bluuebunny/crossref_metadata_2025 huggingface dataset:
When zero is not zero: The problem of ambiguous baseline conditions in fMRI
Authors: Stark Craig E. L. , Squire Larry R.
Published: July 2002

By using blocked and rapid event-related functional MRI studies of memory, we explored the implications of using rest periods as a baseline condition in functional MRI studies. Activity in the medial temporal lobe (as well as in other brain regions) was substantially higher during rest than during several alternative baseline conditions. The effect of this elevated activity during rest was to reduce, eliminate, or even reverse the sign of the activity during task conditions relevant to memory functions. The results demonstrate that periods of rest are associated with significant cognitive activity and, therefore, provide a nonoptimal baseline for memory tasks. These results were observed not only when relatively long blocks of rest were used (experiment 1), but also when rest consisted of the short null trials typically used in rapid event-related designs (experiment 2). The findings have important implications for the design and interpretation of a wide range of fMRI studies of cognition.
Deamidation of human proteins
Authors: Robinson N. E. , Robinson A. B.
Published: July 2002

Deamidation of asparaginyl and glutaminyl residues causes time-dependent changes in charge and conformation of peptides and proteins. Quantitative and experimentally verified predictive calculations of the deamidation rates of 1,371 asparaginyl residues in a representative collection of 126 human proteins have been performed. These rates suggest that deamidation is a biologically relevant phenomenon in a remarkably large percentage of human proteins.
A targeted mutation in the IL-4Rα gene protects mice against autoimmune diabetes
Authors: Radu Dorel L. , Noben-Trauth Nancy , Hu-Li Jane , Paul William E. , Bona Constantin A.
Published: July 2002

Autoimmune insulin-dependent diabetes mellitus (IDDM) occurs spontaneously in mice-bearing transgenes encoding the influenza hemagglutinin under the control of the rat insulin promoter and a T cell receptor specific for an hemagglutinin peptide associated with I-Ed. Such “double transgenic” mice expressing wild-type or targeted IL-4Rα genes were examined for the onset of IDDM. Eight of 11 mice homozygous for wild-type IL-4Rα were hyperglycemic by 8 weeks of age, whereas only 1 of 16 mice homozygous for the targeted allele were hyperglycemic at this time. Most 1L-4Rα−/− mice remained normoglycemic to 36 weeks of age. Although only 10% of double transgenic mice homozygous for the wild-type IL-4Rα allele survived to 30 weeks, 80% of mice homozygous for the targeted allele did so. Heterozygous mice displayed an intermediate frequency of diabetes. Even as late as 270 days of age, mice homozygous for the targeted allele had no insulitis or only peri-insulitis. Thus, the inability to respond to IL-4 and/or IL-13 protects mice against IDDM in this model of autoimmunity.
Glucose and cAMP regulate the L-type pyruvate kinase gene by phosphorylation/dephosphorylation of the carbohydrate response element binding protein
Authors: Kawaguchi Takumi , Takenoshita Makoto , Kabashima Tsutomu , Uyeda Kosaku
Published: July 2002

Recently we purified and identified a previously uncharacterized transcription factor from rat liver binding to the carbohydrate responsive element of the L-type pyruvate kinase (L-PK) gene. This factor was named carbohydrate responsive element binding protein (ChREBP). ChREBP, essential for L-PK gene transcription, is activated by high glucose and inhibited by cAMP. Here, we demonstrated that (
i
) nuclear localization signal and basic helix-loop-helix/leucine-zipper domains of ChREBP were essential for the transcription, and (
ii
) these domains were the targets of regulation by cAMP and glucose. Among three cAMP-dependent protein kinase phosphorylation sites, Ser
196
and Thr
666
were the target sites. Phosphorylation of the former resulted in inactivation of nuclear import, and that of the latter resulted in loss of the DNA-binding activity and L-PK transcription. On the other hand, glucose activated the nuclear import by dephosphorylation of Ser
196
in the cytoplasm and also stimulated the DNA-binding activity by dephosphorylation of Thr
666
in the nucleus. These results thus reveal mechanisms for regulation of ChREBP and the L-PK transcription by excess carbohydrate and cAMP.
A novel type of RNA editing occurs in the mitochondrial tRNAs of the centipede
Lithobius forficatus
Authors: Lavrov Dennis V. , Brown Wesley M. , Boore Jeffrey L.
Published: July 2002

We determined the complete mtDNA sequence of the centipede
Lithobius forficatus
and found that only one of the 22
inferred tRNA genes encodes a fully paired aminoacyl acceptor stem. The
other 21 genes encode tRNAs with up to five mismatches in these stems,
and some of these overlap extensively with the downstream genes.
Because a well-paired acceptor stem is required for proper tRNA
functioning, RNA editing in the products of these genes was suspected.
We investigated this hypothesis by studying cDNA sequences from eight
tRNAs and found the editing of up to 5 nt at their 3′ ends. This
editing appears to occur by a novel mechanism with the 5′ end of the
acceptor stem being used as a template for the
de novo
synthesis of the 3′ end, presumably by an RNA-dependent RNA polymerase.
In addition, unusual secondary structures for several tRNAs were found,
including those lacking a TΨC (T) or a dihydrouridine (D) arm, and
having an unusual number of base pairs in the acceptor or anticodon
stems.
Identification of a developmental chemoattractant in
Myxococcus xanthus
through metabolic engineering
Authors: Kearns Daniel B. , Venot Andre , Bonner Pamela J. , Stevens Brian , Boons Geert-Jan , Shimkets Lawrence J.
Published: July 2002

