bluuebunny/crossref_metadata_2025 huggingface dataset:
Poly(ADP-ribose) glycohydrolase mediates oxidative and excitotoxic neuronal death
Authors: Ying Weihai , Sevigny Mary B. , Chen Yongmei , Swanson Raymond A.
Published: July 2002

Excessive activation of poly(ADP-ribose) polymerase 1 (PARP1) leads to NAD+depletion and cell death during ischemia and other conditions that generate extensive DNA damage. When activated by DNA strand breaks, PARP1 uses NAD+as substrate to form ADP-ribose polymers on specific acceptor proteins. These polymers are in turn rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG), a ubiquitously expressed exo- and endoglycohydrolase. In this study, we examined the role of PARG in the PARP1-mediated cell death pathway. Mouse neuron and astrocyte cultures were exposed to hydrogen peroxide,N-methyl-d-aspartate (NMDA), or the DNA alkylating agent,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Cell death in each condition was markedly reduced by the PARP1 inhibitor benzamide and equally reduced by the PARG inhibitors gallotannin and nobotanin B. The PARP1 inhibitor benzamide and the PARG inhibitor gallotannin both prevented the NAD+depletion that otherwise results from PARP1 activation by MNNG or H2O2. However, these agents had opposite effects on protein poly(ADP-ribosyl)ation. Immunostaining for poly(ADP-ribose) on Western blots and neuron cultures showed benzamide to decrease and gallotannin to increase poly(ADP-ribose) accumulation during MNNG exposure. These results suggest that PARG inhibitors do not inhibit PARP1 directly, but instead prevent PARP1-mediated cell death by slowing the turnover of poly(ADP-ribose) and thus slowing NAD+consumption. PARG appears to be a necessary component of the PARP-mediated cell death pathway, and PARG inhibitors may have promise as neuroprotective agents.
Identification of the proton pathway in bacterial reaction centers: Both protons associated with reduction of Q
B
to Q
B
H
2
share a common entry point
Authors: Ädelroth Pia , Paddock Mark L. , Sagle Laura B. , Feher George , Okamura Melvin Y.
Published: July 2002

The reaction center from
Rhodobacter sphaeroides
uses light energy for the reduction and protonation of a quinone
molecule, Q
B
. This process involves the transfer of two
protons from the aqueous solution to the protein-bound Q
B
molecule. The second proton, H
+
(2), is supplied to
Q
B
by Glu-L212, an internal residue protonated in response
to formation of Q
A
−
and Q
B
−
. In this
work, the pathway for H
+
(2) to Glu-L212 was studied by
measuring the effects of divalent metal ion binding on the protonation
of Glu-L212, which was assayed by two types of processes. One was
proton uptake from solution after the one-electron reduction of
Q
A
(DQ
A
→D
+
Q
A
−
)
and Q
B
(DQ
B
→D
+
Q
B
−
), studied by
using pH-sensitive dyes. The other was the electron transfer
k
AB
(1)
(Q
A
−
Q
B
→Q
A
Q
B
−
).
At pH 8.5, binding of Zn
2+
, Cd
2+
, or
Ni
2+
reduced the rates of proton uptake upon
Q
A
−
and Q
B
−
formation as well as
k
AB
(1)
by ≈an order of magnitude,
resulting in similar final values, indicating that there is a common
rate-limiting step. Because D
+
Q
A
−
is
formed 10
5
-fold faster than the induced proton uptake, the
observed rate decrease must be caused by an inhibition of the proton
transfer. The Glu-L212→Gln mutant reaction centers displayed greatly
reduced amplitudes of proton uptake and exhibited no changes in rates
of proton uptake or electron transfer upon Zn
2+
binding.
Therefore, metal binding specifically decreased the rate of proton
transfer to Glu-L212, because the observed rates were decreased only
when proton uptake by Glu-L212 was required. The entry point for the
second proton H
+
(2) was thus identified to be the same as
for the first proton H
+
(1), close to the metal binding
region Asp-H124, His-H126, and His-H128.
Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals
Authors: Moskovitz Jackob , Bar-Noy Shoshana , Williams Wesley M. , Requena Jesus , Berlett Barbara S. , Stadtman Earl R.
Published: July 2002

Oxidation of proteins by reactive oxygen species is associated with aging, oxidative stress, and many diseases. Although free and protein-bound methionine residues are particularly sensitive to oxidation to methionine sulfoxide derivatives, these oxidations are readily repaired by the action of methionine sulfoxide reductase (MsrA). To gain a better understanding of the biological roles of MsrA in metabolism, we have created a strain of mouse that lacks the
MsrA
gene. Compared with the wild type, this mutant: (
i
) exhibits enhanced sensitivity to oxidative stress (exposure to 100% oxygen); (
ii
) has a shorter lifespan under both normal and hyperoxic conditions; (
iii
) develops an atypical (tip-toe) walking pattern after 6 months of age; (
iv
) accumulates higher tissue levels of oxidized protein (carbonyl derivatives) under oxidative stress; and (
v
) is less able to up-regulate expression of thioredoxin reductase under oxidative stress. It thus seems that MsrA may play an important role in aging and neurological disorders.
3′ deletions cause aniridia by preventing
PAX6
gene expression
Authors: Lauderdale James D. , Wilensky Jonathan S. , Oliver Edward R. , Walton David S. , Glaser Tom
Published: July 2002

