bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional consequences of caspase activation in cardiac myocytes
Authors: Communal Catherine , Sumandea Marius , de Tombe Pieter , Narula Jagat , Solaro R. John , Hajjar Roger J.
Published: July 2002

Cardiomyocyte apoptosis is present in many cardiac disease states, including heart failure and ischemic heart disease. Apoptosis is associated with the activation of caspases that mediate the cleavage of vital and structural proteins. However, the functional contribution of apoptosis to these conditions is not known. Furthermore, in cardiac myocytes, apoptosis may not be complete, allowing the cells to persist for a prolonged period within the myocardium. Therefore, we examined whether caspase-3 cleaved cardiac myofibrillar proteins and, if so, whether it affects contractile function. The effects of caspase-3 were studied
in vitro
on individual components of the cardiac myofilament including α-actin, α-actinin, myosin heavy chain, myosin light chain 1/2, tropomyosin, cardiac troponins (T, I, C), and the trimeric troponin complex. Exposure of the myofibrillar protein (listed above) to caspase-3 for 4 h resulted in the cleavage of α-actin and α-actinin, but not myosin heavy chain, myosin light chain 1/2, and tropomyosin, into three fragments (30, 20, and 15 kDa) and one major fragment (45 kDa), respectively. When cTnT, cTnI, and cTnC were incubated individually with caspase-3, there was no detectable cleavage. However, when the recombinant troponin complex was exposed to caspase-3, cTnT was cleaved, resulting in fragments of 25 kDa. Furthermore, rat cardiac myofilaments exposed to caspase-3 exhibited similar patterns of myofibrillar protein cleavage. Treatment with the caspase inhibitor DEVD-CHO or z-VAD-fmk abolished the cleavage. Myofilaments, isolated from adult rat ventricular myocytes after induction of apoptotic pathway by using β-adrenergic stimulation, displayed a similar pattern of actin and TnT cleavage. Exposure of skinned fiber to caspase-3 decreased maximal Ca
2+
-activated force and myofibrillar ATPase activity. Our results indicate that caspase-3 cleaved myofibrillar proteins, resulting in an impaired force/Ca
2+
relationship and myofibrillar ATPase activity. Induction of apoptosis in cardiac cells was associated with similar cleavage of myofilaments. Therefore, activation of apoptotic pathways may lead to contractile dysfunction before cell death.
Neonatal hepatic steatosis by disruption of the adenosine kinase gene
Authors: Boison Detlev , Scheurer Louis , Zumsteg Valérie , Rülicke Thomas , Litynski Piotr , Fowler Brian , Brandner Sebastian , Mohler Hanns
Published: July 2002

Neonatal hepatic steatosis (OMIM 228100) is a fatal condition of unknown etiology characterized by a pale and yellow liver and early postnatal mortality. In the present study, a deficit in adenosine-dependent metabolism is proposed as a causative factor. Physiologically, adenosine is efficiently metabolized to AMP by adenosine kinase (ADK), an enzyme highly expressed in liver. ADK not only ensures normal adenine nucleotide levels but also is essential for maintainingS-adenosylmethionine-dependent transmethylation processes, where adenosine, an obligatory product, has to be constantly removed. HomozygousAdk−/−mutants developed normally during embryogenesis. However, within 4 days after birth they displayed microvesicular hepatic steatosis and died within 14 days with fatty liver. Adenine nucleotides were decreased andS-adenosylhomocysteine, a potent inhibitor of transmethylation reactions, was increased in the mutant liver. Thus, a deficiency in adenosine metabolism is identified as a powerful contributor to the development of neonatal hepatic steatosis, providing a model for the rapid development of postnatally lethal fatty liver.
Avicins: Triterpenoid saponins from
Acacia victoriae
(Bentham) induce apoptosis by mitochondrial perturbation
Authors: Haridas Valsala , Higuchi Masahiro , Jayatilake Gamini S. , Bailey David , Mujoo Kalpana , Blake Mary E. , Arntzen Charles J. , Gutterman Jordan U.
Published: July 2002

