bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular aging, destabilization, and cancer.
Authors: Rubin H , Chow M , Yao A
Published: July 2002

Three major characteristics of aging in animals are a slowdown of cell proliferation, an increase in residual bodies associated with age pigments, and a marked increase in the likelihood of neoplastic transformation. The 28 L subline of the NIH 3T3 line of mouse embryo fibroblasts exhibits all these characteristics when held at confluence for extended periods. The impairment of proliferation is the first behavioral characteristic detected in low density subcultures from the confluent cultures, and it persists through many cell generations of exponential multiplication. There is an equal degree of growth impairment among replicate cultures (lineages) recovered after each of 2 successive rounds of confluence, although heterogeneity appears after the third round. The growth impairment pervades the entire cell population of each lineage. The degree and duration of impairment increase with repeated rounds of confluence. A marked increase of residual bodies characteristic of age pigments occurs in the cytoplasm of all the cells kept under prolonged confluence. Neoplastic transformation first appears as foci of multilayered cells on a monolayered background of nontransformed cells. The transformed cells arise at different times in the lineages and originate from a very small fraction of the population. The transformed cells selectively overgrow the entire population in successive rounds of confluence leading to an increase in saturation density of each lineage at different times. Under cloning conditions, isolated colonies of transformed cells develop more slowly than colonies of nontransformed cells but eventually reach a higher population density. The regularity of persistent growth impairment among the lineages and the appearance of large numbers of residual bodies in all the cells of each population are more characteristic of an epigenetic process than of specific local mutations. although random chromosomal lesions cannot be ruled out. By contrast, the low frequency and stochastic character of neoplastic transformation are consistent with a conventional genetic origin. The advent in long-term confluent NIH 3T3 cultures of three cardinal characteristics of cellular aging in vivo recommends it as a model for aging cells.
Comparative interleukin (IL-2)/interferon IFN-gamma and IL-4/IL-10 responses during acute infection of macaques inoculated with attenuated nef-truncated or pathogenic SICmac251 virus.
Authors: Benveniste O , Vaslin B , Le Grand R , Cheret A , Matheux F , Theodoro F , Cranage M P , Dormont D
Published: July 2002

Comparison of immune responses to infection by a pathogenic or a nonpathogenic immunodeficiency virus in macaques may provide insights into pathogenetic events leading to simian AIDS. This work is aimed at exploring cytokine expression during infection by simian immunodeficiency virus (SIV). We used semiquantitative reverse transcription-PCR to monitor interleukin (IL)-2/interferon (IFN)-gamma (Th1-like), and IL-4/IL-10 (Th2-like) expression in unmanipulated peripheral blood mononuclear cells (PBMCs), during the acute phase of infection of eight cynomolgus macaques (Macaca fascicularis) with a pathogenic primary isolate of SIVmac251 (full-length nef), and of four other cynomolgus macaques by an attenuated molecular clone of SIVmac251 (nef-truncated). All the monkeys became infected, as clearly shown by the presence of infected PBMCs and by seroconversion. Nevertheless, PBMC-associated virus loads and p27 antigenemia in monkeys infected by the attenuated virus clone remained lower than those observed in animals infected with the pathogenic SIVmac251 isolate. A rise of IL-10 mRNA expression occurred in both groups of monkeys coincident with the peak of viral replication. In monkeys infected with the pathogenic SIVmac251, IL-2, IL-4, and IFN-gamma mRNAs were either weakly detectable or undetectable. On the contrary, animals infected by the attenuated virus exhibited an overexpression of these cytokine mRNAs during the first weeks after inoculation. The lack of expression of these cytokines in monkeys infected with the pathogenic primary isolate may reflect early immunodeficiency.
Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance.
Authors: Levine M , Conry-Cantilena C , Wang Y , Welch R W , Washko P W , Dhariwal K R , Park J B , Lazarev A , Graumlich J F , King J , Cantilena L R
Published: July 2002

Determinants of the recommended dietary allowance (RDA) for vitamin C include the relationship between vitamin C dose and steady-state plasma concentration, bioavailability, urinary excretion, cell concentration, and potential adverse effects. Because current data are inadequate, an in-hospital depletion-repletion study was conducted. Seven healthy volunteers were hospitalized for 4-6 months and consumed a diet containing <5 mg of vitamin C daily. Steady-state plasma and tissue concentrations were determined at seven daily doses of vitamin C from 30 to 2500 mg. Vitamin C steady-state plasma concentrations as a function of dose displayed sigmoid kinetics. The steep portion of the curve occurred between the 30- and 100-mg daily dose, the current RDA of 60 mg daily was on the lower third of the curve, the first dose beyond the sigmoid portion of the curve was 200 mg daily, and complete plasma saturation occurred at 1000 mg daily. Neutrophils, monocytes, and lymphocytes saturated at 100 mg daily and contained concentrations at least 14-fold higher than plasma. Bioavailability was complete for 200 mg of vitamin C as a single dose. No vitamin C was excreted in urine of six of seven volunteers until the 100-mg dose. At single doses of 500 mg and higher, bioavailability declined and the absorbed amount was excreted. Oxalate and urate excretion were elevated at 1000 mg of vitamin C daily compared to lower doses. Based on these data and Institute of Medicine criteria, the current RDA of 60 mg daily should be increased to 200 mg daily, which can be obtained from fruits and vegetables. Safe doses of vitamin C are less than 1000 mg daily, and vitamin C daily doses above 400 mg have no evident value.
Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells
Authors: de Murcia Josiane Ménissier , Niedergang Claude , Trucco Carlotta , Ricoul Michèle , Dutrillaux Bernard , Mark Manuel , Oliver F. Javier , Masson Murielle , Dierich Andrée , LeMeur Marianne , Walztinger Caroline , Chambon Pierre , de Murcia Gilbert
Published: July 2002

