolamine. Our flow cytometric strategy may be useful for isolation of other mutants where mislocalization of fluorescent compounds can be detected.
The mechanism of proton pumping by cytochrome
c
oxidase
Authors: Michel Hartmut
Published: July 2002

Cytochrome
c
oxidase catalyzes the reduction of oxygen to water that is accompanied by pumping of four protons across the mitochondrial or bacterial membrane. Triggered by the results of recent x-ray crystallographic analyses, published data concerning the coupling of individual electron transfer steps to proton pumping are reanalyzed: Conversion of the conventional oxoferryl intermediate F to the fully oxidized form O is connected to pumping of only one proton. Most likely one proton is already pumped during the double reduction of O, and only three protons during conversion of the “peroxy” forms P to O via the oxoferryl form F. Based on the available structural, spectroscopic, and mutagenesis data, a detailed mechanistic model, carefully considering electrostatic interactions, is presented. In this model, each of the four reductions of heme
a
during the catalytic cycle is coupled to the uptake of one proton via the D-pathway. These protons, but never more than two, are temporarily stored in the regions of the heme
a
and
a
3
propionates and are driven to the outside (“pumped”) by electrostatic repulsion from protons entering the active site during turnover. The first proton is pumped by uptake of one proton via the K-pathway during reduction, the second and third proton during the P → F transition when the D-pathway and the active site become directly connected, and the fourth one upon conversion of F to O. Atomic structures are assigned to each intermediate including F′ with an alternative route to O.
Suppression of cell transformation by the cyclin-dependent kinase inhibitor p57
KIP2
requires binding to proliferating cell nuclear antigen
Authors: Watanabe Hiroyuki , Pan Zhen-Qiang , Schreiber-Agus Nicole , DePinho Ronald A. , Hurwitz Jerard , Xiong Yue
Published: July 2002

Proper control of the mammalian cell cycle requires the function of cyclin-dependent kinase (CDK) inhibitors. The p21 family currently includes three distinct genes, p21, p27
Kip1
, and p57
Kip2
, that share a common N-terminal domain for binding to and inhibiting the kinase activity of CDK-cyclin complexes. The p21 protein also binds to proliferating cell nuclear antigen (PCNA) through a separate C-terminal domain affecting DNA replication and repair. The p27 and p57 proteins also each contain unique C-terminal domains whose functions are unknown. Here we show that the human p57 protein, like p21, contains a PCNA-binding domain within its C terminus that, when separated from its N-terminal CDK-cyclin binding domain, can prevent DNA replication
in vitro
and S phase entry
in vivo
. Disruption of either CDK/cyclin or PCNA binding partially reduced p57’s ability to suppress myc/RAS-mediated transformation in primary cells, while loss of both inhibitory functions completely eliminated p57’s suppressive activity. Thus, control of cell cycle and suppression of cell transformation by p57 require both CDK and PCNA inhibitory activity, and disruption of either or both functions may lead to uncontrolled cell growth.
A unique glucose-dependent apoptotic pathway induced by c-Myc
Authors: Shim Hyunsuk , Chun Yoon S. , Lewis Brian C. , Dang Chi V.
Published: July 2002

The lactate dehydrogenase A (
LDH-A
) gene, whose product participates in normal anaerobic glycolysis and is frequently increased in human cancers, has been identified as a c-Myc-responsive gene. It was of interest, therefore, to compare the effect of glucose deprivation in c-Myc-transformed and nontransformed cells. We observed that glucose deprivation or treatment with the glucose antimetabolite 2-deoxyglucose caused nontransformed cells to arrest in the G
0
/G
1
phase of the cell cycle. In contrast, c-Myc-transformed fibroblasts, lymphoblastoid, or lung carcinoma cells underwent extensive apoptosis. Ectopic expression of
LDH-A
alone in Rat1a fibroblasts was sufficient to induce apoptosis with glucose deprivation but not with serum withdrawal, suggesting that LDH-A mediates the unique apoptotic effect of c-Myc when glycolysis is blocked. The apoptosis caused by glucose deprivation was blocked by Bcl-2 expression but appeared to be independent of wild-type p53 activity. These studies provide insights on the coupling of glucose metabolism and the cell cycle in c-Myc-transformed cells and may in the future be exploited for cancer therapeutics.
A poly(A) binding protein functions in the chloroplast as a message-specific translation factor
Authors: Yohn Christopher B. , Cohen Amybeth , Danon Avihai , Mayfield Stephen P.
Published: July 2002

