bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction between astrocytes and adult subventricular zone precursors stimulates neurogenesis
Authors: Lim Daniel A. , Alvarez-Buylla Arturo
Published: July 2002

Neurogenesis continues in the mammalian subventricular zone (SVZ) throughout life. However, the signaling and cell–cell interactions required for adult SVZ neurogenesis are not known.
In vivo
, migratory neuroblasts (type A cells) and putative precursors (type C cells) are in intimate contact with astrocytes (type B cells). Type B cells also contact each other. We reconstituted SVZ cell–cell interactions in a culture system free of serum or exogenous growth factors. Culturing dissociated postnatal or adult SVZ cells on astrocyte monolayers—but not other substrates—supported extensive neurogenesis similar to that observed
in vivo
. SVZ precursors proliferated rapidly on astrocytes to form colonies containing up to 100 type A neuroblasts. By fractionating the SVZ cell dissociates with differential adhesion to immobilized polylysine, we show that neuronal colony-forming precursors were concentrated in a fraction enriched for type B and C cells. Pure type A cells could migrate in chains but did not give rise to neuronal colonies. Because astrocyte-conditioned medium alone was not sufficient to support SVZ neurogenesis, direct cell–cell contact between astrocytes and SVZ neuronal precursors may be necessary for the production of type A cells.
Damage to photosystem II in symbiotic dinoflagellates: A determinant of coral bleaching
Authors: Warner Mark E. , Fitt William K. , Schmidt Gregory W.
Published: July 2002

Coral bleaching has been defined as a general phenomenon, whereby reef corals turn visibly pale because of the loss of their symbiotic dinoflagellates and/or algal pigments during periods of exposure to elevated seawater temperatures. During the summer of 1997, seawater temperatures in the Florida Keys remained at or above 30°C for more than 6 weeks, and extensive coral bleaching was observed. Bleached colonies of the dominant Caribbean reef-building species,
Montastrea faveolata
and
Montastrea franksi
, were sampled over a depth gradient from 1 to 17 m during this period of elevated temperature and contained lower densities of symbiotic dinoflagellates in deeper corals than seen in previous “nonbleaching” years. Fluorescence analysis by pulse-amplitude modulation fluorometry revealed severe damage to photosystem II (PSII) in remaining symbionts within the corals, with greater damage indicated at deeper depths. Dinoflagellates with the greatest loss in PSII activity also showed a significant decline in the D1 reaction center protein of PSII, as measured by immunoblot analysis. Laboratory experiments on the temperature-sensitive species
Montastrea annularis
, as well as temperature-sensitive and temperature-tolerant cultured symbiotic dinoflagellates, confirmed the temperature-dependent loss of PSII activity and concomitant decrease in D1 reaction center protein seen in symbionts collected from corals naturally bleached on the reef. In addition, variation in PSII repair was detected, indicating that perturbation of PSII protein turnover rates during photoinhibition at elevated temperatures underlies the physiological collapse of symbionts in corals susceptible to heat-induced bleaching.
The violaxanthin cycle protects plants from photooxidative damage by more than one mechanism
Authors: Havaux Michel , Niyogi Krishna K.
Published: July 2002

When light energy absorbed by plants becomes excessive relative to the capacity of photosynthesis, the xanthophyll violaxanthin is reversibly deepoxidized to zeaxanthin (violaxanthin cycle). The protective function of this phenomenon was investigated in a mutant of
Arabidopsis thaliana
,
npq1
, that has no functional violaxanthin deepoxidase. Two major consequences of the
npq1
mutation are the absence of zeaxanthin formation in strong light and the partial inhibition of the quenching of singlet excited chlorophylls in the photosystem II light-harvesting complexes. Prolonged exposure of whole plants to bright light resulted in a limited photoinhibition of photosystem II in both
npq1
and wild-type leaves, although CO
2
fixation and the linear electron transport in
npq1
plants were reduced substantially. Lipid peroxidation was more pronounced in
npq1
compared with the wild type, as measured by chlorophyll thermoluminescence, ethane production, and the total hydroperoxy fatty acids content. Lipid peroxidation was amplified markedly under chilling stress, and photooxidative damage ultimately resulted in leaf bleaching and tissue necrosis in
npq1
. The
npq4
mutant, which possesses a normal violaxanthin cycle but has a limited capacity of quenching singlet excited chlorophylls, was rather tolerant to lipid peroxidation. The double mutant,
npq4 npq1
, which differs from
npq4
only by the absence of the violaxanthin cycle, exhibited an increased susceptibility to photooxidative damage, similar to that of
npq1
. Our results demonstrate that the violaxanthin cycle specifically protects thylakoid membrane lipids against photooxidation. Part of this protection involves a mechanism other than quenching of singlet excited chlorophylls.
Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology
Authors: Trigatti Bernardo , Rayburn Helen , Viñals Marisa , Braun Anne , Miettinen Helena , Penman Marsha , Hertz Miki , Schrenzel Mark , Amigo Ludwig , Rigotti Attilio , Krieger Monty
Published: July 2002

