bluuebunny/crossref_metadata_2025 huggingface dataset:
Bax degradation by the ubiquitin/proteasome-dependent pathway: Involvement in tumor survival and progression
Authors: Li Benyi , Dou Q. Ping
Published: July 2002

Previously we reported that proteasome inhibitors were able to overcome Bcl-2-mediated protection from apoptosis. Here we show that inhibition of the proteasome activity in Bcl-2-overexpressing cells accumulates the proapoptotic Bax protein to mitochondria/cytoplasm, where it interacts to Bcl-2 protein. This event was followed by release of mitochondrial cytochrome
c
into the cytosol and activation of caspase-mediated apoptosis. In contrast, proteasome inhibition did not induce any apparent changes in Bcl-2 protein levels. In addition, treatment with a proteasome inhibitor increased levels of ubiquitinated forms of Bax protein, without any effects on Bax mRNA expression. We also established a cell-free Bax degradation assay in which an
in vitro
-translated,
35
S-labeled Bax protein can be degraded by a tumor cell protein extract, inhibitable by addition of a proteasome inhibitor or depletion of the proteasome or ATP. The Bax degradation activity can be reconstituted in the proteasome-depleted supernatant by addition of a purified 20S proteasome or proteasome-enriched fraction. Finally, by using tissue samples of human prostate adenocarcinoma, we demonstrated that increased levels of Bax degradation correlated well with decreased levels of Bax protein and increased Gleason scores of prostate cancer. Our studies strongly suggest that ubiquitin/proteasome-mediated Bax degradation is a novel survival mechanism in human cancer cells and that selective targeting of this pathway should provide a unique approach for treatment of human cancers, especially those overexpressing Bcl-2.
An essential amino acid residue in the protein translocation channel revealed by targeted random mutagenesis of SecY
Authors: Mori Hiroyuki , Ito Koreaki
Published: July 2002

The SecY/Sec61α family of membrane proteins are the central
subunits of the putative protein translocation channel. We introduced
random mutations into a segment of
Escherichia coli
SecY
within its cytoplasmic domain 5, which was shown previously to be
important for the SecA-dependent translocation activity. Mutations were
classified into those retaining function and those gaining a
dominant-interfering ability caused by a loss of function. These
analyses showed that Arg-357, Pro-358, Gly-359, and Thr-362 are
functionally important; Arg-357, conserved in almost all organisms, was
identified as an indispensable residue.
Cerebral metabolic and cognitive decline in persons at genetic risk for Alzheimer's disease
Authors: Small Gary W. , Ercoli Linda M. , Silverman Daniel H. S. , Huang S.-C. , Komo Scott , Bookheimer Susan Y. , Lavretsky Helen , Miller Karen , Siddarth Prabha , Rasgon Natalie L. , Mazziotta John C. , Saxena Sanjaya , Wu H. M. , Mega Michael S. , Cummings Jeffrey L. , Saunders Ann M. , Pericak-Vance Margaret A. , Roses Allen D. , Barrio Jorge R. , Phelps Michael E.
Published: July 2002

The major known genetic risk for Alzheimer's disease (AD), apolipoprotein E-4 (APOE-4), is associated with lowered parietal, temporal, and posterior cingulate cerebral glucose metabolism in patients with a clinical diagnosis of AD. To determine cognitive and metabolic decline patterns according to genetic risk, we investigated cerebral metabolic rates by using positron emission tomography in middle-aged and older nondemented persons with normal memory performance. A single copy of the APOE-4 allele was associated with lowered inferior parietal, lateral temporal, and posterior cingulate metabolism, which predicted cognitive decline after 2 years of longitudinal follow-up. For the 20 nondemented subjects followed longitudinally, memory performance scores did not decline significantly, but cortical metabolic rates did. In APOE-4 carriers, a 4% left posterior cingulate metabolic decline was observed, and inferior parietal and lateral temporal regions demonstrated the greatest magnitude (5%) of metabolic decline after 2 years. These results indicate that the combination of cerebral metabolic rates and genetic risk factors provides a means for preclinical AD detection that will assist in response monitoring during experimental treatments.
Dissection of the ion-induced folding of the hammerhead ribozyme using
19
F NMR
Authors: Hammann Christian , Norman David G. , Lilley David M. J.
Published: July 2002

We have used
19
F NMR to analyze the metal ion-induced
folding of the hammerhead ribozyme by selective incorporation of
5fluorouridine. We have studied the chemical shift and linewidths
of
19
F resonances of 5-fluorouridine at the 4 and 7
positions in the ribozyme core as a function of added Mg
2+
.
The data fit well to a simple two-state model whereby the formation of
domain 1 is induced by the noncooperative binding of Mg
2+
with an association constant in the range of 100 to 500
M
−1
, depending on the concentration of monovalent ions
present. The results are in excellent agreement with data reporting on
changes in the global shape of the ribozyme. However, the NMR
experiments exploit reporters located in the center of the RNA sections
undergoing the folding transitions, thereby allowing the assignment of
specific nucleotides to the separate stages. The results define the
folding pathway at high resolution and provide a time scale for the
first transition in the millisecond range.
Regulation of transcription by H1 phosphorylation in
Tetrahymena
is position independent and requires clustered sites
Authors: Dou Yali , Gorovsky Martin A.
Published: July 2002