High-affinity binding of a set of proteins with specificity for the 5′ untranslated region (UTR) of the
Chlamydomonas reinhardtii
chloroplast
psbA
mRNA correlates with light-regulated translational activation of this message. We have isolated a cDNA encoding the main
psbA
RNA binding protein, RB47, and identified this protein as a member of the poly(A) binding protein family. Poly(A) binding proteins are a family of eukaryotic, cytoplasmic proteins thought to bind poly(A) tails of mRNAs and play a role in translational regulation.
In vitro
translation of RNA transcribed from the RB47 cDNA produces a precursor protein that is efficiently transported into the chloroplast and processed to the mature 47-kDa protein. RB47 expressed and purified from
Escherichia coli
binds to the
psbA
5′ UTR with similar specificity and affinity as RB47 isolated from
C. reinhardtii
chloroplasts. The identification of a normally cytoplasmic translation factor in the chloroplast suggests that the prokaryotic-like chloroplast translation machinery utilizes a eukaryotic-like initiation factor to regulate the translation of a key chloroplast mRNA. These data also suggest that poly(A) binding proteins may play a wider role in translation regulation than previously appreciated.
Negative regulation of the proteolytic activation of a developmental transcription factor in
Bacillus subtilis
Authors: Resnekov Orna , Losick Richard
Published: July 2002

The sporulation transcription factor σ
K
of
Bacillus subtilis
is controlled by a signal transduction pathway that operates at the level of the proteolytic processing of the inactive precursor protein pro-σ
K
. The conversion of pro-σ
K
to σ
K
requires the putative processing enzyme SpoIVFB and is governed by the regulatory proteins SpoIVFA and BofA. We engineered vegetative cells to carry out processing of pro-σ
K
by inducing the synthesis of the proprotein, a modified form of the putative processing enzyme, and its two regulators during growth. The results showed that (
i
) modified SpoIVFB was the only sporulation protein necessary to achieve processing of pro-σ
K
; (
ii
) SpoIVFA stimulated processing, apparently by protecting the processing enzyme from degradation; (
iii
) BofA inhibited processing in a manner that did not involve degradation of SpoIVFB; and (
iv
) the inhibition of SpoIVFB by BofA was dependent on SpoIVFA. We conclude that BofA and SpoIVFA act synergistically and are the only two sporulation proteins needed to inhibit the function of SpoIVFB. Our results are consistent with the idea that activation of pro-σ
K
occurs by a reversal of the BofA/SpoIVFA-mediated inhibition of the processing enzyme.
Crystal structure of MTCP-1: Implications for role of TCL-1 and MTCP-1 in T cell malignancies
Authors: Fu Zheng-Qing , Du Bois Garrett C. , Song Sherry P. , Kulikovskaya Irina , Virgilio Laura , Rothstein Jay L. , Croce Carlo M. , Weber Irene T. , Harrison Robert W.
Published: July 2002