The high density lipoprotein (HDL) receptor SR-BI (scavenger receptor class B type I) mediates the selective uptake of plasma HDL cholesterol by the liver and steroidogenic tissues. As a consequence, SR-BI can influence plasma HDL cholesterol levels, HDL structure, biliary cholesterol concentrations, and the uptake, storage, and utilization of cholesterol by steroid hormone-producing cells. Here we used homozygous null SR-BI knockout mice to show that SR-BI is required for maintaining normal biliary cholesterol levels, oocyte development, and female fertility. We also used SR-BI/apolipoprotein E double homozygous knockout mice to show that SR-BI can protect against early-onset atherosclerosis. Although the mechanisms underlying the effects of SR-BI loss on reproduction and atherosclerosis have not been established, potential causes include changes in (
i
) plasma lipoprotein levels and/or structure, (
ii
) cholesterol flux into or out of peripheral tissues (ovary, aortic wall), and (
iii
) reverse cholesterol transport, as indicated by the significant reduction of gallbladder bile cholesterol levels in SR-BI and SR-BI/apolipoprotein E double knockout mice relative to controls. If SR-BI has similar activities in humans, it may become an attractive target for therapeutic intervention in a variety of diseases.
The essential
Staphylococcus aureus
gene
fmhB
is involved in the first step of peptidoglycan pentaglycine interpeptide formation
Authors: Rohrer S. , Ehlert K. , Tschierske M. , Labischinski H. , Berger-Bächi B.
Published: July 2002

The factor catalyzing the first step in the synthesis of the characteristic pentaglycine interpeptide in
Staphylococcus aureus
peptidoglycan was found to be encoded by the essential gene
fmhB
. We have analyzed murein composition and structure synthesized when
fmhB
expression is reduced. The endogenous
fmhB
promoter was substituted with the xylose regulon from
Staphylococcus xylosus
, which allowed glucose-controlled repression of
fmhB
transcription. Repression of
fmhB
reduced growth and triggered a drastic accumulation of uncrosslinked, unmodified muropeptide monomer precursors at the expense of the oligomeric fraction, leading to a substantial decrease in overall peptidoglycan crosslinking. The composition of the predominant muropeptide was confirmed by MS to be
N-
acetylglucosamine-(β-1,4)-
N-
acetylmuramic acid(-
l
-Ala-
d
-iGln-
l
-Lys-
d
-Ala-
d
-Ala), proving that FmhB is involved in the attachment of the first glycine to the pentaglycine interpeptide. This interpeptide plays an important role in crosslinking and stability of the
S. aureus
cell wall, acts as an anchor for cell wall-associated proteins, determinants of pathogenicity, and is essential for the expression of methicillin resistance. Any shortening of the pentaglycine side chain reduces or even abolishes methicillin resistance, as occurred with
fmhB
repression. Because of its key role FmhB is a potential target for novel antibacterial agents that could control the threat of emerging multiresistant
S. aureus
.
Structure-based design of
Taq
DNA polymerases with improved properties of dideoxynucleotide incorporation
Authors: Li Ying , Mitaxov Vesselin , Waksman Gabriel
Published: July 2002

The
Taq
DNA polymerase is the most commonly used enzyme in DNA sequencing. However, all versions of
Taq
polymerase are deficient in two respects: (
i
) these enzymes incorporate each of the four dideoxynucleoside 5′ triphosphates (ddNTPs) at widely different rates during sequencing (ddGTP, for example, is incorporated 10 times faster than the other three ddNTPs), and (
ii
) these enzymes show uneven band-intensity or peak-height patterns in radio-labeled or dye-labeled DNA sequence profiles, respectively. We have determined the crystal structures of all four ddNTP-trapped closed ternary complexes of the large fragment of the
Taq
DNA polymerase (Klentaq1). The ddGTP-trapped complex structure differs from the other three ternary complex structures by a large shift in the position of the side chain of residue 660 in the O helix, resulting in additional hydrogen bonds being formed between the guanidinium group of this residue and the base of ddGTP. When Arg-660 is mutated to Asp, Ser, Phe, Tyr, or Leu, the enzyme has a marked and selective reduction in ddGTP incorporation rate. As a result, the G track generated during DNA sequencing by these
Taq
polymerase variants does not terminate prematurely, and higher molecular-mass G bands are detected. Another property of these
Taq
polymerase variants is that the sequencing patterns produced by these enzymes are remarkably even in band-intensity and peak-height distribution, thus resulting in a significant improvement in the accuracy of DNA sequencing.
Regulation of the Src family tyrosine kinase Blk through E6AP-mediated ubiquitination
Authors: Oda Hideaki , Kumar Sushant , Howley Peter M.
Published: July 2002

The Src family of nonreceptor tyrosine kinases are important regulators of a variety of cellular processes, including cytoskeletal organization, cell–cell contact, and cell–matrix adhesion. Activation of Src family kinases also can induce DNA synthesis and cellular proliferation; therefore, tight regulation of their kinase activities is important for the cell to maintain proliferative control. Posttranslational phosphorylation and dephosphorylation are recognized as the principle modifications by which the activities of the Src family of tyrosine kinases are regulated. We have discovered that this family of kinases also is regulated by ubiquitin-mediated proteolysis. Studies aimed at the identification of cellular targets for E6AP, an E3 ubiquitin protein ligase involved in ubquitin-mediated degradation, led us to the identification of members of the Src family kinases as potential substrates for E6AP. We have found that E6AP can bind to several of the Src family tyrosine kinases. Here we show that activated Blk is preferentially degraded by the ubiquitin–proteasome pathway and that its ubiquitination is mediated by E6AP. Identification of members of the Src tyrosine kinase family as substrates of the E6AP ubiquitin-protein ligase implicates a role for the ubiquitin pathway in regulating the activities of individual members of this important family of signaling molecules.
The hsp70-associating protein Hap46 binds to DNA and stimulates transcription
Authors: Zeiner Matthias , Niyaz Yilmaz , Gehring Ulrich
Published: July 2002