Poly(ADP-ribose) polymerase [PARP; NAD
+
ADP-ribosyltransferase; NAD
+
: poly(adenosine-diphosphate-
d
-ribosyl)-acceptor ADP-
d
-ribosyltransferase, EC
2.4.2.30
] is a zinc-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents. To determine its biological function, we have inactivated both alleles by gene targeting in mice. Treatment of
PARP
−/−
mice either by the alkylating agent
N
-methyl-
N
-nitrosourea (MNU) or by γ-irradiation revealed an extreme sensitivity and a high genomic instability to both agents. Following whole body γ-irradiation (8 Gy) mutant mice died rapidly from acute radiation toxicity to the small intestine. Mice-derived PARP
−/−
cells displayed a high sensitivity to MNU exposure: a G
2
/M arrest in mouse embryonic fibroblasts and a rapid apoptotic response and a p53 accumulation were observed in splenocytes. Altogether these results demonstrate that PARP is a survival factor playing an essential and positive role during DNA damage recovery.
Association of the mammalian helicase MAH with the pre-mRNA splicing complex
Authors: Molnar Gyongyi M. , Crozat Anne , Kraeft Stine-Kathrein , Dou Qing Ping , Chen Lan Bo , Pardee Arthur B.
Published: July 2002

Conversion of pre-mRNAs into mature mRNAs includes several consecutive enzymatic modification steps that are carried out in the spliceosomes. Helicases have been shown to contribute to these catalytic processes both in yeast and in mammalian cells. Our results identify the mammalian protein MAH (matrix-associated helicase) as a new helicase present in the spliceosome complex. Sequence comparison describes MAH as the first higher eukaryotic member of the helicase superfamily I, with demonstrated enzymatic activity. Because MAH does not bind small nuclear ribonucleoproteins (snRNPs), it appears to be a non-snRNP binding factor of the splicing complex. In conclusion, our data suggest the involvement of MAH in processing of pre-mRNAs in mammalian cells.
Genetic influence on neurogenesis in the dentate gyrus of adult mice
Authors: Kempermann Gerd , Kuhn H. Georg , Gage Fred H.
Published: July 2002

To address genetic influences on hippocampal neurogenesis in adult mice, we compared C57BL/6, BALB/c, CD1(ICR), and 129Sv/J mice to examine proliferation, survival, and differentiation of newborn cells in the dentate gyrus. Proliferation was highest in C57BL/6; the survival rate of newborn cells was highest in CD1. In all strains ≈60% of surviving newborn cells had a neuronal phenotype, but 129/SvJ produced more astrocytes. Over 6 days C57BL/6 produced 0.36% of their total granule cell number of 239,000 as new neurons, BALB/c 0.30% of 242,000, CD1 (ICR) 0.32% of 351,000, and 129/SvJ 0.16% of 280,000. These results show that different aspects of adult hippocampal neurogenesis are differentially influenced by the genetic background.
Genetic basis of tolerance to O
2
deprivation in
Drosophila melanogaster
Authors: Haddad Gabriel G. , Sun Yi-an , Wyman Robert J. , Xu Tian
Published: July 2002

The ability to tolerate a low-O
2
environment varies widely among species in the animal kingdom. Some animals, such as
Drosophila melanogaster
, can tolerate anoxia for prolonged periods without apparent tissue injury. To determine the genetic basis of the cellular responses to low O
2
, we performed a genetic screen in
Drosophila
to identify loci that are responsible for anoxia resistance. Four X-linked, anoxia-sensitive mutants belonging to three complementation groups were isolated after screening more than 10,000 mutagenized flies. The identified recessive and dominant mutations showed marked delay in recovery from O
2
deprivation. In addition, electrophysiologic studies demonstrated that polysynaptic transmission in the central nervous system of the mutant flies was abnormally long during recovery from anoxia. These studies show that anoxic tolerance can be genetically dissected.
ER-27319, an acridone-related compound, inhibits release of antigen-induced allergic mediators from mast cells by selective inhibition of Fcɛ receptor I-mediated activation of Syk
Authors: Moriya Katsuhiro , Rivera Juan , Odom Sandra , Sakuma Yoshinori , Muramato Kenzo , Yoshiuchi Tatsuya , Miyamoto Mitsuaki , Yamada Koji
Published: July 2002