Aniridia is a panocular human eye malformation caused by
heterozygous null mutations within
PAX6
, a paired-box
transcription factor, or cytogenetic deletions of chromosome 11p13 that
encompass
PAX6
. Chromosomal rearrangements also have
been described that disrupt 11p13 but spare the
PAX6
transcription unit in two families with aniridia. These presumably
cause a loss of gene expression, by removing positive
cis
regulatory elements or juxtaposing negative DNA
sequences. We report two submicroscopic
de novo
deletions of 11p13 that cause aniridia but are located >11 kb from the
3′ end of
PAX6
. The clinical manifestations are
indistinguishable from cases with chain-terminating mutations in the
coding region. Using human × mouse retinoblastoma somatic cell
hybrids, we show that
PAX6
is transcribed only from the
normal allele but not from the deleted chromosome 11 homolog. Our
findings suggest that remote 3′ regulatory elements are required for
initiation of
PAX6
expression.
Traumatic brain damage prevented by the non-N-methyl-D-aspartate antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f] quinoxaline.
Authors: Bernert H , Turski L
Published: July 2002

The mechanisms of neuronal degeneration following traumatic head injury are not well understood and no adequate treatment is currently available for the prevention of traumatic brain damage in humans. Traumatic head injury leads to primary (at impact) and secondary (distant) damage to the brain. Mechanical percussion of the rat cortex mimics primary damage seen after traumatic head injury in humans; no animal model mimicking the secondary damage following traumatic head injury has yet been established. Rats subjected to percussion trauma of the cortex showed primary damage in the cortex and secondary damage in the hippocampus. Morphometric analysis demonstrated that both cortical and hippocampal damage was mitigated by pretreatment with either the N-methyl-D-aspartate (NMDA) antagonist 3-((+/-)- 2-carboxypiperazin-4-yl)-propyl-1-phosphonate (CPP) or the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX). Neither treatment prevented primary damage in the cortex when therapy was started after trauma. Surprisingly, delayed treatment of rats with NBQX, but not with CPP, beginning between 1 and 7 hr after trauma prevented hippocampal damage. No protection was seen when therapy with NBQX was started 10 hr after trauma. These data indicate that both NMDA- and non-NMDA-dependent mechanisms contribute to the development of primary damage in the cortex, whereas non-NMDA mechanisms are involved in the evolution of secondary damage in the hippocampus in rats subjected to traumatic head injury. The wide therapeutic time-window documented for NBQX suggests that antagonism at non-NMDA receptors may offer a novel therapeutic approach for preventing deterioration of the brain after head injury.
Role of the p21 cyclin-dependent kinase inhibitor in limiting intimal cell proliferation in response to arterial injury.
Authors: Yang Z Y , Simari R D , Perkins N D , San H , Gordon D , Nabel G J , Nabel E G
Published: July 2002

Arterial injury induces a series of proliferative, vasoactive, and inflammatory responses that lead to vascular proliferative diseases, including atherosclerosis and restenosis. Although several factors have been defined which stimulate this process in vivo, the role of specific cellular gene products in limiting this response is not well understood. The p21 cyclin-dependent kinase inhibitor affects cell cycle progression, senescence, and differentiation in transformed cells, but its expression in injured blood vessels has not been investigated. In this study, we report that p21 protein is induced in porcine arteries following balloon catheter injury and suggest that p21 is likely to play a role in limiting arterial cell proliferation in vivo. Vascular endothelial and smooth muscle cell growth was arrested through the ability of p21 to inhibit progression through the G1 phase of the cell cycle. Following injury to porcine arteries, p21 gene product was detected in the neointima and correlated inversely with the location and kinetics of intimal cell proliferation. Direct gene transfer of p21 using an adenoviral vector into balloon injured porcine arteries inhibited the development of intimal hyperplasia. Taken together, these findings suggest that p21, and possibly related cyclin-dependent kinase inhibitors, may normally regulate cellular proliferation following arterial injury, and strategies to increase its expression may prove therapeutically beneficial in vascular diseases.
Clusters of charged residues in protein three-dimensional structures.
Authors: Zhu Z Y , Karlin S
Published: July 2002

Statistically significant charge clusters (basic, acidic, or of mixed charge) in tertiary protein structures are identified by new methods from a large representative collection of protein structures. About 10% of protein structures show at least one charge cluster, mostly of mixed type involving about equally anionic and cationic residues. Positive charge clusters are very rare. Negative (or histidine-acidic) charge clusters often coordinate calcium, or magnesium or zinc ions [e.g., thermolysin (PDB code: 3tln), mannose-binding protein (2msb), aminopeptidase (1amp)]. Mixed-charge clusters are prominent at interchain contacts where they stabilize quaternary protein formation [e.g., glutathione S-transferase (2gst), catalase (8act), and fructose-1,6-bisphosphate aldolase (1fba)]. They are also involved in protein-protein interaction and in substrate binding. For example, the mixed-charge cluster of aspartate carbamoyl-transferase (8atc) envelops the aspartate carbonyl substrate in a flexible manner (alternating tense and relaxed states) where charge associations can vary from weak to strong. Other proteins with charge clusters include the P450 cytochrome family (BM-3, Terp, Cam), several flavocytochromes, neuraminidase, hemagglutinin, the photosynthetic reaction center, and annexin. In each case in Table 2 we discuss the possible role of the charge clusters with respect to protein structure and function.
Telomeric repeats (TTAGGC)n are sufficient for chromosome capping function in Caenorhabditis elegans.
Authors: Wicky C , Villeneuve A M , Lauper N , Codourey L , Tobler H , Müller F
Published: July 2002