Two related oncogenes,
TCL-1
and
MTCP-1
, are overexpressed in T cell prolymphocytic leukemias as a result of chromosomal rearrangements that involve the translocation of one T cell receptor gene to either chromosome 14q32 or Xq28. The crystal structure of human recombinant MTCP-1 protein has been determined at 2.0 Å resolution by using multiwavelength anomalous dispersion data from selenomethionine-enriched protein and refined to an
R
factor of 0.21. MTCP-1 folds into a compact eight-stranded β barrel structure with a short helix between the fourth and fifth strands. The topology is unique. The structure of TCL-1 has been predicted by molecular modeling based on 40% amino acid sequence identity with MTCP-1. The identical residues are clustered inside the barrel and on the surface at one side of the barrel. The overall structure of MTCP-1 superficially resembles the structures of proteins in the lipocalin family and calycin superfamily. These proteins have diverse functions, including transport of retinol, fatty acids, chromophores, pheromones, synthesis of prostaglandin, immune modulation, and cell regulation. However, MTCP-1 differs in the topology of the β strands. The structural similarity suggests that MTCP-1 and TCL-1 form a unique family of β barrel proteins that is predicted to bind small hydrophobic ligands and function in cell regulation.
Light-induced expression of fatty acid desaturase genes
Authors: Kis Mihály , Zsiros Otto , Farkas Tibor , Wada Hajime , Nagy Ferenc , Gombos Zoltán
Published: July 2002

In cyanobacterial cells, fatty acid desaturation is one of the crucial steps in the acclimation processes to low-temperature conditions. The expression of all the four acyl lipid desaturase genes of
Synechocystis
PCC 6803 was studied as a function of temperature and separately as a function of light. We used cells grown at 25°C in light-activated heterotrophic growth conditions. In these cells, the production of α-linolenic acid and 18:4 fatty acids was negligible and the synthesis of γ-linolenic acid was remarkably suppressed compared with those of the cells grown photoautotrophically. The cells grown in the light in the presence of glucose showed no difference in fatty acid composition compared with cells grown photoautotrophically. The level of
desC
mRNA for Δ9 desaturase was not affected by either the temperature or the light. It was constitutively expressed at 25°C with and without illumination. The level of
desB
transcripts was negligible in the dark-grown cells and was enhanced about 10-fold by exposure of the cells to light. The maximum level of expression occurred within 15 min. The level of
desA
and
desD
mRNAs was higher in dark-grown cells than that of
desB
mRNA for ω3 desaturase. However, the induction of both
desA
and
desD
mRNAs for Δ12 and Δ6 desaturases, respectively, was enhanced by light about 10-fold. Rifampicin, chloramphenicol, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea completely blocked the induction of the expression of
desA
,
desB
, and
desD
. Consequently, we suggest the regulatory role of light via photosynthetic processes in the induction of the expression of acyl lipid desaturases.
Evidence for keratinocyte stem cells
in vitro
: Long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes
Authors: Kolodka Taduesz M. , Garlick Jonathan A. , Taichman Lorne B.
Published: July 2002

Epidermis is renewed by a population of stem cells that have been defined
in vivo
by slow turnover, label retention, position in the epidermis, and enrichment in β
1
integrin, and
in vitro
by clonogenic growth, prolonged serial passage, and rapid adherence to extracellular matrix. The goal of this study is to determine whether clonogenic cells with long-term growth potential
in vitro
persist
in vivo
and give rise to a fully differentiated epidermis. Human keratinocytes were genetically labeled in culture by transduction with a retrovirus encoding the
lacZ
gene and grafted to athymic mice. Analysis of the cultures before grafting showed that 21.1–27.8% of clonogenic cells with the capacity for >30 generations were successfully transduced.
In vivo
, β-galactosidase (β-gal) positive cells participated in the formation of a fully differentiated epithelium and were detected throughout the 40-week postgraft period, initially as loosely scattered clusters and later as distinct vertical columns. Viable cells recovered from excised grafts were seeded at clonal densities and 23.3–33.3% of the colonies thus formed were β-gal positive. In addition, no evidence of transgene inactivation was obtained: all keratinocyte colonies recovered from grafted tissue that were β-gal negative also lacked the
lacZ
transgene. These results show that cells with long-term growth properties
in vitro
do indeed persist
in vivo
and form a fully differentiated epidermis, thereby exhibiting the properties of stem cells.
From HER2/Neu signal cascade to androgen receptor and its coactivators: A novel pathway by induction of androgen target genes through MAP kinase in prostate cancer cells
Authors: Yeh Shuyuan , Lin Hui-Kuan , Kang Hong-Yo , Thin Tin Htwe , Lin Ming-Fong , Chang Chawnshang
Published: July 2002