In
Tetrahymena
cells, constitutive phosphorylation of histone H1 phenocopies the loss of H1 from chromatin. Regulation of transcription by H1 phosphorylation is achieved by altering the overall charges of a small domain. Here, we further explore the electrostatic properties of this domain and the mechanism by which it regulates transcription. We demonstrate that the regulatory effect of the clustered charges does not require any long-range interaction and is position independent. However, when the same number of charges was dispersed throughout the H1 molecule, the effect became undetectable. The results are explained by a nucleation-propagation model and provide
in vivo
evidence that the synergy of the clustered positive charges plays a role in histone function and gene regulation.
RNA editing activity is associated with splicing factors in lnRNP particles: The nuclear pre-mRNA processing machinery
Authors: Raitskin Oleg , Cho Dan-Sung C. , Sperling Joseph , Nishikura Kazuko , Sperling Ruth
Published: July 2002

Multiple members of the ADAR (adenosine deaminases acting on RNA)
gene family are involved in A-to-I RNA editing. It has been speculated
that they may form a large multicomponent protein complex. Possible
candidates for such complexes are large nuclear ribonucleoprotein
(lnRNP) particles. The lnRNP particles consist mainly of four
spliceosomal subunits that assemble together with the pre-mRNA to form
a large particle and thus are viewed as the naturally assembled
pre-mRNA processing machinery. Here we investigated the presence of
ADARs in lnRNP particles by Western blot analysis using anti-ADAR
antibodies and by indirect immunoprecipitation. Both ADAR1 and ADAR2
were found associated with the spliceosomal components Sm and SR
proteins within the lnRNP particles. The two ADARs, associated with
lnRNP particles, were enzymatically active in site-selective A-to-I RNA
editing. We demonstrate the association of ADAR RNA editing enzymes
with physiological supramolecular complexes, the lnRNP particles.
Localized expression of a
dpp
/
BMP2
/
4
ortholog in a coral embryo
Authors: Hayward David C. , Samuel Gabrielle , Pontynen Patricia C. , Catmull Julian , Saint Robert , Miller David J. , Ball Eldon E.
Published: July 2002

As the closest outgroup to the Bilateria, the Phylum Cnidaria is likely to be critical to understanding the origins and evolution of body axes. Proteins of the decapentaplegic (DPP)/bone morphogenetic protein (BMP) 2/4 subfamily are central to the specification of the dorsoventral (D/V) axis in bilateral animals, albeit with an axis inversion between arthropods and chordates. We show that a
dpp
/
BMP2
/
4
ortholog (
bmp2
/
4-Am
) is present in the reef-building scleractinian coral,
Acropora millepora
(Class Anthozoa) and that it is capable of causing phenotypic effects in
Drosophila
that mimic those of the endogenous
dpp
gene. We also show that, during coral embryonic development,
bmp2
/
4-Am
expression is localized in an ectodermal region adjacent to the blastopore. Thus, a representative of the DPP/BMP2/4 subfamily of ligands was present in the common ancestor of diploblastic and triploblastic animals where it was probably expressed in a localized fashion during development. A localized source of DPP/BMP2/4 may have already been used in axis formation in this ancestor, or it may have provided a means by which an axis could evolve in triploblastic animals.
Holins kill without warning
Authors: Gründling Angelika , Manson Michael D. , Young Ry
Published: July 2002

Holins comprise the most diverse functional group of proteins known. They are small bacteriophage-encoded proteins that accumulate during the period of late-protein synthesis after infection and cause lysis of the host cell at a precise genetically programmed time. It is unknown how holins achieve temporal precision, but a conserved feature of their function is that energy poisons subvert the normal scheduling mechanism and instantly trigger membrane disruption. On this basis, timing has been proposed to involve a progressive decrease in the energized state of the membrane until a critical triggering level is reached. Here, we report that membrane integrity is not compromised after the induction of holin synthesis until seconds before lysis. The proton motive force was monitored by the rotation of individual cells tethered by a single flagellum. The results suggest an alternative explanation for the lysis “clock,” in which holin concentrations build to a critical level that leads to formation of an oligomeric complex that disrupts the membrane.
How cuckoldry can decrease the opportunity for sexual selection: Data and theory from a genetic parentage analysis of the sand goby,
Pomatoschistus minutus
Authors: Jones Adam G. , Walker DeEtte , Kvarnemo Charlotta , Lindström Kai , Avise John C.
Published: July 2002

Alternative mating strategies are common in nature and are generally thought to increase the intensity of sexual selection. However, cuckoldry can theoretically decrease the opportunity for sexual selection, particularly in highly polygamous species. We address here the influence of sneaking (fertilization thievery) on the opportunity for sexual selection in the sand goby
Pomatoschistus minutus
, a marine fish species in which males build and defend nests. Our microsatellite-based analysis of the mating system in a natural sand goby population shows high rates of sneaking and multiple mating by males. Sneaker males had fertilized eggs in ≈50% of the assayed nests, and multiple sneakers sometimes fertilized eggs from a single female. Successful males had received eggs from 2 to 6 females per nest (mean = 3.4). We developed a simple mathematical model showing that sneaking in this polygynous sand goby population almost certainly decreases the opportunity for sexual selection, an outcome that contrasts with the usual effects of cuckoldry in socially monogamous animals. These results highlight a more complex and interesting relationship between cuckoldry rates and the intensity of sexual selection than previously assumed in much of the literature on animal mating systems.
Quinine blocks specific gap junction channel subtypes
Authors: Srinivas Miduturu , Hopperstad Matthew G. , Spray David C.
Published: July 2002