Telomeres are specialized structures located at the ends of linear eukaryotic chromosomes that ensure their complete replication and protect them from fusion and degradation. We report here the characterization of the telomeres of the nematode Caenorhabditis elegans. We show that the chromosomes terminate in 4-9 kb of tandem repeats of the sequence TTAGGC. Furthermore, we have isolated clones corresponding to 11 of the 12 C. elegans telomeres. Their subtelomeric sequences are all different from each other, demonstrating that the terminal TTAGGC repeats are sufficient for general chromosomal capping functions. Finally, we demonstrate that the me8 meiotic mutant, which is defective in X chromosome crossing over and segregation, bears a terminal deficiency, that was healed by the addition of telomeric repeats, presumably by the activity of a telomerase enzyme. The 11 cloned telomeres represent an important advance for the completion of the physical map and for the determination of the entire sequence of the C. elegans genome.
Differential inhibition of the Fas- and granule-mediated cytolysis pathways by the orthopoxvirus cytokine response modifier A/SPI-2 and SPI-1 protein.
Authors: Macen J L , Garner R S , Musy P Y , Brooks M A , Turner P C , Moyer R W , McFadden G , Bleackley R C
Published: July 2002

Cytotoxic T lymphocytes are important effectors of antiviral immunity, and they induce target cell death either by secretion of cytoplasmic granules containing perforin and granzymes or by signaling through the Fas cell surface antigen. Although it is not known whether the granule-mediated and Fas-mediated cytolytic mechanisms share common components, proteinase activity has been implicated as an important feature of both pathways. The orthopoxviruses cowpox virus and rabbitpox virus each encode three members of the serpin family of proteinase inhibitors, designated SPI-1, SPI-2, and SPI-3. Of these, SPI-2 (also referred to as cytokine response modifier A in cowpox virus) has been shown to inhibit the proteolytic activity of both members of the interleukin 1 beta converting enzyme family and granzyme B. We report here that cells infected with cowpox or rabbitpox viruses exhibit resistance to cytolysis by either cytolytic mechanism. Whereas mutation of the cytokine response modifier A/SPI-2 gene was necessary to relieve inhibition of Fasmediated cytolysis, in some cell types mutation of SPI-1, in addition to cytokine response modifier A/SPI-2, was necessary to completely abrogate inhibition. In contrast, viral inhibition of granule-mediated killing was unaffected by mutation of cytokine response modifier A/SPI-2 alone, and it was relieved only when both the cytokine response modifier A/SPI-2 and SPI-1 genes were inactivated. These results suggest that an interleukin 1 beta converting enzyme-like enzymatic activity is involved in both killing mechanisms and indicate that two viral proteins, SPI-1 and cytokine response modifier A/SPI-2, are necessary to inhibit both cytolysis pathways.
DNA rearrangement mediated by inverted repeats.
Authors: Bi X , Liu L F
Published: July 2002

Inverted repeats of DNA are widespread in the genomes of eukaryotes and prokaryotes and can mediate genome rearrangement. We studied rearrangement mediated by plasmid-borne inverted repeats in Escherichia coli. We show that inverted repeats can mediate an efficient and recA-independent recombination event. Surprisingly, the product of this recombination is not that of simple inversion between the inverted repeats, but almost exclusively an unusual head-to-head dimer with complex DNA rearrangement. Moreover, this recombination is dramatically reduced by increasing the distance separating the repeats. These results can be readily explained by a model involving reciprocal switching of the leading and lagging strands of DNA replication within the inverted repeats, which leads to the formation of a Holliday junction. Reciprocal strand switching during DNA replication might be a common mechanism for genome rearrangement associated with inverted duplication.
The Drosophila p70s6k homolog exhibits conserved regulatory elements and rapamycin sensitivity.
Authors: Stewart M J , Berry C O , Zilberman F , Thomas G , Kozma S C
Published: July 2002

The protein p70s6k/p85s6k lies on a mitogen-stimulated signaling pathway and plays a key role in G1 progression of the cell cycle. Activation of this enzyme is mediated by a complex set of phosphorylation events, which has largely contributed to the difficulty in identifying the upstream kinases that mediate p70s6k activation. Genetics has proved a powerful complementary approach for such problems, providing an alternative means to identify components of signaling cascades and their functional end targets. As a first step toward implementing such an approach, we have cloned cDNAs encoding the Drosophila melanogaster p70s6k homolog (Dp70s6k). Dp70s6k is encoded by a single gene, which generates three mRNA transcripts and exhibits an overall identity of 78% in the catalytic domain with its mammalian counterpart. Importantly, this high identity extends beyond the catalytic domain to the N terminus, linker region, and the autoinhibitory domain. Furthermore, all the critical phosphorylation sites required for mammalian p70s6k activation are conserved within these same domains of Dp70s6k. Chief amongst these conserved sites are those associated with the selective rapamycin-induced p70s6k dephosphorylation and inactivation. Consistent with this observation, analysis of total S6 kinase activity in fractionated Drosophila Schneider line 2 cell extracts reveals two peaks of activity, only one of which is rapamycin sensitive. By employing a monospecific polyclonal antibody generated against Dp70s6k, we show that the cloned DP70s6k cDNA has identity with only the rapamycin sensitive peak, suggesting that this biological system would be useful in determining not only the mechanism of p70s6k activation, but also in elucidating the mechanism by which rapamycin acts to inhibit cell growth.
Sex ratio adjustment in relation to paternal attractiveness in a wild bird population.
Authors: Ellegren H , Gustafsson L , Sheldon B C
Published: July 2002