Anticancer agents target various subcellular components and trigger
apoptosis in chemosensitive cells. We have recently reported
the tumor cell growth inhibitory properties of a mixture of
triterpenoid saponins obtained from an Australian desert tree
(Leguminosae)
Acacia victoriae
(Bentham). Here we report
the purification of this mixture into two biologically pure components
called avicins that contain an acacic acid core with two acyclic
monoterpene units connected by a quinovose sugar. We demonstrate that
the mixture of triterpenoid saponins and avicins induce
apoptosis in the Jurkat human T cell line by affecting the
mitochondrial function. Avicin G induced cytochrome
c
release within 30–120 min in whole cells and within a minute in the
cell-free system. Caspase inhibitors DEVD or zVAD-fmk had no effect on
cytochrome
c
release, suggesting the direct action of
avicin G on the mitochondria. Activation of caspase-3 and total
cleavage of poly(ADP-ribose) polymerase (PARP) occurred between 2 and
6 h posttreatment with avicins by zVAD-fmk. Interestingly, in the
treated cells no significant changes in the membrane potential preceded
or accompanied cytochrome
c
release. A small decrease in
the generation of reactive oxygen species (ROS) was measured. The study
of these evolutionarily ancient compounds may represent an interesting
paradigm for the application of chemical ecology and chemical biology
to human health.
Genomewide identification of proteins secreted by the Hrp type III protein secretion system of
Pseudomonas syringae
pv.
tomato
DC3000
Authors: Petnicki-Ocwieja Tanja , Schneider David J. , Tam Vincent C. , Chancey Scott T. , Shan Libo , Jamir Yashitola , Schechter Lisa M. , Janes Misty D. , Buell C. Robin , Tang Xiaoyan , Collmer Alan , Alfano James R.
Published: July 2002

The ability of
Pseudomonas
syringae
pv.
tomato
DC3000 to be pathogenic on plants depends on the Hrp (hypersensitive response and pathogenicity) type III protein secretion system and the effector proteins it translocates into plant cells. Through iterative application of experimental and computational techniques, the DC3000 effector inventory has been substantially enlarged. Five homologs of known avirulence (Avr) proteins and five effector candidates, encoded by genes with putative Hrp promoters and signatures of horizontal acquisition, were demonstrated to be secreted in culture and/or translocated into
Arabidopsis
in a Hrp-dependent manner. These 10 Hrp-dependent outer proteins (Hops) were designated HopPtoC (AvrPpiC2 homolog), HopPtoD1 and HopPtoD2 (AvrPphD homologs), HopPtoK (AvrRps4 homolog), HopPtoJ (AvrXv3 homolog), HopPtoE, HopPtoG, HopPtoH, HopPtoI, and HopPtoS1 (an ADP-ribosyltransferase homolog). Analysis of the enlarged collection of proteins traveling the Hrp pathway in
P. syringae
revealed an export-associated pattern of equivalent solvent-exposed amino acids in the N-terminal five positions, a lack of Asp or Glu residues in the first 12 positions, and amphipathicity in the first 50 positions. These characteristics were used to search the unfinished DC3000 genome, yielding 32 additional candidate effector genes that predicted proteins with Hrp export signals and that also possessed signatures of horizontal acquisition. Among these were genes encoding additional ADP-ribosyltransferases, a homolog of SrfC (a candidate effector in
Salmonella
enterica
), a catalase, and a glucokinase. One ADP-ribosyltransferase and the SrfC homolog were tested and shown to be secreted in a Hrp-dependent manner. These proteins, designated HopPtoS2 and HopPtoL, respectively, bring the DC3000 Hrp-secreted protein inventory to 22.
A downstream element in the human β-globin promoter: Evidence of extended sequence-specific transcription factor IID contacts
Authors: Lewis Brian A. , Kim Tae-Kyung , Orkin Stuart H.
Published: July 2002