We investigated the ubiquitously expressed hsp70-associating protein Hap46, which is also called RAP46 and is homologous to BAG-1, for activities independent of hsp70 interactions. We observed
in vitro
binding to various DNA fragments but detected no apparent sequence specificity. Deletion of the amino-terminal decapeptide, which contains two clusters of three basic amino acids each, abolished the DNA-binding ability of Hap46. Similarly, exchange of either of these positively charged clusters for three alanines resulted in loss of DNA binding. Using a fusion of Hap46 and green fluorescent protein, we found preferential accumulation in cell nuclei on heat stress as compared with unstressed cells. The repressive effect of heat shock on overall transcriptional activity in human DU145 carcinoma cells was largely prevented when Hap46 was overexpressed by transfection. Such overproduction of Hap46 also resulted in enhanced expression of specific reporter gene constructs and in increased levels of mRNAs specific for hsp70 and hsp40 after temperature stress.
In vitro
transcription with nuclear extracts was stimulated greatly by Hap46. Like DNA binding, transcriptional enhancement required amino-terminally located basic amino acid residues but not the carboxyl-terminal portion of Hap46 known to participate in hsp70 interaction. Our results show that Hap46 is a bifunctional protein that can interact with both hsp70s and DNA, employing different portions of the molecule. They also suggest that Hap46 is involved in temperature-sensitive regulation of transcription, acting as a general transcriptional activator.
Regeneration of a germinal layer in the adult mammalian brain
Authors: Doetsch Fiona , García-Verdugo Jose Manuel , Alvarez-Buylla Arturo
Published: July 2002

Neuronal precursors reside in the subventricular zone (SVZ) of adult mammals. This region is composed of a network of chains of migrating neuroblasts ensheathed by astrocytes and juxtaposed by clusters of immature precursors (type C cells). Here we show that after antimitotic treatment with cytosine-β-
d
-arabinofuranoside, neuroblasts and type C cells are eliminated but some astrocytes remain. Remarkably, the SVZ network rapidly regenerates. Soon after cytosine-β-
d
-arabinofuranoside treatment astrocytes divide. Two days later, type C cells reappear, followed at 4.5 days by migrating neuroblasts. By 10 days the SVZ network is fully regenerated, and the orientation and organization of chains of migrating neuroblasts resemble that of normal mice. This regeneration reveals an unexpected plasticity in the adult central nervous system and should provide a model system to study the early stages of neurogenesis in the adult brain.
Identification of an ATP-binding cassette transporter involved in bicarbonate uptake in the cyanobacterium
Synechococcus
sp. strain PCC 7942
Authors: Omata Tatsuo , Price G. Dean , Badger Murray R. , Okamura Masato , Gohta Satoshi , Ogawa Teruo
Published: July 2002

Exposure of cells of cyanobacteria (blue–green algae) grown under high-CO
2
conditions to inorganic C-limitation induces transcription of particular genes and expression of high-affinity CO
2
and HCO
3
−
transport systems. Among the low-CO
2
-inducible transcription units of
Synechococcus
sp. strain PCC 7942 is the
cmpABCD
operon, encoding an ATP-binding cassette transporter similar to the nitrate/nitrite transporter of the same cyanobacterium. A nitrogen-regulated promoter was used to selectively induce expression of the
cmpABCD
genes by growth of transgenic cells on nitrate under high CO
2
conditions. Measurements of the initial rate of HCO
3
−
uptake after onset of light, and of the steady-state rate of HCO
3
−
uptake in the light, showed that the controlled induction of the
cmp
genes resulted in selective expression of high-affinity HCO
3
−
transport activity. The forced expression of
cmpABCD
did not significantly increase the CO
2
uptake capabilities of the cells. These findings demonstrated that the
cmpABCD
genes encode a high-affinity HCO
3
−
transporter. A deletion mutant of
cmpAB
(M42) retained low CO
2
-inducible activity of HCO
3
−
transport, indicating the occurrence of HCO
3
−
transporter(s) distinct from the one encoded by
cmpABCD
. HCO
3
−
uptake by low-CO
2
-induced M42 cells showed lower affinity for external HCO
3
−
than for wild-type cells under the same conditions, showing that the HCO
3
−
transporter encoded by
cmpABCD
has the highest affinity for HCO
3
−
among the HCO
3
−
transporters present in the cyanobacterium. This appears to be the first unambiguous identification and description of a primary active HCO
3
−
transporter.
Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage
Authors: Khosravi Rami , Maya Ruth , Gottlieb Tanya , Oren Moshe , Shiloh Yosef , Shkedy Dganit
Published: July 2002

The p53 tumor-suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. This process is associated with posttranslational modifications of p53, some of which are mediated by the ATM protein kinase. However, these modifications alone may not account in full for p53 stabilization. p53's stability and activity are negatively regulated by the oncoprotein MDM2, whose gene is activated by p53. Conceivably, p53 function may be modulated by modifications of MDM2 as well. We show here that after treatment of cells with ionizing radiation or a radiomimetic chemical, but not UV radiation, MDM2 is phosphorylated rapidly in an ATM-dependent manner. This phosphorylation is independent of p53 and the DNA-dependent protein kinase. Furthermore, MDM2 is directly phosphorylated by ATM
in vitro
. These findings suggest that in response to DNA strand breaks, ATM may promote p53 activity and stability by mediating simultaneous phosphorylation of both partners of the p53-MDM2 autoregulatory feedback loop.
Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly
Lutzomyia longipalpis
Authors: Charlab Rosane , Valenzuela Jesus G. , Rowton Edgar D. , Ribeiro José M. C.
Published: July 2002