When the relative fitness of sons and daughters differs, sex-allocation theory predicts that it would be adaptive for individuals to adjust their investment in different sexes of offspring. Sex ratio adjustment by females in response to the sexual attractiveness of their mate would be an example of this. In vertebrates the existence of this form of sex ratio adjustment is controversial and may be confounded with sex-biased mortality, particularly in sexually size-dimorphic species. Here we use PCR amplification of a conserved W-chromosome-linked gene to show that the sex ratio within broods of a natural population of sexually size-monomorphic collared flycatchers Ficedula albicollis is related to the size of their father's forehead patch, a heritable secondary sexual character implicated in female choice. Experimental manipulations of paternal investment, which influence the size of his character in future breeding attempts, result in corresponding changes in the sex ratio of offspring born to males in those breeding attempts. In contrast, manipulations of maternal investment have no effect on future sex ratios, and there is no relationship between variables predicting the reproductive value of the brood and nestling sex ratio. Analysis of recruitment of offspring reveals similar patterns of sex ratio bias. The results suggest that female collared flycatchers be able to adjust the sex ratio of eggs ovulated in response to the phenotype of their mate. This finding is most consistent with "genetic quality" models of sexual selection.
An
in vivo
pathway for disulfide bond isomerization in
Escherichia coli
Authors: Rietsch Arne , Belin Dominique , Martin Nancy , Beckwith Jonathan
Published: July 2002

Biochemical studies have shown that the periplasmic protein
disulfide oxidoreductase DsbC can isomerize aberrant disulfide bonds.
Here we present the first evidence for an
in vivo
role
of DsbC in disulfide bond isomerization. Furthermore, our data suggest
that the enzymes DsbA and DsbC play distinct roles in the cell in
disulfide bond formation and isomerization, respectively. We have shown
that mutants in
dsbC
display a defect in disulfide bond
formation specific for proteins with multiple disulfide bonds. The
defect can be complemented by the addition of reduced
dithiothreitol to the medium, suggesting that absence of DsbC
results in accumulation of misoxidized proteins. Mutations in the
dipZ
and
trxA
genes have similar
phenotypes. We propose that DipZ, a cytoplasmic membrane protein with a
thioredoxin-like domain, and thioredoxin, the product of the
trxA
gene, are components of a pathway for maintaining
DsbC active as a protein disulfide bond isomerase.
The DNA damage response in DNA-dependent protein kinase-deficient SCID mouse cells: Replication protein A hyperphosphorylation and p53 induction
Authors: Fried Laura M. , Koumenis Constantinos , Peterson Scott R. , Green Susannah L. , van Zijl Pierre , Allalunis-Turner Joan , Chen David J. , Fishel Richard , Giaccia Amato J. , Brown J. Martin , Kirchgessner Cordula U.
Published: July 2002

Severe combined immunodeficient (SCID) mice display an increased
sensitivity to ionizing radiation compared with the parental, C.B-17,
strain due to a deficiency in DNA double-strand break repair. The
catalytic subunit of DNA-dependent protein kinase
(DNA-PK
CS
) has previously been identified as a strong
candidate for the SCID gene. DNA-PK phosphorylates many proteins
in vitro
, including p53 and replication protein A (RPA),
two proteins involved in the response of cells to DNA damage. To
determine whether p53 and RPA are also substrates of DNA-PK
in
vivo
following DNA damage, we compared the response of SCID and
MO59J (human DNA-PK
cs
-deficient glioblastoma) cells with
their respective wild-type parents following ionizing radiation. Our
findings indicate that (
i
) p53 levels are increased in
SCID cells following ionizing radiation, and (
ii
) RPA
p34 is hyperphosphorylated in both SCID cells and MO59J cells following
ionizing radiation. The hyperphosphorylation of RPA p34
in
vivo
is concordant with a decrease in the binding of RPA to
single-stranded DNA in crude extracts derived from both C.B-17 and SCID
cells. These results suggest that DNA-PK is not the only kinase capable
of phosphorylating RPA. We conclude that the DNA damage response
involving p53 and RPA is not associated with the defect in DNA repair
in SCID cells and that the physiological substrate(s) for DNA-PK
essential for DNA repair has not yet been identified.
Conversion of DNA gyrase into a conventional type II topoisomerase
Authors: Kampranis Sotirios C. , Maxwell Anthony
Published: July 2002