Engagement of the mast cell high-affinity receptor for immunoglobulin E (IgE), FcɛRI, induces tyrosine phosphorylation of Syk, a non-receptor tyrosine kinase, that has been demonstrated as critical for degranulation. Herein we describe a synthetic compound, ER-27319, as a potent and selective inhibitor of antigen or anti-IgE-mediated degranulation of rodent and human mast cells. ER-27319 affected neither Lyn kinase activity nor the antigen-induced phosphorylation of the FcɛRI but did effectively inhibit the tyrosine phosphorylation of Syk and thus its activity. As a consequence, tyrosine phosphorylation of phospholipase C-γ1, generation of inositol phosphates, release of arachidonic acid, and secretion of histamine and tumor necrosis factor α were also inhibited. ER-27319 did not inhibit the anti-CD3-induced tyrosine phosphorylation of phospholipase C-γ1 in Jurkat T cells, demonstrating a specificity for Syk-induced signals. In contrast the tyrosine phosphorylation and activation of Syk, induced byin vitroincubation with the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of FcɛRI γ subunit or by antigen activation of RBL-2H3 cells, was specifically inhibited by ER-27319. However, when ER-27319 was added to immunoprecipitated Syk, derived from activated cells, no effect was seen on Syk activity. ER-27319 did not inhibit the tyrosine phosphorylation of Syk induced by activation in the presence of Igβ ITAM or the anti-IgM-induced phosphorylation of Syk in human peripheral B cells. Therefore, ER-27319 selectively interferes with the FcɛRI γ phospho-ITAM activation of Sykin vitroand in intact cells. These results confirm the importance of Syk in FcɛRI-mediated responses in mast cells and demonstrate the mast cell selectivity and therapeutic potential of ER-27319 in the treatment of allergic disease.
Studies on the enzymatic and transcriptional activity of the dimerization cofactor for hepatocyte nuclear factor 1
Authors: Johnen Georg , Kaufman Seymour
Published: July 2002

The relationship between the enzymatic and the transcriptional activity of the bifunctional protein pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor 1 (DCoH) has been elucidated by site-directed mutagenesis. DCoH dimers harbor a binding site for hepatocyte nuclear factor 1 (HNF1), two active centers that bind pterins, and a saddle-shaped surface that resembles nucleic acid binding domains. Two domains of the protein have been selectively targeted to determine if a change in one activity affects the other. No strong correlation has been found, supporting the idea that carbinolamine dehydratase activity is not required for HNF1 binding
in vitro
or transcriptional coactivation
in vivo
. Double mutations in the active center, however, influence the
in vivo
transcriptional activity but not HNF1 binding. This finding suggests that some active center residues also are used during transcription, possibly for binding of another (macro)molecule. Several mutations in the saddle led to a surprising increase in transcription, therefore linking this domain to transcriptional regulation as well. The transcriptional function of DCoH therefore is composed of two parts, HNF1 binding and another contributing effect that involves the active site and, indirectly, the saddle.
U
S
9, a stable lysine-less herpes simplex virus 1 protein, is ubiquitinated before packaging into virions and associates with proteasomes
Authors: Brandimarti Renato , Roizman Bernard
Published: July 2002

The U
S
9 gene of herpes simplex virus 1 encodes a virion tegument protein with a predicted
M
r
of 10,000. Earlier studies have shown that the gene is not essential for viral replication in cells in culture. We report that (
i
) U
S
9 forms in denaturing polyacrylamide gels multiple overlapping bands ranging in
M
r
from 12,000 to 25,000; (
ii
) the protein recovered from infected cells or purified virions reacts with anti-ubiquitin antibodies; (
iii
) autoradiographic images of U
S
9 protein immunoprecipitated from cells infected with [
35
S]methionine-labeled virus indicate that the protein is stable for at least 4 h after entry into cells (the protein was also stable for at least 4 h after a 1-h labeling interval 12 h after infection); (
iv
) antibody to subunit 12 of proteasomes pulls down U
S
9 protein from herpes simplex virus-infected cell lysates; and (
v
) the U
S
9 gene is highly conserved among the members of the alpha subfamily of herpes viruses, and the U
S
9 gene product lacks lysines. We conclude that U
S
9 is a lysine-less, ubiquitinated protein that interacts with the ubiquitin-dependent pathway for degradation of proteins and that this function may be initiated at the time of entry of the virus into the cell.
Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: Enhancement by angiotensin II
Authors: Pagano Patrick J. , Clark Justin K. , Cifuentes-Pagano M. Eugenia , Clark Stacie M. , Callis Gayle M. , Quinn Mark T.
Published: July 2002

Superoxide anion (O
2
−
) plays a key role in the endogenous suppression of endothelium-derived nitric oxide (NO) bioactivity and has been implicated in the development of hypertension. In previous studies, we found that O
2
−
is produced predominantly in the adventitia of isolated rabbit aorta and acts as a barrier to NO. In the present studies, we characterize the enzyme responsible for O
2
−
production in the adventitia and show that this enzyme is a constitutively active NADPH oxidase with similar composition as the phagocyte NADPH oxidase. Constitutive O
2
−
-generating activity was localized to aortic adventitial fibroblasts and was enhanced by the potent vasoconstrictor angiotensin II. Immunohistochemistry of aortic sections demonstrated the presence of p22
phox
, gp91
phox
, p47
phox
, and p67
phox
localized exclusively in rabbit aortic adventitia, coincident with the site of staining for O
2
−
production. Furthermore, immunodepletion of p67
phox
from adventitial fibroblast particulates resulted in the loss of NADPH oxidase activity, which could be restored by the addition of recombinant p67
phox
. Further study into the regulation of this adventitial source of O
2
−
is important in elucidating the mechanisms regulating the bioactivity of NO and may contribute to our understanding of the pathogenesis of hypertension.
High-precision position-specific isotope analysis
Authors: Corso Thomas N. , Brenna J. Thomas
Published: July 2002