Overexpression of the
HER2
/
Neu
protooncogene has been linked to the progression of breast cancer. Here we demonstrate that the growth of prostate cancer LNCaP cells can also be increased by the stable transfection of
HER2
/
Neu
. Using AG879, a HER2/Neu inhibitor, and PD98059, a MAP kinase inhibitor, as well as MAP kinase phosphatase-1 (MPK-1), in the transfection assay, we found that HER2/Neu could induce prostate-specific antigen (PSA), a marker for the progression of prostate cancer, through the MAP kinase pathway at a low androgen level. Reporter assays and mammalian two-hybrid assays further suggest this HER2/Neu-induced androgen receptor (AR) transactivation may function through the promotion of interaction between AR and AR coactivators, such as ARA70. Furthermore, we found this HER2/Neu → MAP kinase → AR-ARAs → PSA pathway could not be blocked completely by hydroxyflutamide, an antiandrogen used in the treatment of prostate cancer. Together, these data provide a novel pathway from HER2/Neu to AR transactivation, and they may represent one of the reasons for the PSA re-elevation and hormone resistance during androgen ablation therapy in prostate cancer patients.
Evolution of the fungal self-fertile reproductive life style from self-sterile ancestors
Authors: Yun Sung-Hwan , Berbee Mary L. , Yoder O. C. , Turgeon B. Gillian
Published: July 2002

In most fungal ascomycetes, mating is controlled by a single locus (
MAT
). Fungi requiring a partner to mate are heterothallic (self-sterile); those not requiring a partner are homothallic (self-fertile). Structural analyses of
MAT
sequences from homothallic and heterothallic
Cochliobolus
species support the hypothesis that heterothallism is ancestral. Homothallic species carry both
MAT
genes in a single nucleus, usually closely linked or fused, in contrast to heterothallic species, which have alternate
MAT
genes in different nuclei. The structural organization of
MAT
from all heterothallic species examined is highly conserved; in contrast, the organization of
MAT
in each homothallic species is unique. The mechanism of conversion from heterothallism to homothallism is a recombination event between islands of identity in otherwise dissimilar
MAT
sequences. Expression of a fused
MAT
gene from a homothallic species confers self-fertility on a
MAT
-null strain of a heterothallic species, suggesting that
MAT
alone is sufficient to change reproductive life style.
Microsatellites provide evidence for Y chromosome diversity among the founders of the New World
Authors: Ruiz-Linares Andrés , Ortíz-Barrientos Daniel , Figueroa Mauricio , Mesa Natalia , Múnera Juan G. , Bedoya Gabriel , Vélez Iván D. , García Luis F. , Pérez-Lezaun Anna , Bertranpetit Jaume , Feldman Marcus W. , Goldstein David B.
Published: July 2002

Recently, Y chromosome markers have begun to be used to study Native American origins. Available data have been interpreted as indicating that the colonizers of the New World carried a single founder haplotype. However, these early studies have been based on a few, mostly complex polymorphisms of insufficient resolution to determine whether observed diversity stems from admixture or diversity among the colonizers. Because the interpretation of Y chromosomal variation in the New World depends on founding diversity, it is important to develop marker systems with finer resolution. Here we evaluate the hypothesis of a single-founder Y haplotype for Amerinds by using 11 Y-specific markers in five Colombian Amerind populations. Two of these markers (DYS271, DYS287) are reliable indicators of admixture and detected three non-Amerind chromosomes in our sample. Two other markers (DYS199, M19) are single-nucleotide polymorphisms mostly restricted to Native Americans. The relatedness of chromosomes defined by these two markers was evaluated by constructing haplotypes with seven microsatellite loci (DYS388 to 394). The microsatellite backgrounds found on the two haplogroups defined by marker DYS199 demonstrate the existence of at least two Amerind founder haplotypes, one of them (carrying allele DYS199 T) largely restricted to Native Americans. The estimated age and distribution of these haplogroups places them among the founders of the New World.
Synthesis andin vivomurine evaluation of Na4[1-(1′-B10H9)-6-SHB10H8] as a potential agent for boron neutron capture therapy
Authors: Feakes Debra A. , Waller R. Corey , Hathaway Deborah K. , Morton Veronica S.
Published: July 2002