Fruiting body formation of
Myxococcus xanthus
requires the ordered migration of tens of thousands of cells by using a form of surface motility known as gliding and chemical signal(s) that have yet to be elucidated. Directed movement is regulated by phosphatidylethanolamine (PE) purified from
M. xanthus
cell membranes. Because the purified PE preparation contains a remarkably diverse mixture of fatty acids, metabolic engineering was used to elucidate the biologically active fatty acid component. The mutational block in an
esg
mutant, which renders it defective in producing primers for branched-chain fatty acid biosynthesis, was bypassed with one of a series of primers that enriches for a particular family of branched-chain fatty acids. Each PE enrichment was observed for chemotactic activity by using an excitation assay and for fatty acid content. The excitation activity of a PE preparation was generally proportional with the concentration of the fatty acid 16:1ω5c. 1,2-
O
-Bis[11-(
Z
)-hexadecenoyl]-
sn
-glycero-3-phosphoethanolamine (PE-16:1ω5c/16:1ω5c) was synthesized and elicited an excitation peak at 2 ng. This peak activity occurred at a 1,000-fold lower concentration than dilauroyl PE (PE-12:0/12:0) and the peak magnitude was 2-fold higher. PE containing 16:1ω5c is likely to play a role in development because it is active at physiological concentrations and only under developmental conditions.
Diet and the evolution of the earliest human ancestors
Authors: Teaford Mark F. , Ungar Peter S.
Published: July 2002

Over the past decade, discussions of the evolution of the earliest
human ancestors have focused on the locomotion of the
australopithecines. Recent discoveries in a broad range of disciplines
have raised important questions about the influence of ecological
factors in early human evolution. Here we trace the cranial and dental
traits of the early australopithecines through time, to show that
between 4.4 million and 2.3 million years ago, the dietary capabilities
of the earliest hominids changed dramatically, leaving them well suited
for life in a variety of habitats and able to cope with significant
changes in resource availability associated with long-term and
short-term climatic fluctuations.
Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase
Authors: Yamada Kazuhiro , Chen Zhoutao , Rozen Rima , Matthews Rowena G.
Published: July 2002

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the
conversion of methylenetetrahydrofolate to methyltetrahydrofolate, the
major methyl donor for the conversion of homocysteine to methionine.
Two common polymorphisms of the human enzyme have been
identified: 677C>T, which leads to the substitution of Ala-222 by
valine, and 1298A>C, which leads to the replacement of Glu-429 by
alanine; the former polymorphism is the most frequent genetic cause of
mild hyperhomocysteinemia, a risk factor for cardiovascular disease. By
using a baculovirus expression system, recombinant human MTHFR has been
expressed at high levels and purified to homogeneity in quantities
suitable for biochemical characterization. The Glu429Ala protein has
biochemical properties that are indistinguishable from the wild-type
enzyme. The Ala222Val MTHFR, however, has an enhanced propensity to
dissociate into monomers and to lose its FAD cofactor on dilution; the
resulting loss of activity is slowed in the presence of
methyltetrahydrofolate or adenosylmethionine. This biochemical
phenotype is in good agreement with predictions made on the basis of
studies comparing wild-type
Escherichia coli
MTHFR with
a mutant, Ala177Val, homologous to the Ala222Val mutant human enzyme
[Guenther, B. D.,
et al.
(1999)
Nat.
Struct. Biol.
6, 359–365].
A large carbon sink in the woody biomass of Northern forests
Authors: Myneni R. B. , Dong J. , Tucker C. J. , Kaufmann R. K. , Kauppi P. E. , Liski J. , Zhou L. , Alexeyev V. , Hughes M. K.
Published: July 2002

The terrestrial carbon sink, as of yet unidentified, represents
15–30% of annual global emissions of carbon from fossil fuels and
industrial activities. Some of the missing carbon is sequestered
in vegetation biomass and, under the Kyoto Protocol of the United
Nations Framework Convention on Climate Change, industrialized nations
can use certain forest biomass sinks to meet their greenhouse gas
emissions reduction commitments. Therefore, we analyzed 19 years of
data from remote-sensing spacecraft and forest inventories to identify
the size and location of such sinks. The results, which cover the years
1981–1999, reveal a picture of biomass carbon gains in Eurasian boreal
and North American temperate forests and losses in some Canadian boreal
forests. For the 1.42 billion hectares of Northern forests, roughly
above the 30th parallel, we estimate the biomass sink to be 0.68
± 0.34 billion tons carbon per year, of which nearly 70% is in
Eurasia, in proportion to its forest area and in disproportion to its
biomass carbon pool. The relatively high spatial resolution of these
estimates permits direct validation with ground data and contributes to
a monitoring program of forest biomass sinks under the Kyoto protocol.
Feeding acetyl-
l
-carnitine and lipoic acid to old rats significantly improves metabolic function while decreasing oxidative stress
Authors: Hagen Tory M. , Liu Jiankang , Lykkesfeldt Jens , Wehr Carol M. , Ingersoll Russell T. , Vinarsky Vladimir , Bartholomew James C. , Ames Bruce N.
Published: July 2002