Intramolecular carbon isotope distributions reflect details of the
origin of organic compounds and may record the status of complex
systems, such as environmental or physiological states. A strategy is
reported here for high-precision determination of
13
C/
12
C ratios at specific positions in
organic compounds separated from complex mixtures. Free radical
fragmentation of methyl palmitate, a test compound, is induced by an
open tube furnace. Two series of peaks corresponding to bond breaking
from each end of the molecule are analyzed by isotope ratio mass
spectrometry and yield precisions of SD(δ-
13
C) < 0.4‰.
Isotope labeling in the carboxyl, terminal, and methyl positions
demonstrates the absence of rearrangement during activation and
fragmentation. Negligible isotopic fractionation was observed as degree
of fragmentation was adjusted by changing pyrolysis temperature.
[1-
13
C]methyl palmitate with overall
δ-
13
C = 4.06‰, yielded values of +457‰ for the
carboxyl position, in agreement with expectations from the dilution,
and an average of −27.95‰ for the rest of the molecule,
corresponding to −27.46‰ for the olefin series. These data
demonstrate the feasibility of automated high-precision
position-specific analysis of carbon for molecules contained in complex
mixtures.
Identification of PN1, a predominant voltage-dependent sodium channel expressed principally in peripheral neurons
Authors: Toledo-Aral Juan J. , Moss Brenda L. , He Zhi-Jun , Koszowski Adam G. , Whisenand Teri , Levinson Simon R. , Wolf John J. , Silos-Santiago Inmaculada , Halegoua Simon , Mandel Gail
Published: July 2002

Membrane excitability in different tissues is due, in large part, to the selective expression of distinct genes encoding the voltage-dependent sodium channel. Although the predominant sodium channels in brain, skeletal muscle, and cardiac muscle have been identified, the major sodium channel types responsible for excitability within the peripheral nervous system have remained elusive. We now describe the deduced primary structure of a sodium channel, peripheral nerve type 1 (PN1), which is expressed at high levels throughout the peripheral nervous system and is targeted to nerve terminals of cultured dorsal root ganglion neurons. Studies using cultured PC12 cells indicate that both expression and targeting of PN1 is induced by treatment of the cells with nerve growth factor. The preferential localization suggests that the PN1 sodium channel plays a specific role in nerve excitability.
Neurotrophic immunophilin ligands stimulate structural and functional recovery in neurodegenerative animal models
Authors: Steiner Joseph P. , Hamilton Gregory S. , Ross Douglas T. , Valentine Heather L. , Guo Hongzhi , Connolly Maureen A. , Liang Shi , Ramsey Cynthia , Li Jia-He J. , Huang Wei , Howorth Pamela , Soni Rajat , Fuller Michael , Sauer Hans , Nowotnik Alison C. , Suzdak Peter D.
Published: July 2002

Although immunosuppressant immunophilin ligands promote neurite outgrowth
in vitro
, their neurotrophic activities are clearly independent of their immunosuppressive activity. In the present report, a novel nonimmunosuppressive immunophilin ligand, GPI-1046 (3-(3-pyridyl)-1-propyl (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidinecarboxylate) is described.
In vitro
, GPI-1046 bound to FK506 binding protein-12 and elicited neurite outgrowth from sensory neuronal cultures with picomolar potency with maximal effects comparable to nerve growth factor.
In vivo
, GPI-1046 stimulated the regeneration of lesioned sciatic nerve axons and myelin levels. In the central nervous system, GPI-1046 promoted protection and/or sprouting of serotonin-containing nerve fibers in somatosensory cortex following parachloroamphetamine treatment. GPI-1046 also induced regenerative sprouting from spared nigrostriatal dopaminergic neurons following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice or 6-hydroxydopamine (6-OHDA) toxicity in rats. The rotational abnormality in 6-OHDA treated rats was alleviated by GPI-1046. These neurotrophic actions in multiple models suggest therapeutic utility for GPI-1046 in neurodegenerative diseases.
Mutagenic effects of a single and an exact number of α particles in mammalian cells
Authors: Hei Tom K. , Wu Li-Jun , Liu Su-Xian , Vannais Diane , Waldren Charles A. , Randers-Pehrson Gerhard
Published: July 2002

One of the main uncertainties in risk estimation for environmental radon exposure using lung cancer data from underground miners is the extrapolation from high- to low-dose exposure where multiple traversal is extremely rare. The biological effects of a single α particle are currently unknown. Using the recently available microbeam source at the Radiological Research Accelerator Facility at Columbia University, we examined the frequencies and molecular spectrum of S1
−
mutants induced in human–hamster hybrid (A
L
) cells by either a single or an exact number of α particles. Exponentially growing cells were stained briefly with a nontoxic concentration of Hoechst dye for image analysis, and the location of individual cells was computer-monitored. The nucleus of each cell was irradiated with either 1, 2, 4, or 8 α particles at a linear energy transfer of 90 keV/μm consistent with the energy spectrum of domestic radon exposure. Although single-particle traversal was only slightly cytotoxic to A
L
cells (survival fraction ≈ 0.82), it was highly mutagenic, and the induced mutant fraction averaged 110 mutants per 10
5
survivors. In addition, both toxicity and mutant induction were dose-dependent. Multiplex PCR analysis of mutant DNA showed that the proportion of mutants with multilocus deletions increased with the number of particle traversals. These data provide direct evidence that a single α particle traversing a nucleus will have a high probability of resulting in a mutation and highlight the need for radiation protection at low doses.
Insulin-like growth factor II stimulates cell proliferation through the insulin receptor
Authors: Morrione Andrea , Valentinis Barbara , Xu Shi-qiong , Yumet Gladys , Louvi Angeliki , Efstratiadis Argiris , Baserga Renato
Published: July 2002