We demonstrate that the antimalarial drug quinine specifically reduces currents through gap junctions formed by some connexins (Cx) in transfected mammalian cells, but does not affect other gap junction types. Quinine blocked Cx36 and Cx50 junctional currents in a reversible and concentration-dependent manner with half maximal blocking concentrations of 32 and 73 μM, respectively; Hill coefficients for block by quinine were about 2 for both connexins. In contrast, quinine did not substantially block gap junction channels formed by Cx26, Cx32, Cx40, and Cx43, and only moderately affected Cx45 junctions. To determine the location of the binding site of quinine (pKa = 8.7), we investigated the effect of quinine at various external and internal pH values and the effect of a permanently charged quaternary derivative of quinine. Our results indicate that the binding site for quinine is intracellular, possibly within the pore. Single-channel studies indicated that exposure to quinine induced slow transitions between open and fully closed states that decreased open probability of the channel. Quinine thus offers a potentially useful method to block certain types of gap junction channels, including those between neurons that are formed by Cx36. Moreover, quinine derivatives that are excluded from other types of membrane channels may provide molecules with connexin-specific as well as connexin-selective blocking activity.
Hindering auxiliary anchors are potent modulators of peptide binding and selection by I-Akclass II molecules
Authors: Latek Robert R. , Petzold Shirley J. , Unanue Emil R.
Published: July 2002

Selection of particular antigen-derived peptides by class II MHC molecules determines the population of complexes represented on the antigen-presenting cell surface and available for T cell receptor engagement. This discriminating selection process results from unique interactions between the spectrum of peptides generated during antigen processing and the MHC molecules. Here, we examined the selection of peptides by the class II MHC, I-Ak. Our results indicate that although peptide primary anchors are key in MHC binding, auxiliary anchors are a powerful regulatory component in the selection of peptides by I-Ak. Study of the segments surrounding the dominant hen egg white lysozome(48-61) epitope demonstrates that auxiliary anchors also are involved in determining the binding register of I-Akalong an extended peptide. In addition, we found that unique combinations of auxiliary anchors can act in concert to modulate the binding of peptides to I-Ak.
Genetic variation in aldosterone synthase predicts plasma glucose levels
Authors: Ranade Koustubh , Wu Kwan Dun , Risch Neil , Olivier Michael , Pei Dee , Hsiao Chin-Fu , Chuang Lee-Ming , Ho Low-Tone , Jorgenson Eric , Pesich Robert , Chen Yii-Der I. , Dzau Victor , Lin Alfred , Olshen Richard A. , Curb David , Cox David R. , Botstein David
Published: July 2002

The mineralocorticoid hormone, aldosterone, is known to play a role in sodium homeostasis. We serendipitously found, however, highly significant association between single-nucleotide polymorphisms in the aldosterone synthase gene and plasma glucose levels in a large population of Chinese and Japanese origin. Two polymorphisms—one in the putative promoter (T-344C) and another resulting in a lysine/arginine substitution at amino acid 173, which are in complete linkage disequilibrium in this population—were associated with fasting plasma glucose levels (
P
= 0.000017) and those 60 (
P
= 0.017) and 120 (
P
= 0.0019) min after an oral glucose challenge. A C/T variant in intron 1, between these polymorphisms, was not associated with glucose levels. Arg-173 and -344C homozygotes were most likely to be diabetic [odds ratio 2.51; 95% confidence interval (C.I.) 1.39–3.92;
P
= 0.0015] and have impaired fasting glucose levels (odds ratio 3.53; 95% C.I. 2.02–5.5;
P
= 0.0000036). These results suggest a new role for aldosterone in glucose homeostasis.
Evidence from disruption of the lmcpb gene array of Leishmania mexicana that cysteine proteinases are virulence factors.
Authors: Mottram J C , Souza A E , Hutchison J E , Carter R , Frame M J , Coombs G H
Published: July 2002

The mammalian form of the protozoan parasite Leishmania mexicana contains high activity of a cysteine proteinase (LmCPb) encoded on a tandem array of 19 genes (lmcpb). Homozygous null mutants for lmcpb have been produced by targeted gene disruption. All life-cycle stages of the mutant can be cultured in vitro, demonstrating that the gene is not essential for growth or differentiation of the parasite. However, the mutant exhibits a marked phenotype affecting virulence-- its infectivity to macrophages is reduced by 80%. The mutants are as efficient as wild-type parasites in invading macrophages but they only survive in a small proportion of the cells. However, those parasites that successfully infect these macrophages grow normally. Despite their reduced virulence, the mutants are still able to produce subcutaneous lesions in mice, albeit at a slower rate than wild-type parasites. The product of a single copy of lmcpb re-expressed in the null mutant was enzymatically active and restored infectivity toward macrophages to wild-type levels. Double null mutants created for lmcpb and lmcpa (another cathepsin L-like cysteine proteinase) have a similar phenotype to the lmcpb null mutant, showing that LmCPa does not compensate for the loss of LmCPb.
A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin 2 gene regulation.
Authors: Rothenberg E V , Ward S B
Published: July 2002