Reaction of the normal isomer of [B20H18]2−and the protected thiol anion, [SC(O)OC(CH3)3]−, produces an unexpected isomer of [B20H17SC(O)OC(CH3)3]4−directly and in good yield. The isomer produced under mild conditions is characterized by an apical–apical boron atom intercage connection as well as the location of the thiol substituent on an equatorial belt adjacent to the terminal boron apex. Although the formation of this isomer from nucleophilic attack of the normal isomer of [B20H18]2−has not been reported previously, the isomeric assignment has been unambiguously confirmed by one-dimensional and two-dimensional11B NMR spectroscopy. Deprotection of the thiol substituent under acidic conditions produces a protonated intermediate, [B20H18SH]3−, which can be deprotonated with a suitable base to yield the desired product, [B20H17SH]4−. The sodium salt of the resulting [B20H17SH]4−ion has been encapsulated in small, unilamellar liposomes, which are capable of delivering their contents selectively to tumorsin vivo, and investigated as a potential agent for boron neutron capture therapy. The biodistribution of boron was determined after intravenous injection of the liposomal suspension into BALB/c mice bearing EMT6 mammary adenocarcinoma. At low injected doses, the tumor boron concentration increased throughout the time-course experiment, resulting in a maximum observed boron concentration of 46.7 μg of B per g of tumor at 48 h and a tumor to blood boron ratio of 7.7. The boron concentration obtained in the tumor corresponds to 22.2% injected dose (i.d.) per g of tissue, a value analogous to the most promising polyhedral borane anions investigated for liposomal delivery and subsequent application in boron neutron capture therapy.
ZEB represses transcription through interaction with the corepressor CtBP
Authors: Postigo Antonio A. , Dean Douglas C.
Published: July 2002

ZEB is an active transcriptional repressor that regulates lymphocyte and muscle differentiation in vertebrates. Its homologue in
Drosophila
(
zfh-1
) is also essential for differentiation of somatic and cardiac muscle. Here, we demonstrate that ZEB and
zfh-1
interact with the corepressor CtBP to repress transcription. ZEB and
zfh-1
, both contain the sequence PLDLS in the same region of the repressor domain, and we demonstrate that this sequence binds CtBP-1 and -2. In vertebrate species, ZEB contains two additional CtBP-like binding sites (variations of the PLDLS sequence) that also bind CtBP proteins and are required for full repressor activity. The three sites have an additive effect, and mutation of all three sites is necessary to abolish both binding to CtBP and repressor activity. Finally, we demonstrate that the interaction of CtBP with ZEB at the promoter is necessary for repressor activity.
A method for high-sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry
Authors: Keough T. , Youngquist R. S. , Lacey M. P.
Published: July 2002