The saliva of blood-sucking arthropods contains powerful pharmacologically active substances and may be a vaccine target against some vector-borne diseases. Subtractive cloning combined with biochemical approaches was used to discover activities in the salivary glands of the hematophagous fly
Lutzomyia longipalpis
. Sequences of nine full-length cDNA clones were obtained, five of which are possibly associated with blood-meal acquisition, each having cDNA similarity to: (
i
) the bed bug
Cimex lectularius
apyrase, (
ii
) a 5′-nucleotidase/phosphodiesterase, (
iii
) a hyaluronidase, (
iv
) a protein containing a carbohydrate-recognition domain (CRD), and (
v
) a RGD-containing peptide with no significant matches to known proteins in the
blast
databases. Following these findings, we observed that the salivary apyrase activity of
L. longipalpis
is indeed similar to that of
Cimex
apyrase in its metal requirements. The predicted isoelectric point of the putative apyrase matches the value found for
Lutzomyia
salivary apyrase. A 5′-nucleotidase, as well as hyaluronidase activity, was found in the salivary glands, and the CRD-containing cDNA matches the N-terminal sequence of the HPLC-purified salivary anticlotting protein. A cDNA similar to α-amylase was discovered and salivary enzymatic activity demonstrated for the first time in a blood-sucking arthropod. Full-length clones were also found coding for three proteins of unknown function matching, respectively, the N-terminal sequence of an abundant salivary protein, having similarity to the CAP superfamily of proteins and the
Drosophila
yellow protein. Finally, two partial sequences are reported that match possible housekeeping genes. Subtractive cloning will considerably enhance efforts to unravel the salivary pharmacopeia of blood-sucking arthropods.
Psr1, a nuclear localized protein that regulates phosphorus metabolism in
Chlamydomonas
Authors: Wykoff Dennis D. , Grossman Arthur R. , Weeks Donald P. , Usuda Hideaki , Shimogawara Kosuke
Published: July 2002

Understanding the ways in which phosphorus metabolism is regulated in photosynthetic eukaryotes is critical for optimizing crop productivity and managing aquatic ecosystems in which phosphorus can be a major source of pollution. Here we describe a gene encoding a regulator of phosphorus metabolism, designated
Psr1
(phosphorus starvation response), from a photosynthetic eukaryote. The Psr1 protein is critical for acclimation of the unicellular green alga
Chlamydomonas reinhardtii
to phosphorus starvation. The N-terminal half of Psr1 contains a region similar to myb DNA-binding domains and the C-terminal half possesses glutamine-rich sequences characteristic of transcriptional activators. The level of Psr1 increases at least 10-fold upon phosphate starvation, and immunocytochemical studies demonstrate that this protein is nuclear-localized under both nutrient-replete and phosphorus-starvation conditions. Finally, Psr1 and angiosperm proteins have domains that are similar, suggesting a possible role for Psr1 homologs in the control of phosphorus metabolism in vascular plants. With the identification of regulators such as Psr1 it may become possible to engineer photosynthetic organisms for more efficient utilization of phosphorus and to establish better practices for the management of agricultural lands and natural ecosystems.
A subcortical pathway to the right amygdala mediating “unseen” fear
Authors: Morris J. S. , Öhman A. , Dolan R. J.
Published: July 2002

Neuroimaging studies have shown differential amygdala responses to
masked (“unseen”) emotional stimuli. How visual signals related
to such unseen stimuli access the amygdala is unknown. A possible
pathway, involving the superior colliculus and pulvinar, is suggested
by observations of patients with striate cortex lesions who show
preserved abilities to localize and discriminate visual stimuli that
are not consciously perceived (“blindsight”). We used measures of
right amygdala neural activity acquired from volunteer subjects viewing
masked fear-conditioned faces to determine whether a colliculo-pulvinar
pathway was engaged during processing of these unseen target stimuli.
Increased connectivity between right amygdala, pulvinar, and superior
colliculus was evident when fear-conditioned faces were unseen rather
than seen. Right amygdala connectivity with fusiform and orbitofrontal
cortices decreased in the same condition. By contrast, the left
amygdala, whose activity did not discriminate seen and unseen
fear-conditioned targets, showed no masking-dependent changes in
connectivity with superior colliculus or pulvinar. These results
suggest that a subcortical pathway to the right amygdala, via midbrain
and thalamus, provides a route for processing behaviorally relevant
unseen visual events in parallel to a cortical route necessary for
conscious identification.
Mouse trp2, the homologue of the human trpc2 pseudogene, encodes mTrp2, a store depletion-activated capacitative Ca
2+
entry channel
Authors: Vannier Brigitte , Peyton Michael , Boulay Gulain , Brown Darren , Qin Ning , Jiang Meisheng , Zhu Xi , Birnbaumer Lutz
Published: July 2002