The interleukin 2 (IL-2) gene is subject to two types of regulation: its expression is T-lymphocyte-specific and it is acutely dependent on specific activation signals. The IL-2 transcriptional apparatus integrates multiple types of biochemical information in determining whether or not the gene will be expressed, using multiple diverse transcription factors that are each optimally activated or inhibited by different signaling pathways. When activation of one or two of these factors is blocked IL-2 expression is completely inhibited. The inability of the other, unaffected factors to work is explained by the striking finding that none of the factors interacts stably with its target site in the IL-2 enhancer unless all the factors are present. Coordinate occupancy of all the sites in the minimal enhancer is apparently maintained by continuous assembly and disassembly cycles that respond to the instantaneous levels of each factor in the nuclear compartment. In addition, the minimal enhancer undergoes specific increases in DNase I accessibility, consistent with dramatic changes in chromatin structure upon activation. Still to be resolved is what interaction(s) conveys T-lineage specificity. In the absence of activating signals, the minimal IL-2 enhancer region in mature T cells is apparently unoccupied, exactly as in non-T lineage cells. However, in a conserved but poorly studied upstream region, we have now mapped several novel sites of DNase I hypersensitivity in vivo that constitutively distinguish IL-2 producer type T cells from cell types that cannot express IL-2. Thus a distinct domain of the IL-2 regulatory sequence may contain sites for competence- or lineage-marking protein contacts.
Relationship between psychostimulant-induced "high" and dopamine transporter occupancy.
Authors: Volkow N D , Wang G J , Fowler J S , Gatley S J , Ding Y S , Logan J , Dewey S L , Hitzemann R , Lieberman J
Published: July 2002

The ability of cocaine to inhibit the dopamine transporter (DAT) appears to be crucial for its reinforcing properties. The potential use of drugs that produce long-lasting inhibition of the DAT as a mean of preventing the "high" and reducing drug-seeking behavior has become a major strategy in medication development. However, neither the relation between the high and DAT inhibition nor the ability to block the high by prior DAT blockade have ever been demonstrated. To evaluate if DAT could prevent the high induced by methylphenidate (MP), a drug which like cocaine inhibits the DAT, we compared the responses in eight non-drug-abusing subjects between the first and the second of two MP doses (0.375 mg/kg, i.v.) given 60 min apart. At 60 min the high from MP has returned to baseline, but 75-80% of the drug remains in brain. Positron-emission tomography and [11C]d-threo-MP were used to estimate DAT occupancies at different times after MP. DAT inhibition by MP did not block or attenuate the high from a second dose of MP given 60 min later, despite a 80% residual transporter occupancy from the first dose. Furthermore some subjects did not perceive a high after single or repeated administration despite significant DAT blockade. These results indicate that DAT occupancy is not sufficient to account for the high, and that for DAT inhibitors to be therapeutically effective, occupancies > 80% may be required.
Crystal structure of the secretory form of membrane-associated human carbonic anhydrase IV at 2.8-Å resolution
Authors: Stams Travis , Nair Satish K. , Okuyama Torayuki , Waheed Abdul , Sly William S. , Christianson David W.
Published: July 2002

It has recently been demonstrated that the C-terminal deletion
mutant of recombinant human carbonic anhydrase IV (G267X CA IV)
converts the normally glycosylphosphatidylinositol-anchored enzyme into
a soluble secretory form which has the same catalytic properties as the
membrane-associated enzyme purified from human tissues. We have
determined the three-dimensional structure of the secretory form of
human CA IV by x-ray crystallographic methods to a resolution of 2.8
Å. Although the zinc binding site and the hydrophobic substrate
binding pocket of CA IV are generally similar to those of other
mammalian isozymes, unique structural differences are found elsewhere
in the active site. Two disufide linkages, Cys-6–Cys-11G and
Cys-23–Cys-203, stabilize the conformation of the N-terminal domain.
The latter disulfide additionally stabilizes an active site loop
containing a
cis
-peptide linkage between Pro-201 and
Thr-202 (this loop contains catalytic residue Thr-199). On the opposite
side of the active site, the Val-131–Asp-136 segment adopts an
extended loop conformation instead of an α-helix conformation as
found in other isozymes. Finally, the C terminus is surrounded by a
substantial electropositive surface potential, which is likely to
stabilize the interaction of CA IV with the negatively charged
phospholipid headgroups of the membrane. These structural features are
unique to CA IV and provide a framework for the design of sulfonamide
inhibitors selective for this particular isozyme.
Progressive entorhinal cortex lesions accelerate hippocampal sprouting and spare spatial memory in rats
Authors: Ramirez Julio J. , McQuilkin Meredith , Carrigan Timothy , MacDonald Katherine , Kelley Melinda S.
Published: July 2002

Accelerating hippocampal sprouting by making unilateral progressive lesions of the entorhinal cortex spared the spatial memory of rats tested for retention of a learned alternation task. Subsequent transection of the sprouted crossed temporodentate pathway (CTD), as well as a simultaneous CTD transection and progressive entorhinal lesion, produced a persistent deficit on the memory task. These results suggest that CTD sprouting, which is homologous to the original perforant path input to the dentate gyrus of the hippocampus, is behaviorally significant and can ameliorate at least some of the memory deficits associated with hippocampal deafferentation.
Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase.
Authors: De Azevedo W F , Mueller-Dieckmann H J , Schulze-Gahmen U , Worland P J , Sausville E , Kim S H
Published: July 2002