R− cells are 3T3-like fibroblasts generated from mouse embryos nullizygous for a targeted disruption of the genes encoding the type 1 insulin-like growth factor (IGF) receptor (IGF1R). These cells fail to proliferate in serum-free medium supplemented with purified growth factors, in contrast to their wild-type counterparts. However, when R− cells overexpress the insulin receptor from a stably integrated plasmid, R−/IR cells, they become capable of growing in serum-free medium supplemented solely with insulin or IGF-II, but not with IGF-I. Moreover, the introduction into R−/IR cells of an additional plasmid expressing IGF-II causes these cells to proliferate in serum-free medium without growth factor supplementation. From these results, we conclude that IGF-II can stimulate cell proliferation not only through its cognate IGF1R but also through the insulin receptor.
In vitro
site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family
Authors: Thorpe Helena M. , Smith Margaret C. M.
Published: July 2002

The genome of the broad host range
Streptomyces
temperate phage, φC31, is known to integrate into the host chromosome via an enzyme that is a member of the resolvase/invertase family of site-specific recombinases. The recombination properties of this novel integrase on the phage and
Streptomyces ambofaciens
attachment sites,
attP
and
attB
, respectively, were investigated in the heterologous host,
Escherichia coli
, and in an
in vitro
assay by using purified integrase. The products of
attP/B
recombination, i.e.,
attL
and
attR
, were identical to those obtained after integration of the prophage in
S. ambofaciens.
In the
in vitro
assay only buffer, purified integrase, and DNAs encoding
attP
and
attB
were required. Recombination occurred irrespective of whether the substrates were supercoiled or linear. A mutant integrase containing an S12F mutation was completely defective in recombination both in
E. coli
and
in vitro
. No recombination was observed between
attB/attB
,
attP/attP, attL/R
, or any combination of
attB
or
attP
with
attL
or
attR
, suggesting that excision of the prophage (
attL/R
recombination) requires an additional phage- or
Streptomyces-
encoded factor. Recombination could occur intramolecularly to cause deletion between appropriately orientated
attP
and
attB
sites. The results show that directionality in φC31 integrase is strictly controlled by nonidentical recombination sites with no requirement to form the topologically defined structures that are more typical of the resolvases/invertases.
The mosquitoAnopheles stephensilimits malaria parasite development with inducible synthesis of nitric oxide
Authors: Luckhart Shirley , Vodovotz Yoram , Cui Liwang , Rosenberg Ronald
Published: July 2002

We have discovered that the mosquitoAnopheles stephensi, a natural vector of human malaria, limits parasite development with inducible synthesis of nitric oxide (NO). Elevated expression ofA. stephensiNO synthase (NOS), which is highly homologous to characterized NOS genes, was detected in the midgut and carcass soon after invasion of the midgut byPlasmodium. Early induction is likely primed by bacterial growth in the blood meal. Later increases inA. stephensiNOS expression and enzyme activity occurred at the beginning of sporozoite release. Circulating levels of nitrite/nitrate, end-products of NO synthesis, were significantly higher inPlasmodium-infected mosquitoes. Dietary provision of the NOS substratel-arginine reducedPlasmodiuminfections inA. stephensi. In contrast, dietary provision of a NOS inhibitor significantly increased parasite numbers in infected mosquitoes, confirming thatA. stephensilimitsPlasmodiumdevelopment with NO.
A stochastic approximation algorithm with Markov chain Monte-Carlo method for incomplete data estimation problems
Authors: Gu Ming Gao , Kong Fan Hui
Published: July 2002

We propose a general procedure for solving incomplete data estimation problems. The procedure can be used to find the maximum likelihood estimate or to solve estimating equations in difficult cases such as estimation with the censored or truncated regression model, the nonlinear structural measurement error model, and the random effects model. The procedure is based on the general principle of stochastic approximation and the Markov chain Monte-Carlo method. Applying the theory on adaptive algorithms, we derive conditions under which the proposed procedure converges. Simulation studies also indicate that the proposed procedure consistently converges to the maximum likelihood estimate for the structural measurement error logistic regression model.
Inactivation of tyrosine hydroxylase by nitration following exposure to peroxynitrite and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)
Authors: Ara Jahan , Przedborski Serge , Naini Ali B. , Jackson-Lewis Vernice , Trifiletti Rosario R. , Horwitz Joel , Ischiropoulos Harry
Published: July 2002

The decrement in dopamine levels exceeds the loss of dopaminergic neurons in Parkinson’s disease (PD) patients and experimental models of PD. This discrepancy is poorly understood and may represent an important event in the pathogenesis of PD. Herein, we report that the rate-limiting enzyme in dopamine synthesis, tyrosine hydroxylase (TH), is a selective target for nitration following exposure of PC12 cells to either peroxynitrite or 1-methyl-4-phenylpyridiniun ion (MPP
+
). Nitration of TH also occurs in mouse striatum after MPTP administration. Nitration of tyrosine residues in TH results in loss of enzymatic activity. In the mouse striatum, tyrosine nitration-mediated loss in TH activity parallels the decline in dopamine levels whereas the levels of TH protein remain unchanged for the first 6 hr post MPTP injection. Striatal TH was not nitrated in mice overexpressing copper/zinc superoxide dismutase after MPTP administration, supporting a critical role for superoxide in TH tyrosine nitration. These results indicate that tyrosine nitration-induced TH inactivation and consequently dopamine synthesis failure, represents an early and thus far unidentified biochemical event in MPTP neurotoxic process. The resemblance of the MPTP model with PD suggests that a similar phenomenon may occur in PD, influencing the severity of parkisonian symptoms.
Presenilin 1 associates with glycogen synthase kinase-3β and its substrate tau
Authors: Takashima Akihiko , Murayama Miyuki , Murayama Ohoshi , Kohno Toshiyuki , Honda Toshiyuki , Yasutake Kaori , Nihonmatsu Naomi , Mercken Marc , Yamaguchi Haruyasu , Sugihara Shiro , Wolozin Benjamin
Published: July 2002