DNA gyrase is unique among topoisomerases in its ability to
introduce negative supercoils into closed-circular DNA. We have
demonstrated that deletion of the C-terminal DNA-binding domain of the
A subunit of gyrase gives rise to an enzyme that cannot supercoil DNA
but relaxes DNA in an ATP-dependent manner. Novobiocin, a competitive
inhibitor of ATP binding by gyrase, inhibits this reaction. The
truncated enzyme, unlike gyrase, does not introduce a
right-handed wrap when bound to DNA and stabilizes DNA crossovers;
characteristics reminiscent of conventional type II topoisomerases.
This new enzyme form can decatenate DNA circles with increased
efficiency compared with intact gyrase and, as a result, can complement
the temperature-sensitive phenotype of a
parC
ts
mutant. Thus these results suggest
that the unique properties of DNA gyrase are attributable to the
wrapping of DNA around the C-terminal DNA-binding domains of the A
subunits and provide an insight into the mechanism of type II
topoisomerases.
Arrest of neuronal migration by excitatory amino acids in hamster developing brain
Authors: Marret Stéphane , Gressens Pierre , Evrard Philippe
Published: July 2002

The influence of the excitotoxic cascade on the developing brain was investigated using ibotenate, a glutamatergic agonist of bothN-methyl-d-aspartate (NMDA) ionotropic receptors and metabotropic receptors. Injected in the neopallium of the golden hamster at the time of production of neurons normally destined for layers IV, III, and II, ibotenate induces arrests of migrating neurons at different distances from the germinative zone within the radial migratory corridors. The resulting cytoarchitectonic patterns include periventricular nodular heterotopias, subcortical band heterotopias, and intracortical arrests of migrating neurons. The radial glial cells and the extracellular matrix are free of detectable damage that could suggest a defect in their guiding role. The migration disorders are prevented by coinjection ofdl-2-amino-7-phosphoheptanoic acid, an NMDA ionotropic antagonist, but are not prevented by coinjection ofl(+)-2-amino-3-phosphonopropionic acid, a metabotropic antagonist. This implies that an excess of ionic influx through the NMDA channels of neurons alters the metabolic pathways supporting neuronal migration. Ibotenate, a unique molecular trigger of the excitotoxic cascade, produces a wide spectrum of abnormal neuronal migration patterns recognized in mammals, including the neocortical deviations encountered in the human brain.
The thyroid hormone-induced tail resorption program during Xenopus laevis metamorphosis.
Authors: Brown D D , Wang Z , Furlow J D , Kanamori A , Schwartzman R A , Remo B F , Pinder A
Published: July 2002

Genes that are up- and down-regulated by thyroid hormone in the tail resorption program of Xenopus laevis have been isolated by a gene expression screen, sequenced, and identified in the GenBank data base. The entire program is estimated to consist of fewer than 35 up-regulated and fewer than 10 down-regulated genes; 17 and 4 of them, respectively, have been isolated and characterized. Up-regulated genes whose function can be predicted on the basis of their sequence include four transcription factors (including one of the thyroid hormone receptors), an extracellular matrix component (fibronectin) and membrane receptor (integrin), four proteinases, a deiodinase that degrades thyroid hormone, and a protein that binds the hypothalamic corticotropin-releasing factor, which has been implicated in controlling thyroid hormone synthesis in Xenopus tadpoles. All four down-regulated genes encode extracellular proteins that are expressed in tadpole epidermis. This survey of the program provides insights into the biology of metamorphosis.
Mapping striate and extrastriate visual areas in human cerebral cortex.
Authors: DeYoe E A , Carman G J , Bandettini P , Glickman S , Wieser J , Cox R , Miller D , Neitz J
Published: July 2002

Functional magnetic resonance imaging (fMRI) was used to identify and map the representation of the visual field in seven areas of human cerebral cortex and to identify at least two additional visually responsive regions. The cortical locations of neurons responding to stimulation along the vertical or horizontal visual field meridia were charted on three-dimensional models of the cortex and on unfolded maps of the cortical surface. These maps were used to identify the borders among areas that would be topographically homologous to areas V1, V2, V3, VP, and parts of V3A and V4 of the macaque monkey. Visually responsive areas homologous to the middle temporal/medial superior temporal area complex and unidentified parietal visual areas were also observed. The topography of the visual areas identified thus far is consistent with the organization in macaque monkeys. However, these and other findings suggest that human and simian cortical organization may begin to differ in extrastriate cortex at, or beyond, V3A and V4.
In vitroselection and evolution of functional proteins by using ribosome display
Authors: Hanes Jozef , Plückthun Andreas
Published: July 2002

We report here a system with which a correctly folded complete protein and its encoding mRNA both remain attached to the ribosome and can be enriched for the ligand-binding properties of the native protein. We have selected a single-chain fragment (scFv) of an antibody 108-fold by five cycles of transcription, translation, antigen-affinity selection, and PCR. The selected scFv fragments all mutatedin vitroby acquiring up to four unrelated amino acid exchanges over the five generations, but they remained fully compatible with antigen binding. Libraries of native folded proteins can now be screened and made to evolve in a cell-free system without any transformation or constraints imposed by the host cell.
P
element insertion-dependent gene activation in the
Drosophila
eye
Authors: Hay Bruce A. , Maile Randy , Rubin Gerald M.
Published: July 2002