Mitochondrial-supported bioenergetics decline and oxidative stress increases during aging. To address whether the dietary addition of acetyl-
l
-carnitine [ALCAR, 1.5% (wt/vol) in the drinking water] and/or (
R
)-α-lipoic acid [LA, 0.5% (wt/wt) in the chow] improved these endpoints, young (2–4 mo) and old (24–28 mo) F344 rats were supplemented for up to 1 mo before death and hepatocyte isolation. ALCAR+LA partially reversed the age-related decline in average mitochondrial membrane potential and significantly increased (
P
= 0.02) hepatocellular O
2
consumption, indicating that mitochondrial-supported cellular metabolism was markedly improved by this feeding regimen. ALCAR+LA also increased ambulatory activity in both young and old rats; moreover, the improvement was significantly greater (
P
= 0.03) in old versus young animals and also greater when compared with old rats fed ALCAR or LA alone. To determine whether ALCAR+LA also affected indices of oxidative stress, ascorbic acid and markers of lipid peroxidation (malondialdehyde) were monitored. The hepatocellular ascorbate level markedly declined with age (
P
= 0.003) but was restored to the level seen in young rats when ALCAR+LA was given. The level of malondialdehyde, which was significantly higher (
P
= 0.0001) in old versus young rats, also declined after ALCAR+LA supplementation and was not significantly different from that of young unsupplemented rats. Feeding ALCAR in combination with LA increased metabolism and lowered oxidative stress more than either compound alone.
G1 arrest and down-regulation of cyclin E/cyclin-dependent kinase 2 by the protein kinase inhibitor staurosporine are dependent on the retinoblastoma protein in the bladder carcinoma cell line 5637.
Authors: Schnier J B , Nishi K , Goodrich D W , Bradbury E M
Published: July 2002

The protein kinase inhibitor staurosporine has been shown to induce G1 phase arrest in normal cells but not in most transformed cells. Staurosporine did not induce G1 phase arrest in the bladder carcinoma cell line 5637 that lacks a functional retinoblastoma protein (pRB-). However, when infected with a pRB-expressing retrovirus [Goodrich, D. W., Chen, Y., Scully, P. & Lee, W.-H. (1992) Cancer Res. 52, 1968-1973], these cells, now pRB+, were arrested by staurosporine in G1 phase. This arrest was accompanied by the accumulation of hypophosphorylated pRB. In both the pRB+ and pRB- cells, cyclin D1-associated kinase activities were reduced on staurosporine treatment. In contrast, cyclin-dependent kinase (CDK) 2 and cyclin E/CDK2 activities were inhibited only in pRB+ cells. Staurosporine treatment did not cause reductions in the protein levels of CDK4, cyclin D1, CDK2, or cyclin E. The CDK inhibitor proteins p21(Waf1/Cip1) and p27 (Kip1) levels increased in staurosporine-treated cells. Immunoprecipitation of CDK2, cyclin E, and p2l from staurosporine-treated pRB+ cells revealed a 2.5- to 3-fold higher ratio of p2l bound to CDK2 compared with staurosporine-treated pRB- cells. In pRB+ cells, p2l was preferentially associated with Thrl6O phosphorylated active CDK2. In pRB- cells, however, p2l was bound preferentially to the unphosphorylated, inactive form of CDK2 even though the phosphorylated form was abundant. This is the first evidence suggesting that G1 arrest by 4 nM staurosporine is dependent on a functional pRB protein. Cell cycle arrest at the pRB- dependent checkpoint may prevent activation of cyclin E/CDK2 by stabilizing its interaction with inhibitor proteins p2l and p27.
Postembryonic segregation of the germ line in sea urchins in relation to indirect development.
Authors: Ransick A , Cameron R A , Davidson E H
Published: July 2002

The four small micromeres of the sea urchin embryo contribute only to the coelomic sacs, which produce major components of the adult body plan during postembryonic development. To test the proposition that the small micromeres are the definitive primordial germ cell lineage of the sea urchin, we deleted their 4th cleavage parents, and raised the deleted embryos through larval life and metamorphosis to sexual maturity. Almost all of the experimental animals produced functional gametes, excluding the possibility that the germ cell lineage arises exclusively and obligatorily from descendants of the small micromeres; rather, the germ cell lineage arises during the postembryonic development of the rudiment. A survey of the literature indicates that there is no known case of an embryonic primordial germ cell lineage in a bilaterian species that displays maximal indirect development.
Monophyletic origin and unique dispersal patterns of domestic fowls.
Authors: Fumihito A , Miyake T , Takada M , Shingu R , Endo T , Gojobori T , Kondo N , Ohno S
Published: July 2002

With the aim of elucidating in greater detail the genealogical origin of the present domestic fowls of the world, we have determined mtDNA sequences of the D-loop regions for a total of 21 birds, of which 12 samples belong to red junglefowl (Gallus gallus) comprising three subspecies (six Gallus gallus gallus, three Gallus gallus spadiceus, and three Gallus gallus bankiva) and nine represent diverse domestic breeds (Gallus gallus domesticus). We also sequenced four green junglefowl (Gallus varius), two Lafayette's junglefowl (Gallus lafayettei), and one grey junglefowl (Gallus sonneratii). We then constructed a phylogenetic tree for these birds by the use of nucleotide sequences, choosing the Japanese quail (Coturnix coturnix japonica) as an outgroup. We found that a continental population of G. g. gallus was the real matriarchic origin of all the domestic poultries examined in this study. It is also of particular interest that there were no discernible differences among G. gallus subspecies; G. g. bankiva was a notable exception. This was because G. g. spadiceus and a continental population of G. g. gallus formed a single cluster in the phylogenetic tree. G. g. bankiva, on the other hand, was a distinct entity, thus deserving its subspecies status. It implies that a continental population of G. g. gallus sufficed as the monophyletic ancestor of all domestic breeds. We also discussed a possible significance of the initial dispersal pattern of the present domestic fowls, using the phylogenetic tree.
Expression of calbindin-D28K in motoneuron hybrid cells after retroviral infection with calbindin-D28K cDNA prevents amyotrophic lateral sclerosis IgG-mediated cytotoxicity.
Authors: Ho B K , Alexianu M E , Colom L V , Mohamed A H , Serrano F , Appel S H
Published: July 2002