Capacitative Ca
2+
entry (CCE) is Ca
2+
entering after stimulation of inositol 1,4,5-trisphosphate (IP3) formation and initiation of Ca
2+
store depletion. One hallmark of CCE is that it can also be triggered merely by store depletion, as occurs after inhibition of internal Ca
2+
pumps with thapsigargin. Evidence has accumulated in support of a role of transient receptor potential (Trp) proteins as structural subunits of a class of Ca
2+
-permeable cation channels activated by agonists that stimulate IP3 formation—very likely through a direct interaction between the IP3 receptor and a Trp subunit of the Ca
2+
entry channel. The role of Trp’s in Ca
2+
entry triggered by store depletion alone is less clear. Only a few of the cloned Trp’s appear to enhance this type of Ca
2+
entry, and when they do, the effect requires special conditions to be observed, which native CCE does not. Here we report the full-length cDNA of mouse
trp2
, the homologue of the human
trp2
pseudogene. Mouse Trp2 is shown to be readily activated not only after stimulation with an agonist but also by store depletion in the absence of an agonist. In contrast to other Trp proteins, Trp2-mediated Ca
2+
entry activated by store depletion is seen under the same conditions that reveal endogenous store depletion-activated Ca
2+
entry, i.e., classical CCE. The findings support the general hypothesis that Trp proteins are subunits of store- and receptor-operated Ca
2+
channels.
Estradiol induces the calcium-dependent translocation of endothelial nitric oxide synthase
Authors: Goetz Regina M. , Thatte Hemant S. , Prabhakar Prakash , Cho Michael R. , Michel Thomas , Golan David E.
Published: July 2002

Although estrogen is known to stimulate nitric oxide synthesis in vascular endothelium, the molecular mechanisms responsible for this effect remain to be elucidated. Using quantitative immunofluorescence imaging approaches, we have investigated the effect of estradiol on the subcellular targeting of endothelial nitric oxide synthase (eNOS) in bovine aortic endothelial cells. In unstimulated endothelial cells, eNOS is predominantly localized at the cell membrane. Within 5 min after the addition of estradiol, most of the eNOS translocates from the membrane to intracellular sites close to the nucleus. On more prolonged exposure to estradiol, most of the eNOS returns to the membrane. This effect of estradiol is evident at a concentration of 1 pM, and a maximal estradiol effect is seen at a concentration of 1 nM. Neither progesterone nor testosterone has any effect on eNOS distribution. After estradiol addition, a transient rise in intracellular Ca
2+
concentration precedes eNOS translocation. Both the Ca
2+
-mobilizing and eNOS-translocating effects of estradiol are completely blocked by the estrogen receptor antagonist ICI 182,780, and the intracellular Ca
2+
chelator 1,2-bis-(
o
-aminophenoxy)ethane-
N,N,N
′
,N
′-tetraacetic acid (BAPTA) prevents estradiol-induced eNOS translocation. Use of the nitric oxide-specific dye diaminofluorescein shows that estradiol treatment increases nitric oxide generation by endothelial cells; this response is blocked by ICI 182,780 and by the eNOS inhibitor
N
ω
-nitro-
l
-arginine. These results show that estradiol induces subcellular translocation of eNOS by a rapid, Ca
2+
-dependent, receptor-mediated mechanism, and they suggest a nongenomic role for estrogen in the modulation of NO-dependent vascular tone.
In vivo
studies of fullerene-based materials using endohedral metallofullerene radiotracers
Authors: Cagle Dawson W. , Kennel Stephen J. , Mirzadeh Saed , Alford J. Michael , Wilson Lon J.
Published: July 2002

Biodistribution studies of a water-soluble radioactive metallofullerene compound have been conducted using BALB/c mice. To this end, a sample containing Ho
x
@C
82
(x = 1, 2) was purified and derivatized to prepare the water-soluble metallofullerol, Ho
x
@C
82
(OH)
y
. This metallofullerol was then neutron-activated (
165
Ho[n,γ]
166
Ho) to prepare the
166
Ho
x
@C
82
(OH)
y
analog as a radiotracer, which was monitored, after intravenous administration, for up to 48 hours by using dissection radioanalysis, and its biodistribution was compared with a control compound, Na
2
[
166
Ho(DTPA)(H
2
O)]. Results showed selective localization of the
166
Ho
x
@C
82
(OH)
y
tracer in the liver but with slow clearance, as well as uptake by bone without clearance. In contrast, excretion of the control compound was nearly quantitative after 1 hour. The fate of
166
Ho was also explored by a metabolism study of
166
Ho
x
@C
82
(OH)
y
in Fischer rats. Results indicated 20% excretion of intact
166
Ho
x
@C
82
(OH)
y
within 5 days. The present findings demonstrate the feasibility of using water-solubilized metallofullerene radiotracers to monitor the fate of fullerene-based materials in animals, and suggest that water-solubilized fullerene materials, in general, may be useful components in drug design.
Mutations in a NIMA-related kinase gene,
Nek1
, cause pleiotropic effects including a progressive polycystic kidney disease in mice
Authors: Upadhya Poornima , Birkenmeier Edward H. , Birkenmeier Connie S. , Barker Jane E.
Published: July 2002

We previously have described a mouse model for polycystic kidney disease (PKD) caused by either of two mutations,
kat
or
kat
2J
, that map to the same locus on chromosome 8. The homozygous mutant animals have a latent onset, slowly progressing form of PKD with renal pathology similar to the human autosomal-dominant PKD. In addition, the mutant animals show pleiotropic effects that include facial dysmorphism, dwarfing, male sterility, anemia, and cystic choroid plexus. We previously fine-mapped the
kat
2J
mutation to a genetic distance of 0.28 ± 0.12 centimorgan between
D8Mit128
and
D8Mit129
. To identify the underlying molecular defect in this locus, we constructed an integrated genetic and physical map of the critical region surrounding the
kat
2J
mutation. Cloning and expression analysis of the transcribed sequences from this region identified
Nek1
, a NIMA (never in mitosis A)-related kinase as a candidate gene. Further analysis of the
Nek1
gene from both
kat
/
kat
and
kat
2J
/
kat
2J
mutant animals identified a partial internal deletion and a single-base insertion as the molecular basis for these mutations. The complex pleiotropic phenotypes seen in the homozygous mutant animals suggest that the NEK1 protein participates in different signaling pathways to regulate diverse cellular processes. Our findings identify a previously unsuspected role for
Nek1
in the kidney and open a new avenue for studying cystogenesis and identifying possible modes of therapy.
Identification of a human centrosomal calmodulin-binding protein that shares homology with pericentrin
Authors: Flory Mark R. , Moser Michael J. , Monnat Raymond J. , Davis Trisha N.
Published: July 2002