A method has been developed for
de novo
peptide sequencing using matrix-assisted laser desorption ionization mass spectrometry. This method will facilitate biological studies that require rapid determination of peptide or protein sequences, e.g., determination of posttranslational modifications, identification of active compounds isolated from combinatorial peptide libraries, and the selective identification of proteins as part of proteome studies. The method involves fast, one-step addition of a sulfonic acid group to the N terminus of tryptic peptides followed by acquisition of postsource decay (PSD) fragment ion spectra. The derivatives are designed to promote efficient charge site-initiated fragmentation of the backbone amide bonds and to selectively enhance the detection of a single fragment ion series that contains the C terminus of the molecule (y-ions). The overall method has been applied to pmol quantities of peptides. The resulting PSD fragment ion spectra often exhibit uninterrupted sequences of 20 or more amino acid residues. However, fragmentation efficiency decreases considerably at amide bonds on the C-terminal side of Pro. The spectra are simple enough that
de novo
sequence tagging is routine. The technique has been successfully applied to peptide mixtures, to high-mass peptides (up to 3,600 Da) and to the unambiguous identification of proteins isolated from two-dimensional gel electrophoresis. The PSD spectra of these derivatized peptides often allow far more selective protein sequence database searches than those obtained from the spectra of native peptides.
A single amino acid near the C terminus of the synaptosomeassociated protein of 25 kDa (SNAP-25) is essential for exocytosis in chromaffin cells
Authors: Criado Manuel , Gil Anabel , Viniegra Salvador , Gutiérrez Luis M.
Published: July 2002

Amperometry in chromaffin cells expressing green fluorescent protein (GFP) fused to synaptosome-associated protein of 25 kDa (SNAP-25) have been used to test the involvement of single amino acids in exocytotic function, overcoming some of the limitations of studies based on Botulinum neurotoxin cleavage, as this occurs at defined sites of the protein. Constructs containing either the whole SNAP-25 polypeptide or several deleted forms lacking its C-terminal domain were heavily overexpressed in transfected cells. All GFP-fusions were located in both the cytoplasm and the plasma membrane. Although a construct containing complete SNAP-25 sustained normal secretion, removal of four or more amino acids of its C terminus greatly altered the overall rate and extent of exocytosis. Further mutational analysis proved that Leu
203
, the fourth residue from the C terminus, is critical for secretion. Kinetics of single granule fusions from cells expressing truncated forms showed slow onset and decay times when compared with control cells expressing full SNAP-25. Thus, these data provide direct evidence for the involvement of a specific residue of SNAP-25 in exocytosis and show that overexpression of GFP-SNAP contructs combined with single vesicle fusion measurements constitutes a powerful approach to dissect the structural elements playing a role in individual steps of the exocytotic cascade.
The Epstein–Barr virus latencyBamHI-Q promoter is positively regulated by STATs and Zta interference with JAK/STAT activation leads to loss ofBamHI-Q promoter activity
Authors: Chen Honglin , Lee Jae Myun , Wang Yilong , Huang Dolly P. , Ambinder Richard F. , Hayward S. Diane
Published: July 2002

In Epstein–Barr virus (EBV)-associated tumors in nonimmunocompromised patients, EBV gene expression is highly restricted. EBV-encoded nuclear antigen (EBNA)-1 is expressed, whereas the immunogenic and proliferative EBNAs are not. This pattern of EBNA expression is generated by usage of theBamHI-Q promoter (Qp). We have determined that the JAK/STAT pathway positively regulates Qp activity. In transient-transfection assays, a Qp–CAT reporter was activated by cotransfected JAK-1 and by treatment of cells with the cytokine IL-6. The ability of Qp to bind signal transducer and activator of transcription (STAT) proteins was directly demonstrated by electrophoretic mobility-shift assay, and mutation of potential STAT-binding sites reduced Qp responsiveness to Janus kinase (JAK)-1. Consistent with a role for STATs in Qp function, Qp using Burkitt’s lymphoma Rael cells and cultured nasopharyngeal carcinoma (NPC) cells contained nuclear STAT protein. We investigated whether the inability to maintain EBV-positive NPC cell lines in culture was related to Qp activity. Passaging of the NPC cell line HK666 led to activation of expression of BZLF1, which encodes Zta and loss of Qp function. Transient expression assays linked Zta expression to the down-regulation of Qp. Cotransfection of Zta reduced Qp activity in reporter assays. This negative regulation required Zta DNA-binding activity. We provide evidence that Zta up-regulation of p53 leads to p53-mediated interference with JAK/STAT activation of Qp. The data imply that JAK/STAT signaling has a role in EBV-associated malignancies.
Rapid GTP binding and hydrolysis by G
q
promoted by receptor and GTPase-activating proteins
Authors: Mukhopadhyay Suchetana , Ross Elliott M.
Published: July 2002