Calbindin-D28K and/or parvalbumin appear to influence the selective vulnerability of motoneurons in amyotrophic lateral sclerosis (ALS). Their immunoreactivity is undetectable in motoneurons readily damaged in human ALS, and in differentiated motoneuron hybrid cells [ventral spinal cord (VSC 4.1 cells)] that undergo calcium-dependent apoptotic cell death in the presence of ALS immunoglobulins. To provide additional evidence for the role of calcium-binding proteins in motoneuron vulnerability, VSC 4.1 cells were infected with a retrovirus carrying calbindin-D28K cDNA under the control of the promoter of the phosphoglycerate kinase gene. Differentiated calbindin-D28K cDNA-infected cells expressed high calbindin-D28K and demonstrated increased resistance to ALS IgG-mediated toxicity. Treatment with calbindin-D28K antisense oligodeoxynucleotides, which significantly decreased calbindin-D28K expression, rendered these cells vulnerable again to ALS IgG toxicity.
A strategy of exon shuffling for making large peptide repertoires displayed on filamentous bacteriophage.
Authors: Fisch I , Kontermann R E , Finnern R , Hartley O , Soler-Gonzalez A S , Griffiths A D , Winter G
Published: July 2002

It has been suggested that recombination and shuffling between exons has been a key feature in the evolution of proteins. We propose that this strategy could also be used for the artificial evolution of proteins in bacteria. As a first step, we illustrate the use of a self-splicing group I intron with inserted lox-Cre recombination site to assemble a very large combinatorial repertoire (> 10(11) members) of peptides from two different exons. Each exon comprised a repertoire of 10 random amino acids residues; after splicing, the repertoires were joined together through a central five-residue spacer to give a combinatorial repertoire of 25-residue peptides. The repertoire was displayed on filamentous bacteriophage by fusion to the pIII phage coat protein and selected by binding to several proteins, including beta-glucuronidase. One of the peptides selected against beta-glucuronidase was chemically synthesized and shown to inhibit the enzymatic activity (inhibition constant: 17 nM); by further exon shuffling, an improved inhibitor was isolated (inhibition constant: 7 nM). Not only does this approach provide the means for making very large peptide repertoires, but we anticipate that by introducing constraints in the sequences of the peptides and of the linker, it may be possible to evolve small folded peptides and proteins.
Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice.
Authors: Lebovitz R M , Zhang H , Vogel H , Cartwright J , Dionne L , Lu N , Huang S , Matzuk M M
Published: July 2002

Manganese superoxide dismutase (SOD2) converts superoxide to oxygen plus hydrogen peroxide and serves as the primary defense against mitochondrial superoxide. Impaired SOD2 activity in humans has been associated with several chronic diseases, including ovarian cancer and type I diabetes, and SOD2 overexpression appears to suppress malignancy in cultured cells. We have produced a line of SOD2 knockout mice (SOD2m1BCM/SOD2m1BCM) that survive up to 3 weeks of age and exhibit several novel pathologic phenotypes including severe anemia, degeneration of neurons in the basal ganglia and brainstem, and progressive motor disturbances characterized by weakness, rapid fatigue, and circling behavior. In addition, SOD2m1BCM/SOD2m1BCM mice older than 7 days exhibit extensive mitochondrial injury within degenerating neurons and cardiac myocytes. Approximately 10% of SOD2m1BCM/SOD2m1BCM mice exhibit markedly enlarged and dilated hearts. These observations indicate that SOD2 deficiency causes increased susceptibility to oxidative mitochondrial injury in central nervous system neurons, cardiac myocytes, and other metabolically active tissues after postnatal exposure to ambient oxygen concentrations. Our SOD2-deficient mice differ from a recently described model in which homozygotes die within the first 5 days of life with severe cardiomyopathy and do not exhibit motor disturbances, central nervous system injury, or ultrastructural evidence of mitochondrial injury.
Studies on locus content mapping.
Authors: Teague J W , Collins A , Morton N E
Published: July 2002

Locus content maps are derived from monosomic or disomic chromosomes broken by radiation, shearing, or other clastogen, the fragments being distributed among clones by dilution or incorporation into the cells of another species and scored for segregation of markers. Locus content maps provide evidence about radiosensitivity of chromosome regions, support for order, and approximate location. Omission of the most aberrant and least informative clones increases efficiency of localization. Correct analysis must allow for preferential retention of certain sequences, monosomy or polysomy of donor chromosomes, and error filtration. Combination of these refinements extracts substantially more information from fewer clones. Because of unmodeled peculiarities in the data, the best analysis does not recover the physical map but roughly localizes markers that may be monomorphic and therefore unsuitable for linkage mapping. As with linkage for polymorphic loci, distance in the composite map should be confirmed by physical methods.
The future of the national laboratories
Authors: Cohen Linda R. , Noll Roger G.
Published: July 2002