The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A novel flavone, (-)-cis-5,7-dihydroxyphenyl-8-[4-(3-hydroxy-1-methyl)piperidinyl] -4H-1-benzopyran-4-one hydrochloride hemihydrate (L868276), is a potent inhibitor of CDKs. A chlorinated form, flavopiridol, is currently in phase I clinical trials as a drug against breast tumors. We determined the crystal structure of a complex between CDK2 and L868276 at 2.33 angstroms resolution and refined to an Rfactor 20.3%. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure. The analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of L868276 to inhibit CDK2 and CDC2.
Islet amyloid formation associated with hyperglycemia in transgenic mice with pancreatic beta cell expression of human islet amyloid polypeptide.
Authors: Verchere C B , D'Alessio D A , Palmiter R D , Weir G C , Bonner-Weir S , Baskin D G , Kahn S E
Published: July 2002

Pancreatic islet amyloid deposits are a characteristic pathologic feature of non-insulin-dependent diabetes mellitus and contain islet amyloid polypeptide (IAPP; amylin). We used transgenic mice that express human IAPP in pancreatic beta cells to explore the potential role of islet amyloid in the pathogenesis of non-insulin-dependent diabetes mellitus. Extensive amyloid deposits were observed in the pancreatic islets of approximately 80% of male transgenic mice > 13 months of age. Islet amyloid deposits were rarely observed in female transgenic mice (11%) and were never seen in nontransgenic animals. Ultrastructural analysis revealed that these deposits were composed of human IAPP-immunoreactive fibrils that accumulated between beta cells and islet capillaries. Strikingly, approximately half of the mice with islet amyloid deposits were hyperglycemic (plasma glucose > 11 mM). In younger (6- to 9-month-old) male transgenic mice, islet amyloid deposits were less commonly observed but were always associated with severe hyperglycemia (plasma glucose > 22 mM). These data indicate that expression of human IAPP in beta cells predisposes male mice to the development of islet amyloid and hyperglycemia. The frequent concordance of islet amyloid with hyperglycemia in these mice suggests an interdependence of these two conditions and supports the hypothesis that islet amyloid may play a role in the development of hyperglycemia.
Cloning and characterization of the mouse vitamin D receptor promoter
Authors: Jehan Frederic , DeLuca Hector F.
Published: July 2002

The gene encoding the mouse vitamin D receptor has been cloned. A new exon 1 has been found that changes the numbering established for the human VDR gene. Exons 2 and 3 in the human VDR gene (coding for the zinc fingers 1 and 2, respectively) are named exons 3 and 4 in the mouse vitamin D receptor. The 1.5-kb 5′-flanking region of the new exon 1 was analyzed and revealed the presence of putative cis-acting elements. Despite the absence of a TATA box, this 5′-flanking region contains several characteristics of a GC-rich promoter including four Sp1 sites present in tandem and two CCAAT boxes. Interestingly, the Sp1 site that is the most proximal to the new exon 1 overlaps a perfect site for Krox-20/24. Krox-20 is a transcription factor involved in brain development, and also in bone remodeling. In luciferase reporter gene expression assays, we showed that sequences from this 5′-flanking region elicit high transactivation activity. Furthermore, in the NIH 3T3 cell line, a 3- to 5-fold increase in response to forskolin treatment (an activator of adenylate cyclase and in turn of protein kinase A pathway) was observed.
Inhibition of the activity of poly(ADP ribose) synthetase reduces ischemia–reperfusion injury in the heart and skeletal muscle
Authors: Thiemermann Christoph , Bowes Joanne , Myint Fiona P. , Vane John R.
Published: July 2002

Reperfusion of the ischemic myocardium results in the generation of oxygen-derived free radicals, NO, and presumably peroxynitrite. These, in turn, may cause strand breaks in DNA, which activate the nuclear enzyme poly(ADP ribose) synthetase (PARS). This results in a rapid depletion of intracellular NAD and ATP. When this reaction is excessive, there is ultimately cell death. Here we demonstrate that 3-aminobenzamide (and several other, chemically distinct, inhibitors of PARS activity) reduces the infarct size caused by ischemia and reperfusion of the heart or skeletal muscle of the rabbit. Inhibition of PARS activity also attenuates the myocardial dysfunction caused by global ischemia and reperfusion in the isolated, perfused heart of the rabbit. In skeletal muscle, inhibition of the activity of neuronal NO synthase reduces infarct size, indicating that the formation of NO contributes to the activation of PARS there. There is no significant neuronal NO synthase activity in the heart, and hence NO synthase inhibitors did not reduce myocardial infarct size. Thus, activation of PARS contributes to the cell death caused by ischemia–reperfusion, and PARS inhibitors may constitute a novel therapy for ischemia–reperfusion injury.
Erythrocyte membrane vesiculation: Model for the molecular mechanism of protein sorting
Authors: Knowles David W. , Tilley Leann , Mohandas Narla , Chasis Joel Anne
Published: July 2002