We describe here the identification and characterization of a functional downstream element in the human adult β-globin promoter. The existence of this element was indicated by two mutations at +22 and +33 downstream of the β-globin transcriptional start site in humans with β-thalassemia.
In vitro
transcriptional analysis of these mutants, plus a third at +13, indicates that all three decrease transcription from the β-globin promoter. Scanning mutagenesis from +10 to +45 indicates that this region contains a functional cis element(s)
in vitro
, and we designated this element the DCE (downstream core element). The DCE functions in concert with the β-globin CATA box and initiator element, as well as in a heterologous, TATA-less context. A second set of mutants indicates that a particular geometry of the DCE and core promoter is necessary for promoter function. Lastly, DCE mutants show reduced affinity for transcription factor IID (TFIID). These data indicate that TFIID makes sequence-specific contacts to the DCE and that TFIID binding is necessary for DCE function.
Identification and characterization of a lysosomal transporter for small neutral amino acids
Authors: Sagné Corinne , Agulhon Cendra , Ravassard Philippe , Darmon Michèle , Hamon Michel , El Mestikawy Salah , Gasnier Bruno , Giros Bruno
Published: July 2002

In eukaryotic cells, lysosomes represent a major site for macromolecule degradation. Hydrolysis products are eventually exported from this acidic organelle into the cytosol through specific transporters. Impairment of this process at either the hydrolysis or the efflux step is responsible of several lysosomal storage diseases. However, most lysosomal transporters, although biochemically characterized, remain unknown at the molecular level. In this study, we report the molecular and functional characterization of a lysosomal amino acid transporter (LYAAT-1), remotely related to a family of H
+
-coupled plasma membrane and synaptic vesicle amino acid transporters. LYAAT-1 is expressed in most rat tissues, with highest levels in the brain where it is present in neurons. Upon overexpression in COS-7 cells, the recombinant protein mediates the accumulation of neutral amino acids, such as γ-aminobutyric acid,
l
-alanine, and
l
-proline, through an H
+
/amino acid symport. Confocal microscopy on brain sections revealed that this transporter colocalizes with cathepsin D, an established lysosomal marker. LYAAT-1 thus appears as a lysosomal transporter that actively exports neutral amino acids from lysosomes by chemiosmotic coupling to the H
+
-ATPase of these organelles. Homology searching in eukaryotic genomes suggests that LYAAT-1 defines a subgroup of lysosomal transporters in the amino acid/auxin permease family.
Induction of the cyclic nucleotide phosphodiesterase PDE4B is essential for LPS-activated TNF-α responses
Authors: Jin S.-L. Catherine , Conti Marco
Published: July 2002

Lipopolysaccharide (LPS) stimulation of the innate immune response requires the activation of signaling cascades that culminate in the synthesis and secretion of proinflammatory cytokines. Given the inhibitory effects of phosphodiesterase (PDE) inhibitors on LPS-induced cytokine production, we have investigated LPS responses in mice deficient in PDE4 (type 4 cAMP-specific PDE)-B and PDE4D. LPS stimulation of mouse peripheral leukocytes induced PDE4B mRNA accumulation and increased PDE4 activity. This response was completely absent in mice deficient in PDE4B but not PDE4D. LPS induction of tumor necrosis factor-α secretion by circulating leukocytes was decreased by approximately 90% in mice deficient in PDE4B but not in mice lacking PDE4D. The impaired LPS response was evident regardless of the LPS dose used for stimulation and was associated with a more than 90% decrease in tumor necrosis factor-α mRNA accumulation. A decreased responsiveness to LPS was also present in other inflammatory cells, including peritoneal and lung macrophages. These findings demonstrate that PDE4B gene activation by LPS constitutes a feedback regulation essential for an efficient immune response.
PML/RARα and FLT3-ITD induce an APL-like disease in a mouse model
Authors: Kelly Louise M. , Kutok Jeffrey L. , Williams Ifor R. , Boulton Christina L. , Amaral Sonia M. , Curley David P. , Ley Timothy J. , Gilliland D. Gary
Published: July 2002