The end of the Cold War has called into question the activities of
the national laboratories and, more generally, the level of support now
given to federal intramural research in the United States. This paper
seeks to analyze the potential role of the laboratories, with
particular attention to the possibility, on the one hand, of
integrating private technology development into the laboratory’s menu
of activities and, on the other hand, of outsourcing traditional
mission activities. We review the economic efficiency arguments for
intramural research and the political conditions that are likely to
constrain the activities of the laboratories, and analyze the early
history of programs intended to promote new technology via cooperative
agreements between the laboratories and private industry. Our analysis
suggests that the laboratories are likely to shrink considerably in
size, and that the federal government faces a significant problem in
deciding how to organize a downsizing of the federal research
establishment.
A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal
Authors: Parks Robin J. , Chen Liane , Anton Martina , Sankar Uma , Rudnicki Michael A. , Graham Frank L.
Published: July 2002

Adenoviruses are attractive vectors for the delivery of foreign
genes into mammalian cells for gene therapy. However, current vectors
retain many viral genes that, when expressed at low levels, contribute
to the induction of a host immune response against transduced cells. We
have developed a helper-dependent packaging system for production of
vectors that have large regions of the genome deleted. Helper viruses
were constructed with packaging signals flanked by
lox
P
sites so that in 293 cells that stably express the Cre recombinase
(293Cre), the packaging signal was efficiently excised, rendering the
helper virus genome unpackageable. However, the helper virus DNA was
replicated at normal levels and could thus express all of the functions
necessary
in trans
for replication and packaging of a
vector genome containing the appropriate cis-acting elements. Serial
passage of the vector in helper virus-infected 293Cre cells resulted in
an ≈10-fold increase in vector titer per passage. The vector could be
partially separated from residual helper virus by cesium chloride
buoyant density centrifugation. Large scale preparations of vector
yielded semi-purified stocks of ≈10
10
transducing
virions/ml, with <0.01% contamination by the E1-deleted helper
virus. This system should have great utility for the generation of
adenovirus-based vectors with increased cloning capacity, increased
safety and reduced immunogenicity.
Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA.
Authors: Cheng J Q , Ruggeri B , Klein W M , Sonoda G , Altomare D A , Watson D K , Testa J R
Published: July 2002

We previously demonstrated that the putative oncogene AKT2 is amplified and overexpressed in some human ovarian carcinomas. We have now identified amplification of AKT2 in approximately 10% of pancreatic carcinomas (2 of 18 cell lines and 1 of 10 primary tumor specimens). The two cell lines with altered AKT2 (PANC1 and ASPC1) exhibited 30-fold and 50-fold amplification of AKT2, respectively, and highly elevated levels of AKT2 RNA and protein. PANC1 cells were transfected with antisense AKT2, and several clones were established after G418 selection. The expression of AKT2 protein in these clones was greatly decreased by the antisense RNA. Furthermore, tumorigenicity in nude mice was markedly reduced in PANC1 cells expressing antisense AKT2 RNA. To examine further whether overexpression of AKT2 plays a significant role in pancreatic tumorigenesis, PANC1 cells and ASPC1 cells, as well as pancreatic carcinoma cells that do not overexpress AKT2 (COLO 357), were transfected with antisense AKT2, and their growth and invasiveness were characterized by a rat tracheal xenotransplant assay. ASPC1 and PANC1 cells expressing antisense AKT2 RNA remained confined to the tracheal lumen, whereas the respective parental cells invaded the tracheal wall. In contrast, no difference was seen in the growth pattern between parental and antisense-treated COLO 357 cells. These data suggest that overexpression of AKT2 contributes to the malignant phenotype of a subset of human ductal pancreatic cancers.
Hippocampal atrophy in recurrent major depression.
Authors: Sheline Y I , Wang P W , Gado M H , Csernansky J G , Vannier M W
Published: July 2002

Hippocampal volumes of subjects with a history of major depressive episodes but currently in remission and with no known medical comorbidity were compared to matched normal controls by using volumetric magnetic resonance images. Subjects with a history of major depression had significantly smaller left and right hippocampal volumes with no differences in total cerebral volumes. The degree of hippocampal volume reduction correlated with total duration of major depression. In addition, large (diameter > or = 4.5 mm)-hippocampal low signal foci (LSF) were found within the hippocampus, and their number also correlated with the total number of days depressed. These results suggest that depression is associated with hippocampal atrophy, perhaps due to a progressive process mediated by glucocorticoid neurotoxicity.
A rat genetic map constructed by representational difference analysis markers with suitability for large-scale typing.
Authors: Toyota M , Canzian F , Ushijima T , Hosoya Y , Kuramoto T , Serikawa T , Imai K , Sugimura T , Nagao M
Published: July 2002

Representational difference analysis (RDA) was applied to isolate chromosomal markers in the rat. Four series of RDA [restriction enzymes, BamHI and HindIII; subtraction of ACI/N (ACI) amplicon from BUF/Nac (BUF) amplicon and vice versa] yielded 131 polymorphic markers; 125 of these markers were mapped to all chromosomes except for chromosome X. This was done by using a mapping panel of 105 ACI x BUF F2 rats. To complement the relative paucity of chromosomal markers in the rat, genetically directed RDA, which allows isolation of polymorphic markers in the specific chromosomal region, was performed. By changing the F2 driver-DNA allele frequency around the region, four markers were isolated from the D1Ncc1 locus. Twenty-five of 27 RDA markers were informative regarding the dot blot analysis of amplicons, hybridizing only with tester amplicons. Dot blot analysis at a high density per unit of area made it possible to process a large number of samples. Quantitative trait loci can now be mapped in the rat genome by processing a large number of samples with RDA markers and then by isolating markers close to the loci of interest by genetically directed RDA.
Early embryonic lethality caused by targeted disruption of the mouse selenocysteine tRNA gene (
Trsp
)
Authors: Bösl Michael R. , Takaku Kazuaki , Oshima Masanobu , Nishimura Susumu , Taketo Makoto M.
Published: July 2002