Budding and vesiculation of erythrocyte membranes occurs by a process involving an uncoupling of the membrane skeleton from the lipid bilayer. Vesicle formation provides an important means whereby protein sorting and trafficking can occur. To understand the mechanism of sorting at the molecular level, we have developed a micropipette technique to quantify the redistribution of fluorescently labeled erythrocyte membrane components during mechanically induced membrane deformation and vesiculation. Our previous studies indicated that the spectrin-based membrane skeleton deforms elastically, producing a constant density gradient during deformation. Our current studies showed that during vesiculation the skeleton did not fragment but rather retracted to the cell body, resulting in a vesicle completely depleted of skeleton. These local changes in skeletal density regulated the sorting of nonskeletal membrane components. Highly mobile membrane components, phosphatidylethanolamine- and glycosylphosphatidylinositol-linked CD59 with no specific skeletal association were enriched in the vesicle. In contrast, two components with known specific skeletal association, band 3 and glycophorin A, were differentially depleted in vesicles. Increasing the skeletal association of glycophorin A by liganding its extrafacial domain reduced the fraction partitioning to the vesicle. We conclude that this technique of bilayer/skeleton uncoupling provides a means with which to study protein sorting driven by changes in local skeletal density. Moreover, it is the interaction of particular membrane components with the spectrin-based skeleton that determines molecular partitioning during protein sorting.
Genes regulated by androgen in the rat ventral prostate
Authors: Wang Zhou , Tufts Rachel , Haleem Riffat , Cai Xiaoyan
Published: July 2002

Genes that are regulated by androgen in the prostate were studied in the rat. Four of the less than 10 genes that are down-regulated by androgen in the ventral prostate of a 7-day castrated rat were identified; their mRNAs decayed with identical kinetics. Twenty-five of the estimated 56 genes that are up-regulated by androgen in the castrated prostate have been isolated. The up-regulated genes fall into two kinetic types. Early genes are significantly up-regulated by 6.5 hr whereas the delayed genes respond mainly after 24 hr from the time of androgen replacement. These androgen-response genes are also regulated in the prostate by castration, indicating that these genes could play important roles in androgen-induced regrowth and/or castration-induced regression of the prostate during hormonal manipulation. A survey of the tissue specificity showed that the androgen-response gene expression program in the prostate is mainly prostate-specific. Total RNA Northern blot analysis detects the expression of about 16 up-regulated genes and 3 down-regulated genes in the prostate only. Four up-regulated genes and one down-regulated gene are regulated by androgen in both the prostate and seminal vesicles but not in other organs. The expression of the remaining androgen-response genes is not limited to the prostate but is only responsive to androgen in the prostate. This survey of the androgen-response gene expression program provides insights into the molecular and cellular mechanisms of androgen action in the prostate.
Characterization of the interaction between DARPP-32 and protein phosphatase 1 (PP-1): DARPP-32 peptides antagonize the interaction of PP-1 with binding proteins
Authors: Kwon Young-Guen , Huang Hsien-Bin , Desdouits Frédéric , Girault Jean-Antoine , Greengard Paul , Nairn Angus C.
Published: July 2002

The catalytic subunit of PP-1 (PP-1C) is potently inhibited (IC
50
, ≈1 nM) by DARPP-32 (
d
opamine- and c
A
MP-
r
egulated
p
hospho
p
rotein,
M
r
32
,000), inhibitor-1, and inhibitor-2. The NH
2
-terminal 50 amino acid residues of DARPP-32 and inhibitor-1 are similar, and phosphorylation of a common threonine residue (Thr-34/Thr-35) is necessary for inhibition of PP-1C. We have characterized further the interaction between DARPP-32 and PP-1C. Using synthetic peptides derived from the NH
2
-terminal region of DARPP-32, residues 6–11, RKKIQF, have been shown to be required for inhibition of PP-1C. Peptides containing this motif were able to antagonize the inhibition of PP-1C by phospho-DARPP-32 and phosphoinhibitor-1. The inhibition of PP-1C by inhibitor-2, but not by okadaic acid, microcystin, or calyculin A, was also attentuated by these antagonist peptides. These results together with results from other studies support a model in which two subdomains of phospho-DARPP-32 interact with PP-1C. The region encompassing phospho-Thr-34 appears to interact with the active site of the enzyme blocking enzyme activity. The region encompassing the RKKIQF motif binds to a domain of PP-1C removed from the active site. Amino acid sequence analysis indicates that basic and hydrophobic features of the RKKIQF motif are conserved in the binding domains of certain PP-1C targeting proteins, suggesting that interaction of inhibitor proteins and targeting proteins may be mutually exclusive.
Nucleotide sequence diversity at the alcohol dehydrogenase 1 locus in wild barley (
Hordeum vulgare
ssp.
spontaneum
): An evaluation of the background selection hypothesis
Authors: Cummings Michael P. , Clegg Michael T.
Published: July 2002

The background selection hypothesis predicts a reduction in nucleotide site diversity and an excess of rare variants, owing to linkage associations with deleterious alleles. This effect is expected to be amplified in species that are predominantly self-fertilizing. To examine the predictions of the background selection hypothesis in self-fertilizing species, we sequenced 1,362 bp of
adh1
, a gene for alcohol dehydrogenase (Adh; alcohol:NAD
+
oxidoreductase, EC
1.1.1.1
), in a sample of 45 accessions of wild barley,
Hordeum vulgare
ssp.
spontaneum
, drawn from throughout the species range. The region sequenced included 786 bp of exon sequence (part of exon 4, all of exons 5–9, and part of exon 10) and 576 bp of intron sequence (all of introns 4–9). There were 19 sites polymorphic for nucleotide substitutions, 8 in introns, and 11 in exons. Of the 11 nucleotide substitutions in codons, 4 were synonymous and 7 were nonsynonymous, occurring uniquely in the sample. There was no evidence of recombination in the region studied, and the estimated effective population size (
N̂

e

) based on synonymous sites was ≈1.8–4.2 × 10
5
. Several tests reveal that the pattern of nonsynonymous substitutions departs significantly from neutral expectations. However, the data do not appear to be consistent with recovery from a population bottleneck, recent population expansion, selective sweep, or strong positive selection. Though several features of the data are consistent with background selection, the distributions of polymorphic synonymous and intron sites are not perturbed toward a significant excess of rare alleles as would be predicted by background selection.
Structural similarities between topoisomerases that cleave one or both DNA strands
Authors: Berger James M. , Fass Deborah , Wang James C. , Harrison Stephen C.
Published: July 2002