Eukaryotic chromosome segregation depends on the mitotic spindle apparatus, a bipolar array of microtubules nucleated from centrosomes. Centrosomal microtubule nucleation requires attachment of γ-tubulin ring complexes to a salt-insoluble centrosomal core, but the factor(s) underlying this attachment remains unknown. In budding yeast, this attachment is provided by the coiled-coil protein Spc110p, which links the yeast γ-tubulin complex to the core of the yeast centrosome. Here, we show that the large coiled-coil protein kendrin is a human orthologue of Spc110p. We identified kendrin by its C-terminal calmodulin-binding site, which shares homology with the Spc110p calmodulin-binding site. Kendrin localizes specifically to centrosomes throughout the cell cycle. N-terminal regions of kendrin share significant sequence homology with pericentrin, a previously identified murine centrosome component known to interact with γ-tubulin. In mitotic human breast carcinoma cells containing abundant centrosome-like structures, kendrin is found only at centrosomes associated with spindle microtubules.
Classification of fusiform neocortical interneurons based on unsupervised clustering
Authors: Cauli Bruno , Porter James T. , Tsuzuki Keisuke , Lambolez Bertrand , Rossier Jean , Quenet Brigitte , Audinat Etienne
Published: July 2002

A classification of fusiform neocortical interneurons (
n
= 60) was performed with an unsupervised cluster analysis based on the comparison of multiple electrophysiological and molecular parameters studied by patch-clamp and single-cell multiplex reverse transcription–PCR in rat neocortical acute slices. The multiplex reverse transcription–PCR protocol was designed to detect simultaneously the expression of GAD65, GAD67, calbindin, parvalbumin, calretinin, neuropeptide Y, vasoactive intestinal peptide (VIP), somatostatin (SS), cholecystokinin, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate,
N
-methyl-
d
-aspartate, and metabotropic glutamate receptor subtypes. Three groups of fusiform interneurons with distinctive features were disclosed by the cluster analysis. The first type of fusiform neuron (
n
= 12), termed regular spiking nonpyramidal (RSNP)-SS cluster, was characterized by a firing pattern of RSNP cells and by a high occurrence of SS. The second type of fusiform neuron (
n
= 32), termed RSNP-VIP cluster, predominantly expressed VIP and also showed firing properties of RSNP neurons with accommodation profiles different from those of RSNP-SS cells. Finally, the last type of fusiform neuron (
n
= 16) contained a majority of irregular spiking-VIPergic neurons. In addition, the analysis of glutamate receptors revealed cell-type-specific expression profiles. This study shows that combinations of multiple independent criteria define distinct neocortical populations of interneurons potentially involved in specific functions.
Recombinase-activating gene (RAG) 2-mediated V(D)J recombination is not essential for tumorigenesis in
Atm
-deficient mice
Authors: Petiniot Lisa K. , Weaver Zoë , Barlow Carrolee , Shen Rhuna , Eckhaus Michael , Steinberg Seth M. , Ried Thomas , Wynshaw-Boris Anthony , Hodes Richard J.
Published: July 2002

The majority of
Atm
-deficient mice die of malignant thymic lymphoma by 4–5 mo of age. Cytogenetic abnormalities in these tumors are consistently identified within the
Tcr α/δ
locus, suggesting that tumorigenesis is secondary to aberrant responses to double-stranded DNA breaks that occur during V(D)J recombination. Since V(D)J recombination is a recombinase-activating gene (RAG)-dependent process, we generated
Rag2
−/−
Atm
−/−
mice to assess the requirement for RAG-dependent recombination in thymic lymphomagenesis. In contrast to expectation, the data presented here indicate that development of malignant thymic lymphoma in
Atm
−/−
mice is not prevented by loss of RAG-2 and thus is not dependent on V(D)J recombination. Malignant thymic lymphomas in
Rag2
−/−
Atm
−/−
mice occurred at a lower frequency and with a longer latency as compared with
Atm
−/−
mice. Importantly, cytogenetic analysis of these tumors indicated that multiple chromosomal abnormalities occurred in each tumor, but that none of these involved the
Tcr α/δ
locus. Nonmalignant peripheral T cells from TCR-transgenic
Rag2
−/−
Atm
−/−
mice also revealed a substantial increase in translocation frequency, suggesting that these translocations are early events in the process of tumorigenesis. These data are consistent with the hypothesis that the major mechanism of tumorigenesis in
Atm
−/−
mice is via chromosomal translocations and other abnormalities that are secondary to aberrant responses to double-stranded DNA breaks. Furthermore, these data suggest that V(D)J recombination is a critical, but not essential, event during which
Atm
-deficient thymocytes are susceptible to developing chromosome aberrations that predispose to malignant transformation.
Acyl-homoserine lactone quorum sensing in Gram-negative bacteria: A signaling mechanism involved in associations with higher organisms
Authors: Parsek Matthew R. , Greenberg E. Peter
Published: July 2002