Selenoprotein biosynthesis is mediated by tRNA
Sec
, which inserts selenocysteine at UGA codons in a complex, context-specific manner. This opal suppressor serves in the conversion of serine to selenocysteine as well. The mouse tRNA
Sec
gene (
Trsp
) maps to a proximal segment of chromosome 7. We constructed mice carrying a targeted deletion of the
Trsp
gene. The heterozygous mutants were viable, fertile, and appeared normal. Although the level of tRNA
Sec
was reduced to about 50%–80% of the wild type in most organs, one of the selenoproteins, glutathione peroxidase, remained unaffected in the levels of its mRNA, protein, and enzyme activity, indicating that the haploid amount of tRNA
Sec
is not limiting in its biosynthesis. In contrast, the homozygous mutants died shortly after implantation, and the embryos were resorbed before 6.5 days post coitum. When the preimplantation embryos were placed in culture, however, the trophoectoderm cells showed outgrowths and the inner cell mass cells of the homozygous embryos were able to proliferate. These results indicate that
Trsp
expression is essential for early development of the embryo, and its lack causes peri-implantation lethality. However, the lethality does not appear to be due to a cell-autonomous function of tRNA
Sec
.
Attenuation of G
i
- and G
q
-mediated signaling by expression of RGS4 or GAIP in mammalian cells
Authors: Huang Chunfa , Hepler John R. , Gilman Alfred G. , Mumby Susanne M.
Published: July 2002

Protein regulators of G protein signaling (RGS proteins) were discovered as negative regulators of heterotrimeric G protein-mediated signal transduction in yeast and worms. Experiments with purified recombinant proteins
in vitro
have established that RGS proteins accelerate the GTPase activity of certain G protein α subunits (the reaction responsible for their deactivation); they can also act as effector antagonists. We demonstrate herein that either of two such RGS proteins, RGS4 or GAIP, attenuated signal transduction mediated by endogenous receptors, G proteins, and effectors when stably expressed as tagged proteins in transfected mammalian cells. The pattern of selectivity observed
in vivo
was similar to that seen
in vitro
. RGS4 and GAIP both attenuated G
i
-mediated inhibition of cAMP synthesis. RGS4 was more effective than GAIP in blocking G
q
-mediated activation of phospholipase Cβ.
Gene identification and DNA sequence analysis in the GC-poor 20 megabase region of human chromosome 21
Authors: Yu Jingwei , Tong Suhong , Shen Yiping , Kao Fa-Ten
Published: July 2002

In contrast to the distal half of the long arm of chromosome 21, the proximal half of approximately 20 megabases of DNA, including 21q11–21 bands, is low in GC content, CpG islands, and identified genes. Despite intensive searches, very few genes and cDNAs have been found in this region. Since the 21q11–21 region is associated with certain Down syndrome pathologies like mental retardation, the identification of relevant genes in this region is important. We used a different approach by constructing microdissection libraries specifically for this region and isolating unique sequence microclones for detailed molecular analysis. We found that this region is enriched with middle and low-copy repetitive sequences, and is also heavily methylated. By sequencing and homology analysis, we identified a significant number of genes/cDNAs, most of which appear to belong to gene families. In addition, we used unique sequence microclones in direct screening of cDNA libraries and isolated 12 cDNAs for this region. Thus, although the 21q11–21 region is gene poor, it is not completely devoid of genes/cDNAs. The presence of high proportions of middle and low-copy repetitive sequences in this region may have evolutionary significance in the genome organization and function of this region. Since 21q11–21 is heavily methylated, the expression of genes in this region may be regulated by a delicate balance of methylation and demethylation, and the presence of an additional copy of chromosome 21 may seriously disturb this balance and cause specific Down syndrome anomalies including mental retardation.
A global budget for fine root biomass, surface area, and nutrient contents
Authors: Jackson R. B. , Mooney H. A. , Schulze E.-D.
Published: July 2002

Global biogeochemical models have improved dramatically in the last decade in their representation of the biosphere. Although leaf area data are an important input to such models and are readily available globally, global root distributions for modeling water and nutrient uptake and carbon cycling have not been available. This analysis provides global distributions for fine root biomass, length, and surface area with depth in the soil, and global estimates of nutrient pools in fine roots. Calculated root surface area is almost always greater than leaf area, more than an order of magnitude so in grasslands. The average C:N:P ratio in living fine roots is 450:11:1, and global fine root carbon is more than 5% of all carbon contained in the atmosphere. Assuming conservatively that fine roots turn over once per year, they represent 33% of global annual net primary productivity.
Evolution of codon usage bias in Drosophila
Authors: Powell Jeffrey R. , Moriyama Etsuko N.
Published: July 2002