Type IA and type II DNA topoisomerases are distinguished by their ability to cleave one or two strands, respectively, of a DNA duplex. Both types have been proposed to use an “enzyme-bridging” mechanism, in which a break is formed in a DNA strand and a gap is opened between the broken pieces to allow passage of a second DNA strand or duplex segment. Although the type IA and type II topoisomerase structures appear overall quite different from one another, unexpected similarities between several structural elements suggest that members of the two subfamilies may use comparable mechanisms to bind and cleave DNA.
A model for MRI contrast enhancement using
T
1
agents
Authors: Ahrens E. T. , Rothbächer U. , Jacobs R. E. , Fraser S. E.
Published: July 2002

Contrast in MRI relies on differences in the local environment of water and is often enhanced by using contrast agents. We present a simple model for evaluating the minimal contrast agent concentration required to produce “satisfactory” contrast enhancement in magnetic resonance images. Previous strategies have been based largely on empirical results for specific systems. The present tissue contrast model (TCM) can be applied to “conventional,” targeted, or biochemically responsive agents. The model results are formulated so that only a small number of parameters are required to analyze a given scenario. The TCM is a particularly useful tool in the development of new classes of magnetic resonance contrast media. These agents will have the ability to target specific cells or tissue, and perhaps be able to report on their physiological status. As an example of the applicability of the TCM, we test it against
in vivo
magnetic resonance microscopy results in frog embryos that have focal cell populations labeled with contrast agent by using calibrated single-cell microinjection techniques.
Rapid deletion of rearranged T cell antigen receptor (TCR) Vα-Jα segment by secondary rearrangement in the thymus: Role of continuous rearrangement of TCR α chain gene and positive selection in the T cell repertoire formation
Authors: Wang Fanping , Huang Ching-Yu , Kanagawa Osami
Published: July 2002

A rearranged T cell receptor (TCR) Vα and Jα gene from a cytochrome
c-
specific T cell hybridoma was introduced into the genomic Jα region. The introduced TCR α chain gene is expressed in a majority of CD3 positive and CD4 CD8 double-negative immature thymocytes. However, only a few percent of the double-positive and single-positive thymocytes express this TCR α chain. This decrease is caused by a rearrangement of TCR α chain locus, which deletes the introduced TCR gene. Analysis of the mice carrying the introduced TCR α chain and the transgenic TCR β chain from the original cytochrome
c-
specific T cell hybridoma revealed that positive selection efficiently rescues double-positive thymocytes from the loss of the introduced TCR α chain gene. In the mice with negatively selecting conditions, T cells expressing the introduced TCR αβ chains were deleted at the double-positive stage. However, a large number of thymocytes escape negative selection by using an endogenous TCR α chain created by secondary rearrangement maintaining normal thymocyte development. These results suggest that secondary rearrangements of the TCR α chain gene play an important role in the formation of the T cell repertoire.
Identification of a transient excision intermediate at the crossroads between DNA polymerase extension and proofreading pathways
Authors: Baker Rosanna P. , Reha-Krantz Linda J.
Published: July 2002

DNA polymerases achieve accurate DNA replication through a delicate balance between primer elongation and proofreading. While insufficient proofreading results in DNA replication errors, indiscriminate removal of correct along with incorrect nucleotides is wasteful and may prevent completion of DNA synthesis. The transition between polymerization and proofreading modes is proposed to be governed by a kinetic barrier that prevents proofreading unless the rate of primer elongation is significantly reduced by the presence of an incorrect base pair at the primer–terminus. We have used mutational analysis, coupled with a sensitive, fluorescence-based assay to characterize intermediate steps in the proofreading pathway. A highly fluorescent complex forms between the bacteriophage T4 DNA polymerase and DNA primer–templates labeled at the 3′ terminus with the base analog 2-aminopurine. Formation of the fluorescent complex appears to be a rate-determining step in the proofreading pathway and is impaired for several mutator T4 DNA polymerases with amino acid substitutions in the exonuclease domain. Although these mutant DNA polymerases are proficient in hydrolysis, their reduced ability to form the fluorescent complex imposes a higher kinetic barrier. As a consequence, the mutant DNA polymerases proofread less frequently, resulting in more DNA replication errors.
Mechanism of nicotinic acetylcholine receptor cluster formation by rapsyn
Authors: Ramarao Manjunath K. , Cohen Jonathan B.
Published: July 2002