Insights into the function of a gene can be gained in multiple ways, including loss-of-function phenotype, sequence similarity, expression pattern, and by the consequences of its misexpression. Analysis of the phenotypes produced by expression of a gene at an abnormal time, place, or level may provide clues to a gene’s function when other approaches are not illuminating. Here we report that an eye-specific, enhancer–promoter present in the
P
element expression vector pGMR is able to drive high level expression in the eye of genes near the site of
P
element insertion. Cell fate determination, differentiation, proliferation, and death are essential for normal eye development. Thus the ability to carry out eye-specific misexpression of a significant fraction of genes in the genome, given the dispensability of the eye for viability and fertility of the adult, should provide a powerful approach for identifying regulators of these processes. To test this idea we carried out two overexpression screens for genes that function to regulate cell death. We screened for insertion-dependent dominant phenotypes in a wild-type background, and for dominant modifiers of a
reaper
overexpression-induced small eye phenotype. Multiple chromosomal loci were identified, including an insertion 5′ to
hid
, a potent inducer of apoptosis, and insertions 5′ to DIAP1, a cell death suppressor. To facilitate the cloning of genes near the
P
element insertion new misexpression vectors were created. A screen with one of these vectors identified
eagle
as a suppressor of a rough eye phenotype associated with overexpression of an activated
Ras1
gene.
Glial cell line-derived neurotrophic factor-dependent RET activation can be mediated by two different cell-surface accessory proteins
Authors: Sanicola M. , Hession C. , Worley D. , Carmillo P. , Ehrenfels C. , Walus L. , Robinson S. , Jaworski G. , Wei H. , Tizard R. , Whitty A. , Pepinsky R. B. , Cate R. L.
Published: July 2002

Glial cell line-derived neurotrophic factor (GDNF)-dependent activation of the tyrosine kinase receptor RET is necessary for kidney and enteric neuron development, and mutations in
RET
are associated with human diseases. Activation of RET by GDNF has been shown to require an accessory component, GDNFR-α (RETL1). We report the isolation and characterization of rat and human cDNAs for a novel cell-surface associated accessory protein, RETL2, that shares 49% identity with RETL1. Both RETL1 and RETL2 can mediate GDNF dependent phosphorylation of RET, but they exhibit different patterns of expression in fetal and adult tissues. The most striking differences in expression observed were in the adult central and peripheral nervous systems. In addition, the mechanisms by which the two accessory proteins facilitate the activation of RET by GDNF are quite distinct.
In vitro
binding experiments with soluble forms of RET, RETL1 and RETL2 demonstrate that while RETL1 binds GDNF tightly to form a membrane-associated complex which can then interact with RET, RETL2 only forms a high affinity complex with GDNF in the presence of RET. This strong RET dependence of the binding of RETL2 to GDNF was confirmed by FACS analysis on RETL1 and RETL2 expressing cells. Together with the recent discovery of a GDNF related protein, neurturin, these data raise the possibility that RETL1 and RETL2 have distinctive roles during development and in the nervous system of the adult.
RETL1
and
RETL2
represent new candidate susceptibility genes and/or modifier loci for RET-associated diseases.
Identification of a new mammalian centrin gene, more closely related to
Saccharomyces cerevisiae
CDC31 gene
Authors: Middendorp Sandrine , Paoletti Anne , Schiebel Elmar , Bornens Michel
Published: July 2002

Among the numerous centrin isoforms identified by two-dimensional gel electrophoresis in human cells, an acidic and slow-migrating isoform is particularly enriched in a centrosome fraction. We report here that this isoform specifically reacts with antibodies raised against
Saccharomyces cerevisiae
Cdc31p and is present, as other centrin isoforms, in the distal lumen of centrioles. It is encoded by a new centrin gene, which we propose to name HsCEN3 (
Homo sapiens
centrin gene 3). This gene is more closely related to the yeast CDC31 gene, and shares less identity with algae centrin than HsCEN1 and HsCEN2. A murine CDC31-related gene was also found that shows 98% identity and 100% similarity with HsCEN3, demonstrating a higher interspecies conservation than the murine centrin gene MmCEN1 (
Mus musculus
centrin gene 1) with either HsCEN1, or HsCEN2. Finally, immunological data suggest that a CDC31-related gene could exist in amphibians and echinoderms as well. All together, our data suggest the existence of two divergent protein subfamilies in the current centrin family, which might be involved in distinct centrosome-associated functions. The possible implication of this new mammalian centrin gene in centrosome duplication is discussed.
Detecting immigration by using multilocus genotypes
Authors: Rannala Bruce , Mountain Joanna L.
Published: July 2002

Immigration is an important force shaping the social structure, evolution, and genetics of populations. A statistical method is presented that uses multilocus genotypes to identify individuals who are immigrants, or have recent immigrant ancestry. The method is appropriate for use with allozymes, microsatellites, or restriction fragment length polymorphisms (RFLPs) and assumes linkage equilibrium among loci. Potential applications include studies of dispersal among natural populations of animals and plants, human evolutionary studies, and typing zoo animals of unknown origin (for use in captive breeding programs). The method is illustrated by analyzing RFLP genotypes in samples of humans from Australian, Japanese, New Guinean, and Senegalese populations. The test has power to detect immigrant ancestors, for these data, up to two generations in the past even though the overall differentiation of allele frequencies among populations is low.
A novel virus in swine is closely related to the human hepatitis E virus
Authors: Meng Xiang-Jin , Purcell Robert H. , Halbur Patrick G. , Lehman James R. , Webb Dale M. , Tsareva Tatiana S. , Haynes Joseph S. , Thacker Brad J. , Emerson Suzanne U.
Published: July 2002