Families bearing mutations in the presenilin 1 (PS1) gene develop Alzheimer’s disease. Previous studies have shown that the Alzheimer-associated mutations in PS1 increase production of amyloid β protein (Aβ
1–42
). We now show that PS1 also regulates phosphorylation of the microtubule-associated protein tau. PS1 directly binds tau and a tau kinase, glycogen synthase kinase 3β (GSK-3β). Deletion studies show that both tau and GSK-3β bind to the same region of PS1, residues 250–298, whereas the binding domain on tau is the microtubule-binding repeat region. The ability of PS1 to bring tau and GSK-3β into close proximity suggests that PS1 may regulate the interaction of tau with GSK-3β. Mutations in PS1 that cause Alzheimer’s disease increase the ability of PS1 to bind GSK-3β and, correspondingly, increase its tau-directed kinase activity. We propose that the increased association of GSK-3β with mutant PS1 leads to increased phosphorylation of tau.
Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation
Authors: Payne Aimee S. , Kelly Edward J. , Gitlin Jonathan D.
Published: July 2002

Wilson disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase. To elucidate the function of the Wilson protein, wild-type and mutant Wilson cDNAs were expressed in a Menkes copper transporter-deficient
mottled
fibroblast cell line defective in copper export. Expression of the wild-type cDNA demonstrated trans
-
Golgi network localization and copper-dependent trafficking of the Wilson protein identical to previous observations for the endogenously expressed protein in hepatocytes. Furthermore, expression of the Wilson cDNA rescued the
mottled
phenotype as evidenced by a reduction in copper accumulation and restoration of cell viability. In contrast, expression of an H1069Q mutant Wilson cDNA did not rescue the
mottled
phenotype, and immunofluorescence studies showed that this mutant Wilson protein was localized in the endoplasmic reticulum. Consistent with these findings, pulse–chase analysis demonstrated a 5-fold decrease in the half-life of the H1069Q mutant as compared with the wild-type protein. Maintenance of these transfected cell lines at 28°C resulted in localization of the H1069Q protein in the trans-Golgi network, suggesting that a temperature-sensitive defect in protein folding followed by degradation constitutes the molecular basis of Wilson disease in patients harboring the H1069Q mutation. Taken together, these studies describe a tractable expression system for elucidating the function and localization of the copper-transporting ATPases in mammalian cells and provide compelling evidence that the Wilson protein can functionally substitute for the Menkes protein, supporting the concept that these proteins use common biochemical mechanisms to effect cellular copper homeostasis.
Neural systems underlying learning and representation of global motion
Authors: Vaina Lucia M. , Belliveau John W. , Roziers Eric B. des , Zeffiro Thomas A.
Published: July 2002

We demonstrate performance-related changes in cortical and cerebellar activity. The largest learning-dependent changes were observed in the anterior lateral cerebellum, where the extent and intensity of activation correlated inversely with psychophysical performance. After learning had occurred (a few minutes), the cerebellar activation almost disappeared; however, it was restored when the subjects were presented with a novel, untrained direction of motion for which psychophysical performance also reverted to chance level. Similar reductions in the extent and intensity of brain activations in relation to learning occurred in the superior colliculus, anterior cingulate, and parts of the extrastriate cortex. The motion direction-sensitive middle temporal visual complex was a notable exception, where there was an expansion of the cortical territory activated by the trained stimulus. Together, these results indicate that the learning and representation of visual motion discrimination are mediated by different, but probably interacting, neuronal subsystems.
Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostate cancer cells
Authors: Ghosh Jagadananda , Myers Charles E.
Published: July 2002

Diets high in fat are associated with an increased risk of prostate cancer, although the molecular mechanism is still unknown. We have previously reported that arachidonic acid, an omega-6 fatty acid common in the Western diet, stimulates proliferation of prostate cancer cells through production of the 5-lipoxygenase metabolite, 5-HETE (5-hydroxyeicosatetraenoic acid). We now show that 5-HETE is also a potent survival factor for human prostate cancer cells. These cells constitutively produce 5-HETE in serum-free medium with no added stimulus. Exogenous arachidonate markedly increases the production of 5-HETE. Inhibition of 5-lipoxygenase by MK886 completely blocks 5-HETE production and induces massive apoptosis in both hormone-responsive (LNCaP) and -nonresponsive (PC3) human prostate cancer cells. This cell death is very rapid: cells treated with MK886 showed mitochondrial permeability transition between 30 and 60 min, externalization of phosphatidylserine within 2 hr, and degradation of DNA to nucleosomal subunits beginning within 2–4 hr posttreatment. Cell death was effectively blocked by the thiol antioxidant,N-acetyl-l-cysteine, but not by androgen, a powerful survival factor for prostate cancer cells. Apoptosis was specific for 5-lipoxygenase—programmed cell death was not observed with inhibitors of 12-lipoxygenase, cyclooxygenase, or cytochrome P450 pathways of arachidonic acid metabolism. Exogenous 5-HETE protects these cells from apoptosis induced by 5-lipoxygenase inhibitors, confirming a critical role of 5-lipoxygenase activity in the survival of these cells. These findings provide a possible molecular mechanism by which dietary fat may influence the progression of prostate cancer.
Presentation of peptide antigens by mouse CD1 requires endosomal localization and protein antigen processing
Authors: Tangri Shabnam , Brossay Laurent , Burdin Nicolas , Lee Delphine J. , Corr Maripat , Kronenberg Mitchell
Published: July 2002