Rapsyn, a peripheral membrane protein of skeletal muscle, clusters nicotinic acetylcholine receptors (nAChRs) at high density in the postsynaptic membrane. The mechanism of nAChR clustering by rapsyn was analyzed by expressing nAChRs in HEK293T cells with various fragments of mouse rapsyn fused to green fluorescent protein. Membrane targeting of rapsyn is conferred solely by its acylated N terminus, as the myristoylated N-terminal 15 amino acids of rapsyn are sufficient to target green fluorescent protein to the plasma membrane. However, neither N-terminal myristoylation nor the conserved N-terminal amino acid sequence is essential. Membrane targeting, self-association, and nAChR clustering are preserved when the first 10 amino acids of rapsyn were replaced by those of
src,
which also contains a consensus sequence for
N
-myristoylation, or by those of GAP43, which contains a palmitoylation sequence. Rapsyn
1–90
, containing two tetratrichopeptide repeats is sufficient for self-association. Rapsyn
1–360,
lacking the cysteine rich domain, clusters nAChRs, while rapsyn
1–287,
containing seven tetratrichopeptide repeats, does not cluster nAChRs. We identified rapsyn
298–331
as a potential coiled-coil domain, and established that mutations disrupting coiled-coil propensity prevent nAChR clustering. Thus the structural domains of rapsyn necessary for membrane targeting, self-association, and nAChR clustering are distinct, with nAChR-rapsyn interaction mediated by a previously unrecognized coiled-coil motif.
Targeted inactivation of
Npt2
in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities
Authors: Beck Laurent , Karaplis Andrew C. , Amizuka Norio , Hewson A. Stacy , Ozawa Hidehiro , Tenenhouse Harriet S.
Published: July 2002

Npt2
encodes a renal-specific, brush-border membrane
Na
+
-phosphate (P
i
) cotransporter that is
expressed in the proximal tubule where the bulk of filtered
P
i
is reabsorbed. Mice deficient in the
Npt2
gene were generated by targeted mutagenesis to define the role of
Npt2
in the overall maintenance of P
i
homeostasis, determine its impact on skeletal development, and clarify
its relationship to autosomal disorders of renal P
i
reabsorption in humans. Homozygous mutants
(
Npt2
−/−
) exhibit increased urinary
P
i
excretion, hypophosphatemia, an appropriate elevation in
the serum concentration of 1,25-dihydroxyvitamin D with attendant
hypercalcemia, hypercalciuria and decreased serum parathyroid hormone
levels, and increased serum alkaline phosphatase activity. These
biochemical features are typical of patients with hereditary
hypophosphatemic rickets with hypercalciuria (HHRH), a Mendelian
disorder of renal P
i
reabsorption. However, unlike HHRH
patients,
Npt2
−/−
mice do not have rickets
or osteomalacia. At weaning,
Npt2
−/−
mice
have poorly developed trabecular bone and retarded secondary
ossification, but, with increasing age, there is a dramatic reversal
and eventual overcompensation of the skeletal phenotype. Our findings
demonstrate that
Npt2
is a major regulator of
P
i
homeostasis and necessary for normal skeletal
development.
The direction of transport through the nuclear pore can be inverted
Authors: Nachury Maxence V. , Weis Karsten
Published: July 2002

Transport of macromolecules across the nuclear envelope is an active process that depends on soluble factors including the GTPase Ran. Ran-GTP is predominantly located in the nucleus and has been shown to regulate cargo binding and release of import and export receptors in their respective target compartments. Recently, it was shown that transport of receptor–cargo complexes across the nuclear pore complex (NPC) does not depend on GTP-hydrolysis by Ran; however, the mechanism of translocation is still poorly understood. Here, we show that the direction of transport through the NPC can be inverted in the presence of high concentrations of cytoplasmic Ran-GTP. Under these conditions, two different classes of export cargoes are transported into the nucleus in the absence of GTP hydrolysis. The inverted transport is very rapid and can be blocked by known inhibitors of nuclear protein export. These results suggest that the NPC functions as a facilitated transport channel, allowing the selective translocation of receptor–cargo complexes. We conclude that the directionality of nucleocytoplasmic transport is determined mainly by the compartmentalized distribution of Ran-GTP.
Widespread accumulation of [
3
H]testosterone in the spinal cord of a wild bird with an elaborate courtship display
Authors: Schultz J. Douglas , Schlinger Barney A.
Published: July 2002

Elaborate courtship displays are relatively common features of the masculine reproductive behavior in birds. However, little is known about their neural and hormonal control. One bird that performs such a display is the golden-collared manakin (
Manacus vitellinus
) of Panamanian forests. Adult males, but not females, perform a physically intense display requiring substantial neuromuscular control of the wings and legs. We tested the hypothesis that steroid sensitivity is a property of neurons in the manakin spinal cord. Males and females were captured from active courtship leks, treated with drugs to block steroidogenesis, injected with
3
H-labeled testosterone, and the spinal cords were removed and processed for autoradiography. Sex steroid-accumulating cells were widely distributed in the spinal cords in each of six males and in one of five females. Cells, including presumptive motoneurons, reached their highest density in the ventral horns of the cervical and lumbosacral enlargements, regions associated with motor control of the wings and legs. These results suggest that neurons in the adult manakin spinal cord can express sex-steroid receptors, but do so less in females than in males. This evidence for androgen sensitivity and sexual dimorphism in the adult avian spinal cord suggests that sex steroids may control diverse behaviors in male birds in part by acting directly on the spinal neural circuits.
How can statistical mechanics contribute to social science?
Authors: Durlauf Steven N.
Published: July 2002

A model of interdependent decision making
has been developed to understand group differences in socioeconomic
behavior such as nonmarital fertility, school attendance, and drug use.
The statistical mechanical structure of the model illustrates how the
physical sciences contain useful tools for the study of socioeconomic
phenomena.