A novel virus, designated swine hepatitis E virus (swine HEV), was identified in pigs. Swine HEV crossreacts with antibody to the human HEV capsid antigen. Swine HEV is a ubiquitous agent and the majority of swine ≥3 months of age in herds from the midwestern United States were seropositive. Young pigs naturally infected by swine HEV were clinically normal but had microscopic evidence of hepatitis, and developed viremia prior to seroconversion. The entire ORFs 2 and 3 were amplified by reverse transcription–PCR from sera of naturally infected pigs. The putative capsid gene (ORF2) of swine HEV shared about 79–80% sequence identity at the nucleotide level and 90–92% identity at the amino acid level with human HEV strains. The small ORF3 of swine HEV had 83–85% nucleotide sequence identity and 77–82% amino acid identity with human HEV strains. Phylogenetic analyses showed that swine HEV is closely related to, but distinct from, human HEV strains. The discovery of swine HEV not only has implications for HEV vaccine development, diagnosis, and biology, but also raises a potential public health concern for zoonosis or xenozoonosis following xenotransplantation with pig organs.
Functional activation in motor cortex reflects the direction and the degree of handedness
Authors: Dassonville Paul , Zhu Xiao-Hong , Ugurbil Kâmil , Kim Seong-Gi , Ashe James
Published: July 2002

Handedness is the clearest example of behavioral lateralization in humans. It is not known whether the obvious asymmetry manifested by hand preference is associated with similar asymmetry in brain activation during movement. We examined the functional activation in cortical motor areas during movement of the dominant and nondominant hand in groups of right-handed and left-handed subjects and found that use of the dominant hand was associated with a greater volume of activation in the contralateral motor cortex. Furthermore, there was a separate relation between the degree of handedness and the extent of functional lateralization in the motor cortex. The patterns of functional activation associated with the direction and degree of handedness suggest that these aspects are independent and are coded separately in the brain.
Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure
Authors: Maniotis Andrew J. , Chen Christopher S. , Ingber Donald E.
Published: July 2002

We report here that living cells and nuclei are hard-wired such that a mechanical tug on cell surface receptors can immediately change the organization of molecular assemblies in the cytoplasm and nucleus. When integrins were pulled by micromanipulating bound microbeads or micropipettes, cytoskeletal filaments reoriented, nuclei distorted, and nucleoli redistributed along the axis of the applied tension field. These effects were specific for integrins, independent of cortical membrane distortion, and were mediated by direct linkages between the cytoskeleton and nucleus. Actin microfilaments mediated force transfer to the nucleus at low strain; however, tearing of the actin gel resulted with greater distortion. In contrast, intermediate filaments effectively mediated force transfer to the nucleus under both conditions. These filament systems also acted as molecular guy wires to mechanically stiffen the nucleus and anchor it in place, whereas microtubules acted to hold open the intermediate filament lattice and to stabilize the nucleus against lateral compression. Molecular connections between integrins, cytoskeletal filaments, and nuclear scaffolds may therefore provide a discrete path for mechanical signal transfer through cells as well as a mechanism for producing integrated changes in cell and nuclear structure in response to changes in extracellular matrix adhesivity or mechanics.
Molecular determinants of inactivation and G protein modulation in the intracellular loop connecting domains I and II of the calcium channel α
1A
subunit
Authors: Herlitze Stefan , Hockerman Gregory H. , Scheuer Todd , Catterall William A.
Published: July 2002

Synaptic transmission is regulated by G protein-coupled receptors
whose activation releases G protein βγ subunits that modulate
presynaptic Ca
2+
channels. The sequence motif QXXER has
been proposed to be involved in the interaction between G protein
βγ subunits and target proteins including adenylyl cyclase 2. This
motif is present in the intracellular loop connecting domains I and II
(L
I-II
) of Ca
2+
channel α
1A
subunits, which are modulated by G proteins, but not in
α
1C
subunits, which are not modulated. Peptides
containing the QXXER motif from adenylate cyclase 2 or from
α
1A
block G protein modulation but a mutant peptide
containing the sequence AXXAA does not, suggesting that the
QXXER-containing peptide from α
1A
can competitively
inhibit Gβγ modulation. Conversion of the R in the QQIER sequence
of α
1A
to E as in α
1C
slows channel
inactivation and shifts the voltage dependence of steady-state
inactivation to more positive membrane potentials. Conversion of the
final E in the QQLEE sequence of α
1C
to R has opposite
effects on voltage-dependent inactivation, although the changes are not
as large as those for α
1A
. Mutation of the QQIER sequence
in α
1A
to QQIEE enhanced G protein modulation, and
mutation to QQLEE as in α
1C
greatly reduced G protein
modulation and increased the rate of reversal of G protein
effects. These results indicate that the QXXER motif in
L
I-II
is an important determinant of both voltage-dependent
inactivation and G protein modulation, and that the amino acid in the
third position of this motif has an unexpectedly large influence on
modulation by Gβγ. Overlap of this motif with the consensus
sequence for binding of Ca
2+
channel β subunits suggests
that this region of L
I-II
is important for three different
modulatory influences on Ca
2+
channel activity.
A novel chimeric Ig heavy chain from a teleost fish shares similarities to IgD
Authors: Wilson Melanie , Bengtén Eva , Miller Norman W. , Clem L. William , Du Pasquier Louis , Warr Gregory W.
Published: July 2002