Receptor-promoted GTP binding and GTPase-activating protein (GAP)-promoted GTP hydrolysis determine the onset and termination of G protein signaling; they coordinately control signal amplitude. The mechanisms whereby cells independently regulate signal kinetics and signal amplitude are therefore central to understanding G protein function. We have used quench-flow kinetic methods to measure the rates of the individual reactions of the agonist-stimulated GTPase cycle for G
q
during steady-state signaling. G
q
and m1 muscarinic cholinergic receptor were co-reconstituted into proteoliposomes with one of two GAPs: phospholipase C (PLC)-β1, the major G
q
-regulated effector protein, and RGS4, a GAP commonly thought to be an inhibitor of G
q
signaling. In this system, the rate constant for GAP-stimulated hydrolysis of Gα
q
-bound GTP at 30°C was 9–12 s
−1
for PLC-β1 and 22–27 s
−1
for RGS4. These rates are 1,000- to 2,000-fold faster than in the absence of a GAP and far faster than measured previously. G
q
can thus hydrolyze bound GTP with deactivation half-times of 25–75 ms at 30°C, commensurate with physiological rates of signal termination. GDP/GTP exchange, which reactivates G
q
, was the principal rate-limiting step for the GTPase cycle and was also faster than previously thought. At physiological concentrations of GTP, exchange was limited by the rate of dissociation of GDP from the receptor–G
q
complex, with a maximal rate of 1.8 s
−1
at 30°C. Comparison of activation and deactivation rates help explain how GDP/GTP exchange balance rapid GTP hydrolysis to maintain steady-state signal amplitude.
Purification, molecular cloning, and expression of 2-hydroxyphytanoyl-CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon–carbon bond cleavage during α-oxidation of 3-methyl-branched fatty acids
Authors: Foulon Veerle , Antonenkov Vasily D. , Croes Kathleen , Waelkens Etienne , Mannaerts Guy P. , Van Veldhoven Paul P. , Casteels Minne
Published: July 2002

In the third step of the α-oxidation of 3-methyl-branched fatty acids such as phytanic acid, a 2-hydroxy-3-methylacyl-CoA is cleaved into formyl-CoA and a 2-methyl-branched fatty aldehyde. The cleavage enzyme was purified from the matrix protein fraction of rat liver peroxisomes and identified as a protein made up of four identical subunits of 63 kDa. Its activity proved to depend on Mg
2+
and thiamine pyrophosphate, a hitherto unrecognized cofactor of α-oxidation. Formyl-CoA and 2-methylpentadecanal were identified as reaction products when the purified enzyme was incubated with 2-hydroxy-3-methylhexadecanoyl-CoA as the substrate. Hence the enzyme catalyzes a carbon–carbon cleavage, and we propose calling it 2-hydroxyphytanoyl-CoA lyase. Sequences derived from tryptic peptides of the purified rat protein were used as queries to recover human expressed sequence tags from the databases. The composite cDNA sequence of the human lyase contained an ORF of 1,734 bases that encodes a polypeptide with a calculated molecular mass of 63,732 Da. Recombinant human protein, expressed in mammalian cells, exhibited lyase activity. The lyase displayed homology to a putative
Caenorhabditis elegans
protein that resembles bacterial oxalyl-CoA decarboxylases. Similarly to the decarboxylases, a thiamine pyrophosphate-binding consensus domain was present in the C-terminal part of the lyase. Although no peroxisome targeting signal, neither 1 nor 2, was apparent, transfection experiments with constructs encoding green fluorescent protein fused to the full-length lyase or its C-terminal pentapeptide indicated that the C terminus of the lyase represents a peroxisome targeting signal 1 variant.