Recent advances in studies of bacterial gene expression have
brought the realization that cell-to-cell communication and community
behavior are critical for successful interactions with higher
organisms. Species-specific cell-to-cell communication is involved in
successful pathogenic or symbiotic interactions of a variety of
bacteria with plant and animal hosts. One type of cell–cell signaling
is acyl-homoserine lactone quorum sensing in Gram-negative bacteria.
This type of quorum sensing represents a dedicated communication system
that enables a given species to sense when it has reached a critical
population density in a host, and to respond by activating expression
of genes necessary for continued success in the host. Acyl-homoserine
lactone signaling in the opportunistic animal and plant pathogen
Pseudomonas aeruginosa
is a model for the relationships
among quorum sensing, pathogenesis, and community behavior. In the
P. aeruginosa
model, quorum sensing is required for
normal biofilm maturation and for virulence. There are multiple
quorum-sensing circuits that control the expression of dozens of
specific genes that represent potential virulence loci.
Ribozyme-mediated inhibition of HIV 1 suggests nucleolar trafficking of HIV-1 RNA
Authors: Michienzi Alessandro , Cagnon Laurence , Bahner Ingrid , Rossi John J.
Published: July 2002

The HIV regulatory proteins Tat and Rev have a nucleolar localization property in human cells. However, no functional role has been attributed to this localization. Recently it has been demonstrated that expression of Rev induces nucleolar relocalization of some protein factors involved in Rev export. Because the function of Rev is to bind HIV RNA and facilitate transport of singly spliced and unspliced RNA to the cytoplasm, it is likely that the nucleolus plays a critical role in HIV-1 RNA export. As a test for trafficking of HIV-1 RNAs into the nucleolus, a hammerhead ribozyme that specifically cleaves HIV-1 RNA was inserted into the body of the U16 small nucleolar RNA, resulting in accumulation of the ribozyme within the nucleoli of human cells. HeLa CD4
+
and T cells expressing this nucleolar localized ribozyme exhibit dramatically suppressed HIV-1 replication. The results presented here suggest a trafficking of HIV-1 RNA through the nucleoli of human cells, thus posing a different paradigm for lentiviral RNA processing.
Global mapping of meiotic recombination hotspots and coldspots in the yeast
Saccharomyces cerevisiae
Authors: Gerton Jennifer L. , DeRisi Joseph , Shroff Robert , Lichten Michael , Brown Patrick O. , Petes Thomas D.
Published: July 2002

In the yeast
Saccharomyces cerevisiae
, meiotic
recombination is initiated by double-strand DNA breaks (DSBs). Meiotic
DSBs occur at relatively high frequencies in some genomic regions
(hotspots) and relatively low frequencies in others (coldspots). We
used DNA microarrays to estimate variation in the level of nearby
meiotic DSBs for all 6,200 yeast genes. Hotspots were nonrandomly
associated with regions of high G + C base composition and certain
transcriptional profiles. Coldspots were nonrandomly associated with
the centromeres and telomeres.
Molecular basis for discriminating between normal and damaged bases by the human alkyladenine glycosylase, AAG
Authors: Lau Albert Y. , Wyatt Michael D. , Glassner Brian J. , Samson Leona D. , Ellenberger Tom
Published: July 2002

The human 3-methyladenine DNA glycosylase [alkyladenine DNA
glycosylase (AAG)] catalyzes the first step of base excision repair by
cleaving damaged bases from DNA. Unlike other DNA glycosylases that are
specific for a particular type of damaged base, AAG excises a
chemically diverse selection of substrate bases damaged by alkylation
or deamination. The 2.1-Å crystal structure of AAG complexed to DNA
containing 1,
N
6
-ethenoadenine suggests how
modified bases can be distinguished from normal DNA bases in the enzyme
active site. Mutational analyses of residues contacting the alkylated
base in the crystal structures suggest that the shape of the damaged
base, its hydrogen-bonding characteristics, and its aromaticity all
contribute to the selective recognition of damage by AAG.
What is the role of non-native intermediates of β-lactoglobulin in protein folding?
Authors: Chikenji George , Kikuchi Macoto
Published: July 2002

The mechanism of α→β transition in folding of
β-lactoglobulin is discussed based on free energy landscape analysis
of a long lattice model. It is found that helical propensity of
β-lactoglobulin is driven by conformational entropy and is
intrinsically coded in its native structure. We propose a view on a
role of folding intermediate, which is “on-pathway” but rich in
non-native structures. The present results suggest that the native
structure topology plays an important role in α→β transition.
Rapid nanopore discrimination between single polynucleotide molecules
Authors: Meller Amit , Nivon Lucas , Brandin Eric , Golovchenko Jene , Branton Daniel
Published: July 2002

A variety of different DNA polymers were electrophoretically driven through the nanopore of an α-hemolysin channel in a lipid bilayer. Single-channel recording of the translocation duration and current flow during traversal of individual polynucleotides yielded a unique pattern of events for each of the several polymers tested. Statistical data derived from this pattern of events demonstrate that in several cases a nanopore can distinguish between polynucleotides of similar length and composition that differ only in sequence. Studies of temperature effects on the translocation process show that translocation duration scales as ∼
T
−2
. A strong correlation exists between the temperature dependence of the event characteristics and the tendency of some polymers to form secondary structure. Because nanopores can rapidly discriminate and characterize unlabeled DNA molecules at low copy number, refinements of the experimental approach demonstrated here could eventually provide a low-cost high-throughput method of analyzing DNA polynucleotides.
Random GFP∷cDNA fusions enable visualization of subcellular structures in cells of
Arabidopsis
at a high frequency
Authors: Cutler Sean R. , Ehrhardt David W. , Griffitts Joel S. , Somerville Chris R.
Published: July 2002