We first review what is known about patterns of codon usage bias inDrosophilaand make the following points: (i)Drosophilagenes are as biased or more biased than those in microorganisms. (ii) The level of bias of genes and even the particular pattern of codon bias can remain phylogenetically invariant for very long periods of evolution. (iii) However, some genes, even very tightly linked genes, can change very greatly in codon bias across species. (iv) Generally G and especially C are favored at synonymous sites in biased genes. (v) With the exception of aspartic acid, all amino acids contribute significantly and about equally to the codon usage bias of a gene. (vi) While most individual amino acids that can use G or C at synonymous sites display a preference for C, there are exceptions: valine and leucine, which prefer G. (vii) Finally, smaller genes tend to be more biased than longer genes. We then examine possible causes of these patterns and discount mutation bias on three bases: there is little evidence of regional mutation bias inDrosophila,mutation bias is likely toward A+T (the opposite of codon usage bias), and not all amino acids display the preference for the same nucleotide in the wobble position. Two lines of evidence support a selection hypothesis based on tRNA pools: highly biased genes tend to be highly and/or rapidly expressed, and the preferred codons in highly biased genes optimally bind the most abundant isoaccepting tRNAs. Finally, we examine the effect of bias on DNA evolution and confirm that genes with high codon usage bias have lower rates of synonymous substitution between species than do genes with low codon usage bias. Surprisingly, we find that genes with higher codon usage bias display higher levels of intraspecific synonymous polymorphism. This may be due to opposing effects of recombination.
A thymidine triphosphate shape analog lacking Watson–Crick pairing ability is replicated with high sequence selectivity
Authors: Moran Sean , Ren Rex X.-F. , Kool Eric T.
Published: July 2002

Compound 1 (F), a nonpolar nucleoside analog that is isosteric with thymidine, has been proposed as a probe for the importance of hydrogen bonds in biological systems. Consistent with its lack of strong H-bond donors or acceptors, F is shown here by thermal denaturation studies to pair very poorly and with no significant selectivity among natural bases in DNA oligonucleotides. We report the synthesis of the 5′-triphosphate derivative of 1 and the study of its ability to be inserted into replicating DNA strands by the Klenow fragment (KF, exo
−
mutant) of
Escherichia coli
DNA polymerase I. We find that this nucleotide derivative (dFTP) is a surprisingly good substrate for KF; steady-state measurements indicate it is inserted into a template opposite adenine with efficiency (
V
max
/
K
m
) only 40-fold lower than dTTP. Moreover, it is inserted opposite A (relative to C, G, or T) with selectivity nearly as high as that observed for dTTP. Elongation of the strand past F in an F–A pair is associated with a brief pause, whereas that beyond A in the inverted A–F pair is not. Combined with data from studies with F in the template strand, the results show that KF can efficiently replicate a base pair (A–F/F–A) that is inherently very unstable, and the replication occurs with very high fidelity despite a lack of inherent base-pairing selectivity. The results suggest that hydrogen bonds may be less important in the fidelity of replication than commonly believed and that nucleotide/template shape complementarity may play a more important role than previously believed.
Inorganic polyphosphate and the induction of
rpoS
expression
Authors: Shiba Toshikazu , Tsutsumi Kaori , Yano Hiroyuki , Ihara Yoshiharu , Kameda Atsushi , Tanaka Kan , Takahashi Hideo , Munekata Masanobu , Rao Narayana N. , Kornberg Arthur
Published: July 2002

Inorganic polyphosphate [poly(P)] levels in
Escherichia coli
were reduced to barely detectable concentrations by expression of the plasmid-borne gene for a potent yeast exopolyphosphatase [poly(P)ase]. As a consequence, resistance to H
2
O
2
was greatly diminished, particularly in
katG
(catalase HPI) mutants, implying a major role for the other catalase, the stationary-phase KatE (HPII), which is
rpoS
dependent. Resistance was restored to wild-type levels by complementation with plasmids expressing
ppk
, the gene for PPK [the polyphosphate kinase that generates poly(P)]. Induction of expression of both
katE
and
rpoS
(the stationary-phase σ factor) was prevented in cells in which the poly(P)ase was overproduced. Inasmuch as this inhibition by poly(P)ase did not affect the levels of the stringent-response guanosine nucleotides (pppGpp and ppGpp) and in view of the capacity of additional
rpoS
expression to suppress the poly(P)ase inhibition of
katE
expression, a role is proposed for poly(P) in inducing the expression of
rpoS
.
Epstein–Barr virus-induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin
Authors: Devergne Odile , Birkenbach Mark , Kieff Elliott
Published: July 2002

The Epstein–Barr virus-induced gene 3 (EBI3) is a novel soluble hematopoietin component related to the p40 subunit of interleukin 12 (IL-12). When EBI3 was expressed in cells, it accumulated in the endoplasmic reticulum and associated with the molecular chaperone calnexin, indicating that subsequent processing and secretion might be dependent on association with a second subunit. Coimmunoprecipitations from lysates and culture media of cells transfected with expression vectors for EBI3 and/or the p35 subunit of IL-12 now reveal a specific association of EBI3 with p35. Coexpression of EBI3 and p35 mutually facilitates their secretion. Most importantly, a large fraction of p35 in extracts of the trophoblast component of a human full-term normal placenta specifically coimmunoprecipitated with EBI3, indicating that EBI3 is in a heterodimer with p35,in vivo. Because EBI3 is expressed in EBV-transformed B lymphocytes, tonsil, spleen, and placental trophoblasts, the EBI3/p35 heterodimer is likely to be an important immunomodulator.
Ethnohistory, genetics, and cancer mortality in Europeans
Authors: Sokal Robert R. , Oden Neal L. , Rosenberg Michael S. , DiGiovanni Donna
Published: July 2002