Mouse CD1(mCD1) molecules have been reported to present two types of antigens: peptides or proteins and the glycolipid α-galactosylceramide. Here, we demonstrate that a protein antigen, chicken ovalbumin (Ova), must be processed to generate peptides presented by mCD1 to CD8+T cells. The processing and mCD1-mediated presentation of chicken Ova depend on endosomal localization because inhibitors of endosomal acidification and endosomal recycling pathways block T cell reactivity. Furthermore, a cytoplasmic tail mutant of mCD1, which disrupts endosomal localization, has a greatly reduced capacity to present Ova to mCD1 restricted cells. Newly synthesized mCD1 molecules, however, are not required for Ova presentation, suggesting that molecules recycling from the cell surface are needed. Because of these data showing that mCD1 trafficks to endosomes, where it can bind peptides derived from exogenous proteins, we conclude that peptide antigen presentation by mCD1 is likely to be a naturally occurring phenomenon. In competition assays, α-galactosylceramide did not inhibit Ova presentation, and presentation of the glycolipid was not inhibited by excess Ova or the peptide epitope derived from it. This suggests that, although both lipid and peptide presentation may occur naturally, mCD1 may interact differently with these two types of antigens.
Otoconin-90, the mammalian otoconial matrix protein, contains two domains of homology to secretory phospholipase A
2
Authors: Wang Yunxia , Kowalski Paul E. , Thalmann Isolde , Ornitz David M. , Mager Dixie L. , Thalmann Rudiger
Published: July 2002

The ability to sense orientation relative to gravity requires dense particles, called otoconia, which are localized in the vestibular macular organs. In mammals, otoconia are composed of proteins (otoconins) and calcium carbonate crystals in a calcite lattice. Little is known about the mechanisms that regulate otoconial biosynthesis. To begin to elucidate these mechanisms, we have partially sequenced and cloned the major protein component of murine otoconia, otoconin-90 (OC90). The amino acid sequence identified an orphan chimeric human cDNA. Because of its similarity to secretory phospholipase A
2
(sPLA
2
), this gene was referred to as

PLA
2
-like (PLA2L)

and enabled the identification of human
Oc90
. Partial murine cDNA and genomic clones were isolated and shown to be specifically expressed in the developing mouse otocyst. The mature mouse OC90 is composed of 453 residues and contains two domains homologous to sPLA
2
. The cloning of
Oc90
will allow an examination of the role of this protein in otoconial biosynthesis and in diseases that affect the vestibular system.
The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding
Authors: Feder John N. , Penny David M. , Irrinki Alivelu , Lee Vince K. , Lebrón José A. , Watson Nicole , Tsuchihashi Zenta , Sigal Elliott , Bjorkman Pamela J. , Schatzman Randall C.
Published: July 2002

We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis called
HFE
. The gene product, a member of the major histocompatibility complex class I-like family, was found to have a mutation, Cys-282 → Tyr (C282Y), in 85% of patient chromosomes. This mutation eliminates the ability of HFE to associate with β
2
-microglobulin (β
2
m) and prevents cell-surface expression. A second mutation that has no effect on β
2
m association, H63D, was found in eight out of nine patients heterozygous for the C282Y mutant. In this report, we demonstrate in cultured 293 cells overexpressing wild-type or mutant HFE proteins that both the wild-type and H63D HFE proteins form stable complexes with the transferrin receptor (TfR). The C282Y mutation nearly completely prevents the association of the mutant HFE protein with the TfR. Studies on cell-associated transferrin at 37°C suggest that the overexpressed wild-type HFE protein decreases the affinity of the TfR for transferrin. The overexpressed H63D protein does not have this effect, providing the first direct evidence for a functional consequence of the H63D mutation. Addition of soluble wild-type HFE/β
2
m heterodimers to cultured cells also decreased the apparent affinity of the TfR for its ligand under steady-state conditions, both in 293 cells and in HeLa cells. Furthermore, at 4°C, the added soluble complex of HFE/β
2
m inhibited binding of transferrin to HeLa cell TfR in a concentration-dependent manner. Scatchard plots of these data indicate that the added heterodimer substantially reduced the affinity of TfR for transferrin. These results establish a molecular link between HFE and a key protein involved in iron transport, the TfR, and raise the possibility that alterations in this regulatory mechanism may play a role in the pathogenesis of hereditary hemochromatosis.
The domain organization ofNaeI endonuclease: Separation of binding and catalysis
Authors: Colandene James D. , Topal Michael D.
Published: July 2002