IgD is considered to be a recently evolved Ig, being previously found only in primates and rodents. Here we describe, from a teleost fish (the channel catfish,Ictalurus punctatus), a novel complex chimeric Ig heavy chain, homologous, in part, to the heavy chain (δ) of IgD. In addition to alternative secretory or membrane-associated C termini, this chimeric molecule contains a rearranged variable domain, the first constant domain of μ, and seven constant domains encoded by a δ gene homolog. Identification of the catfish gene as δ is based on the following properties: sequence relatedness to mammalian δ; a location within the IgH locus that is immediately downstream of the μ gene; separate terminal exons for the secretory and membrane forms; coexpression with the complete μ chain in some but not all B cells. These results (i) suggest that IgD is an ancient immunoglobulin that was present in vertebrates ancestral to both the mammals and the ray-finned fishes, and (ii) raise the possibility that this Ig isotype may have served an as yet unidentified important function early in the evolution of the immune system.
GFRα3 is an orphan member of the GDNF/neurturin/persephin receptor family
Authors: Baloh Robert H. , Gorodinsky Alexander , Golden Judith P. , Tansey Malú G. , Keck Catherine L. , Popescu Nicholas C. , Johnson Eugene M. , Milbrandt Jeffrey
Published: July 2002

GDNF, neurturin, and persephin are transforming growth factor β-related neurotrophic factors known collectively as the GDNF family (GF). GDNF and neurturin signal through a multicomponent receptor complex containing a signaling component (the Ret receptor tyrosine kinase) and either of two glycosyl-phosphatidylinositol-linked binding components (GDNF family receptor α components 1 and 2, GFRα1 or GFRα2), whereas the receptor for persephin is unknown. Herein we describe a third member of the GF coreceptor family called GFRα3 that is encoded by a gene located on human chromosome 5q31.2–32. GFRα3 is not expressed in the central nervous system of the developing or adult animal but is highly expressed in several developing and adult sensory and sympathetic ganglia of the peripheral nervous system. GFRα3 is also expressed at high levels in developing, but not adult, peripheral nerve. GFRα3 is a glycoprotein that is glycosyl-phosphatidylinositol-linked to the cell surface like GFRα1 and GFRα2. Fibroblasts expressing Ret and GFRα3 do not respond to any of the known members of the GDNF family, suggesting that GFRα3 interacts with an unknown ligand or requires a different or additional signaling protein to function.
Crystallographic comparison of the estrogen and progesterone receptor’s ligand binding domains
Authors: Tanenbaum David M. , Wang Yong , Williams Shawn P. , Sigler Paul B.
Published: July 2002

The 2.8-Å crystal structure of the complex formed by estradiol and the human estrogen receptor-α ligand binding domain (hERαLBD) is described and compared with the recently reported structure of the progesterone complex of the human progesterone receptor ligand binding domain, as well as with similar structures of steroid/nuclear receptor LBDs solved elsewhere. The hormone-bound hERαLBD forms a distinctly different and probably more physiologically important dimer interface than its progesterone counterpart. A comparison of the specificity determinants of hormone binding reveals a common structural theme of mutually supported van der Waals and hydrogen-bonded interactions involving highly conserved residues. The previously suggested mechanism by which the estrogen receptor distinguishes estradiol’s unique 3-hydroxy group from the 3-keto function of most other steroids is now described in atomic detail. Mapping of mutagenesis results points to a coactivator-binding surface that includes the region around the “signature sequence” as well as helix 12, where the ligand-dependent conformation of the activation function 2 core is similar in all previously solved steroid/nuclear receptor LBDs. A peculiar crystal packing event displaces helix 12 in the hERαLBD reported here, suggesting a higher degree of dynamic variability than expected for this critical substructure.
Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association
Authors: Steinmetz Eric J. , Brow David A.
Published: July 2002

Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3′-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3′-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element
in vitro
with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation
in vivo
. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function.
Human autoantibodies specific for the α
1A
calcium channel subunit reduce both P-type and Q-type calcium currents in cerebellar neurons
Authors: Pinto Ashwin , Gillard Samantha , Moss Fraser , Whyte Kathryn , Brust Paul , Williams Mark , Stauderman Ken , Harpold Michael , Lang Bethan , Newsom-Davis John , Bleakman David , Lodge David , Boot John
Published: July 2002

The pharmacological properties of voltage-dependent calcium channel (VDCC) subtypes appear mainly to be determined by the α
1
pore-forming subunit but, whether P-and Q-type VDCCs are encoded by the same α
1
gene presently is unresolved. To investigate this, we used IgG antibodies to presynaptic VDCCs at motor nerve terminals that underlie muscle weakness in the autoimmune Lambert–Eaton myasthenic syndrome (LEMS). We first studied their action on changes in intracellular free Ca
2+
concentration [Ca
2+
]
i
in human embryonic kidney (HEK293) cell lines expressing different combinations of human recombinant VDCC subunits. Incubation for 18 h with LEMS IgG (2 mg/ml) caused a significant dose-dependent reduction in the K
+
-stimulated [Ca
2+
]
i
increase in the α
1A
cell line but not in the α
1B
, α
1C
, α
1D
, and α
1E
cell lines, establishing the α
1A
subunit as the target for these autoantibodies. Exploiting this specificity, we incubated cultured rat cerebellar neurones with LEMS IgG and observed a reduction in P-type current in Purkinje cells and both P- and Q-type currents in granule cells. These data are consistent with the hypothesis that the α
1A
gene encodes for the pore-forming subunit of both P-type and Q-type VDCCs.