Geographic variation in cancer rates is thought to be the result of two major factors: environmental agents varying spatially and the attributes, genetic or cultural, of the populations inhabiting the areas studied. These attributes in turn result from the history of the populations in question. We had previously constructed an ethnohistorical database for Europe since 2200 B.C., permitting estimates of the ethnic composition of modern European populations. We were able to show that these estimates correlate with genetic distances. In this study, we wanted to see whether they also correlate with cancer rates. We employed two data sets of cancer mortalities from 42 types of cancer for the European Economic Community and for Central Europe. We subjected spatial differences in cancer mortalities, genetic, ethnohistorical, and geographic distances to matrix permutation tests to determine the magnitude and significance of their association. Our findings are that distances in cancer mortalities are correlated more with ethnohistorical distances than with genetic distances. Possibly the cancer rates may be affected by loci other than the genetic systems available to us, and/or by cultural factors mediated by the ethnohistorical differences. We find it remarkable that patterns of frequently ancient ethnic admixture are still reflected in modern cancer mortalities. Partial correlations with geography suggest that local environmental factors affect the mortalities as well.
Disruption of the m1 receptor gene ablates muscarinic receptor-dependent M current regulation and seizure activity in mice
Authors: Hamilton Susan E. , Loose Michael D. , Qi Ming , Levey Allan I. , Hille Bertil , McKnight G. Stanley , Idzerda Rejean L. , Nathanson Neil M.
Published: July 2002

Muscarinic acetylcholine receptors are members of the G protein-coupled receptor superfamily expressed in neurons, cardiomyocytes, smooth muscle, and a variety of epithelia. Five subtypes of muscarinic acetylcholine receptors have been discovered by molecular cloning, but their pharmacological similarities and frequent colocalization make it difficult to assign functional roles for individual subtypes in specific neuronal responses. We have used gene targeting by homologous recombination in embryonic stem cells to produce mice lacking the m1 receptor. These mice show no obvious behavioral or histological defects, and the m2, m3, and m4 receptors continue to be expressed in brain with no evidence of compensatory induction. However, the robust suppression of the M-current potassium channel activity evoked by muscarinic agonists in sympathetic ganglion neurons is completely lost in m1 mutant mice. In addition, both homozygous and heterozygous mutant mice are highly resistant to the seizures produced by systemic administration of the muscarinic agonist pilocarpine. Thus, the m1 receptor subtype mediates M current modulation in sympathetic neurons and induction of seizure activity in the pilocarpine model of epilepsy.
Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in
Plasmodium falciparum
Authors: Triglia Tony , Menting John G. T. , Wilson Craig , Cowman Alan F.
Published: July 2002

Plasmodium falciparum
causes the most severe form of malaria in humans. An important class of drugs in malaria treatment is the sulfone/sulfonamide group, of which sulfadoxine is the most commonly used. The target of sulfadoxine is the enzyme dihydropteroate synthase (DHPS), and sequencing of the DHPS gene has identified amino acid differences that may be involved in the mechanism of resistance to this drug. In this study we have sequenced the DHPS gene in 10 isolates from Thailand and identified a new allele of DHPS that has a previously unidentified amino acid difference. We have expressed eight alleles of
P. falciparum
PPPK-DHPS in
Escherichia coli
and purified the functional enzymes to homogeneity. Strikingly, the
K
i
for sulfadoxine varies by almost three orders of magnitude from 0.14 μM for the DHPS allele from sensitive isolates to 112 μM for an enzyme expressed in a highly resistant isolate. Comparison of the K
i
of different sulfonamides and the sulfone dapsone has suggested that the amino acid differences in DHPS would confer cross-resistance to these compounds. These results show that the amino acid differences in the DHPS enzyme of sulfadoxine-resistant isolates of
P. falciparum
are central to the mechanism of resistance to sulfones and sulfonamides.
Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor
Authors: Gehm Barry D. , McAndrews Joanne M. , Chien Pei-Yu , Jameson J. Larry
Published: July 2002

The phytochemical resveratrol, which is found in grapes and wine, has been reported to have a variety of anti-inflammatory, anti-platelet, and anti-carcinogenic effects. Based on its structural similarity to diethylstilbestrol, a synthetic estrogen, we examined whether resveratrol might be a phytoestrogen. At concentrations (≈3–10 μM) comparable to those required for its other biological effects, resveratrol inhibited the binding of labeled estradiol to the estrogen receptor and it activated transcription of estrogen-responsive reporter genes transfected into human breast cancer cells. This transcriptional activation was estrogen receptor-dependent, required an estrogen response element in the reporter gene, and was inhibited by specific estrogen antagonists. In some cell types (e.g., MCF-7 cells), resveratrol functioned as a superagonist (i.e., produced a greater maximal transcriptional response than estradiol) whereas in others it produced activation equal to or less than that of estradiol. Resveratrol also increased the expression of native estrogen-regulated genes, and it stimulated the proliferation of estrogen-dependent T47D breast cancer cells. We conclude that resveratrol is a phytoestrogen and that it exhibits variable degrees of estrogen receptor agonism in different test systems. The estrogenic actions of resveratrol broaden the spectrum of its biological actions and may be relevant to the reported cardiovascular benefits of drinking wine.