NaeI is a remarkable type II restriction endonuclease. It must bind two recognition sequences to cleave DNA, forms a covalent protein–DNA intermediate, and is only 1 aa change away from topoisomerase and recombinase activity. The latter activities apparently derive from reactivation of a cryptic DNA ligase active site. Here, we demonstrate thatNaeI has two protease-resistant domains, involving approximately the N-terminal and C-terminal halves of the protein, linked by a protease-accessible region of 30 aa. The domains were purified by cloning. The C-terminal domain was shown by gel mobility-shift assay to have approximately 8-fold lower DNA-binding ability than intactNaeI. Analytical ultracentrifugation showed this domain to be a monomer in solution. The N-terminal domain, which contains the catalytic region defined by random mutagenesis, did not bind DNA and was a mixture of different-sized complexes in solution implying that it mediates self-association. DNA greatly inhibited proteolysis of the linker region. The results identify the DNA-binding domain, imply that DNA cleavage and recognition are independent and separable, and lead us to speculate about a cleft-like structure forNaeI.
ADD1/SREBP1 activates PPARγ through the production of endogenous ligand
Authors: Kim Jae Bum , Wright Harold M. , Wright Margaret , Spiegelman Bruce M.
Published: July 2002

Adipose differentiation is an important part of the energy homeostasis system of higher organisms. Recent data have suggested that this process is controlled by an interplay of transcription factors including PPARγ, the C/EBPs, and ADD1/SREBP1. Although these factors interact functionally to initiate the program of differentiation, there are no data concerning specific mechanisms of interaction. We show here that the expression of ADD1/SREBP1 specifically increases the activity of PPARγ but not other isoforms, PPARα, or PPARδ. This activation occurs through the ligand-binding domain of PPARγ when it is fused to the DNA-binding domain of Gal4. The stimulation of PPARγ by ADD1/SREBP1 does not require coexpression in the same cells; supernatants from cultures that express ADD1/SREBP1 augment the transcriptional activity of PPARγ. Finally, we demonstrate directly that cells expressing ADD1/SREBP1 produce and secrete lipid molecule(s) that bind directly to PPARγ, displacing the binding of radioactive thiazolidinedione ligands. These data establish that ADD1/SREBP1 can control the production of endogenous ligand(s) for PPARγ and suggest a mechanism for coordinating the actions of these adipogenic factors.
Female preference for swords in
Xiphophorus helleri
reflects a bias for large apparent size
Authors: Rosenthal Gil G. , Evans Christopher S.
Published: July 2002

Swordtail fish (Poeciliidae: genus
Xiphophorus
) are a paradigmatic case of sexual selection by sensory exploitation. Female preference for males with a conspicuous “sword” ornament is ancestral, suggesting that male morphology has evolved in response to a preexisting bias. The perceptual mechanisms underlying female mate choice have not been identified, complicating efforts to understand the selection pressures acting on ornament design. We consider two alternative models of receiver behavior, each consistent with previous results. Females could respond either to specific characteristics of the sword or to more general cues, such as the apparent size of potential mates. We showed female swordtails a series of computer-altered video sequences depicting a courting male. Footage of an intact male was preferred strongly to otherwise identical sequences in which portions of the sword had been deleted selectively, but a disembodied courting sword was less attractive than an intact male. There was no difference between responses to an isolated sword and to a swordless male of comparable length, or between an isolated sword and a homogenous background. Female preference for a sworded male was abolished by enlarging the image of a swordless male to compensate for the reduction in length caused by removing the ornament. This pattern of results is consistent with mate choice being mediated by a general preference for large males rather than by specific characters. Similar processes may account for the evolution of exaggerated traits in other systems.
Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the
PR-1
gene
Authors: Zhang Yuelin , Fan Weihua , Kinkema Mark , Li Xin , Dong Xinnian
Published: July 2002

The
Arabidopsis thaliana
NPR1 has been shown to be a key regulator of gene expression during the onset of a plant disease-resistance response known as systemic acquired resistance. The
npr1
mutant plants fail to respond to systemic acquired resistance-inducing signals such as salicylic acid (SA), or express SA-induced pathogenesis-related (
PR
) genes. Using NPR1 as bait in a yeast two-hybrid screen, we identified a subclass of transcription factors in the basic leucine zipper protein family (AHBP-1b and TGA6) and showed that they interact specifically in yeast and
in vitro
with NPR1. Point mutations that abolish the NPR1 function in
A. thaliana
also impair the interactions between NPR1 and the transcription factors in the yeast two-hybrid assay. Furthermore, a gel mobility shift assay showed that the purified transcription factor protein, AHBP-1b, binds specifically to an SA-responsive promoter element of the
A. thaliana PR-1
gene. These data suggest that NPR1 may regulate
PR-1
gene expression by interacting with a subclass of basic leucine zipper protein transcription factors.
Progressive hypertrophy and heart failure in β
1
-adrenergic receptor transgenic mice
Authors: Engelhardt Stefan , Hein Lutz , Wiesmann Frank , Lohse Martin J.
Published: July 2002

Stimulation of cardiac β
1
-adrenergic receptors is the main mechanism that increases heart rate and contractility. Consequently, several pharmacological and gene transfer strategies for the prevention of heart failure aim at improving the function of the cardiac β-adrenergic receptor system, whereas current clinical treatment favors a reduction of cardiac stimulation. To address this controversy, we have generated mice with heart-specific overexpression of β
1
-adrenergic receptors. Their cardiac function was investigated in organ bath experiments as well as
in vivo
by cardiac catheterization and by time-resolved NMR imaging. The transgenic mice had increased cardiac contractility at a young age but also developed marked myocyte hypertrophy (3.5-fold increase in myocyte area). This increase was followed by progressive heart failure with functional and histological deficits typical for humans with heart failure. Contractility was reduced by ≈50% in 35-week-old mice, and ejection fraction was reduced down to a minimum of ≈20%. We conclude that overexpression of β
1
-adrenergic receptors in the heart may lead to a short-lived improvement of cardiac function, but that increased β
1
-adrenergic receptor signalling is ultimately detrimental.