We describe a general approach for identifying components of subcellular structures in a multicellular organism by exploiting the ability to generate thousands of independent transformants in
Arabidopsis thaliana
. A library of
Arabidopsis
cDNAs was constructed so that the cDNAs were inserted at the 3′ end of the green fluorescent protein (GFP) coding sequence. The library was introduced en masse into
Arabidopsis
by
Agrobacterium
-mediated transformation. Fluorescence imaging of 5,700 transgenic plants indicated that ≈2% of lines expressed a fusion protein with a different subcellular distribution than that of soluble GFP. About half of the markers identified were targeted to peroxisomes or other subcellular destinations by non-native coding sequence (i.e., out-of-frame cDNAs). This observation suggests that some targeting signals are of sufficiently low information content that they can be generated frequently by chance. The potential of the approach for identifying markers with unique dynamic processes is demonstrated by the identification of a GFP fusion protein that displays a cell-cycle regulated change in subcellular distribution. Our results indicate that screening GFP-fusion protein libraries is a useful approach for identifying and visualizing components of subcellular structures and their associated dynamics in higher plant cells.
Evolution of Antp-class genes and differential expression ofHydra Hox/paraHoxgenes in anterior patterning
Authors: Gauchat Dominique , Mazet Françoise , Berney Cédric , Schummer Michèl , Kreger Sylvia , Pawlowski Jan , Galliot Brigitte
Published: July 2002

The conservation of developmental functions exerted by Antp-class homeoproteins in protostomes and deuterostomes suggested that homologs with related functions are present in diploblastic animals. Our phylogenetic analyses showed that Antp-class homeodomains belong either to non-Hox or to Hox/paraHox families. Among the 13 non-Hox families, 9 have diploblastic homologs,Msx,Emx,Barx,Evx,Tlx,NK-2, andPrh/Hex,Not, andDlx, reported here. Among the Hox/paraHox, poriferan sequences were not found, and the cnidarian sequences formed at least five distinct cnox families. Two are significantly related to the paraHox Gsx (cnox-2) and the mox (cnox-5) sequences, whereas three display some relatedness to the Hox paralog groups 1 (cnox-1), 9/10 (cnox-3) and the paraHox cdx (cnox-4). IntermediateHox/paraHoxgenes (PG 3 to 8 andlox) did not have clear cnidarian counterparts. InHydra,cnox-1,cnox-2, andcnox-3were not found chromosomally linked within a 150-kb range and displayed specific expression patterns in the adult head. During regeneration,cnox-1was expressed as an early gene whatever the polarity, whereascnox-2was up-regulated later during head but not foot regeneration. Finally,cnox-3expression was reestablished in the adult head once it was fully formed. These results suggest that theHydragenes related to anteriorHox/paraHoxgenes are involved at different stages of apical differentiation. However, the positional information defining the oral/aboral axis inHydracannot be correlated strictly to that characterizing the anterior–posterior axis in vertebrates or arthropods.
CD4+T cells derived from B cell-deficient mice inhibit the establishment of peripheral B cell pools
Authors: Baumgarth Nicole , Jager Gina C. , Herman Ometa C. , Herzenberg Leonard A. , Herzenberg Leonore A.
Published: July 2002

We demonstrate that adoptive transfer of peritoneal cavity B cells fails to replenish the peripheral B-1 cells in adult B cell-deficient (μ−/−) mice but does replenish adult RAG-1−/−mice. We show that this lack of self-replenishment in μ−/−mice is mediated by strongly inhibitory, radiation-sensitive CD4+T cells that also function in cotransfer studies to block the reconstitution of B-1 cells and inhibit accumulation of bone marrow-derived B-2 cells in the periphery in irradiated recipients. CD8+T cells from μ−/−do not mediate this inhibition. The inhibitory CD4+T cells develop early in life, because B-1 cell replenishment occurs normally when B-1 cells are transferred into μ−/−neonates. Thus, we conclude that the presence of B cells in the neonate conditions the CD4+T-cell population to permit the establishment and maintenance of normal B cell pools throughout life.
Fitness effects of advantageous mutations in evolving
Escherichia coli
populations
Authors: Imhof Marianne , Schlötterer Christian
Published: July 2002

The central role of beneficial mutations for adaptive processes in
natural populations is well established. Thus, there has been a
long-standing interest to study the nature of beneficial mutations.
Their low frequency, however, has made this class of mutations almost
inaccessible for systematic studies. In the absence of experimental
data, the distribution of the fitness effects of beneficial mutations
was assumed to resemble that of deleterious mutations. For an
experimental proof of this assumption, we used a novel marker system to
trace adaptive events in an evolving
Escherichia coli
culture and to determine the selective advantage of those beneficial
mutations. Ten parallel cultures were propagated for about 1,000
generations by serial transfer, and 66 adaptive events were identified.
From this data set, we estimate the rate of beneficial mutations to be
4 × 10
−9
per cell and generation. Consistent with an
exponential distribution of the fitness effects, we observed a large
fraction of advantageous mutations with a small effect and only few
with large effect. The mean selection coefficient of advantageous
mutations in our experiment was 0.02.