Acute promyelocytic leukemia (APL) cells invariably express aberrant fusion proteins involving the retinoic acid receptor α (RARα). The most common fusion partner is promyelocytic leukemia protein (PML), which is fused to RARα in the balanced reciprocal chromosomal translocation, t(15;17)(q22:q11). Expression of PML/RARα from the cathepsin G promoter in transgenic mice causes a nonfatal myeloproliferative syndrome in all mice; about 15% go on to develop APL after a long latent period, suggesting that additional mutations are required for the development of APL. A candidate target gene for a second mutation is
FLT3
, because it is mutated in approximately 40% of human APL cases. Activating mutations in FLT3, including internal tandem duplication (ITD) in the juxtamembrane domain, transform hematopoietic cell lines to factor independent growth. FLT3-ITDs also induce a myeloproliferative disease in a murine bone marrow transplant model, but are not sufficient to cause AML. Here, we test the hypothesis that PML/RARα can cooperate with FLT3-ITD to induce an APL-like disease in the mouse. Retroviral transduction of FLT3-ITD into bone marrow cells obtained from PML/RARα transgenic mice results in a short latency APL-like disease with complete penetrance. This disease resembles the APL-like disease that occurs with long latency in the PML/RARα transgenics, suggesting that activating mutations in FLT3 can functionally substitute for the additional mutations that occur during mouse APL progression. The leukemia is transplantable to secondary recipients and is ATRA responsive. These observations document cooperation between PML/RARα and FLT3-ITD in development of the murine APL phenotype.
The role of haustoria in sugar supply during infection of broad bean by the rust fungusUromycesfabae
Authors: Voegele Ralf T. , Struck Christine , Hahn Matthias , Mendgen Kurt
Published: July 2002

Biotrophic plant pathogenic fungi differentiate specialized infection structures within the living cells of their host plants. These haustoria have been linked to nutrient uptake ever since their discovery. We have for the first time to our knowledge shown that the flow of sugars from the hostVicia fabato the rust fungusUromyces fabaeseems to occur largely through the haustorial complex. One of the most abundantly expressed genes in rust haustoria, the expression of which is negligible in other fungal structures, codes for a hexose transporter. Functional expression of the gene termedHXT1inSaccharomyces cerevisiaeandXenopuslaevisoocytes assigned a substrate specificity ford-glucose andd-fructose and indicated a proton symport mechanism. Abs against HXT1p exclusively labeled haustoria in immunofluorescence microscopy and the haustorial plasma membrane in electron microscopy. These results suggest that the fungus concentrates this transporter in haustoria to take advantage of a specialized compartment of the haustorial complex. The extrahaustorial matrix, delimited by the plasma membranes of both host and parasite, constitutes a newly formed apoplastic compartment with qualities distinct from those of the bulk apoplast. This organization might facilitate the competition of the parasite with natural sink organs of the host.
A fatty acid desaturase modulates the activation of defense signaling pathways in plants
Authors: Kachroo Pradeep , Shanklin John , Shah Jyoti , Whittle Edward J. , Klessig Daniel F.
Published: July 2002

Salicylic acid (SA) plays an important role in activating various plant defense responses, including expression of the pathogenesis-related (
PR
) genes and systemic acquired resistance. A critical positive regulator of the SA signaling pathway in
Arabidopsis
is encoded by the
NPR1
gene. However, there is growing evidence that NPR1-independent pathways can also activate
PR
expression and disease resistance. To elucidate the components associated with NPR1-independent defense signaling, we isolated a suppressor of the
npr1–5
allele, designated
ssi2
. The recessive
ssi2
mutation confers constitutive
PR
gene expression, spontaneous lesion formation, and enhanced resistance to
Peronospora parasitica
. In contrast, a subset of defense responses regulated by the jasmonic acid (JA) signaling pathway, including expression of the defensin gene
PDF1.2
and resistance to
Botrytis cinerea,
is impaired in
ssi2
plants. With the use of a map-based approach, the
SSI2
gene was cloned and shown to encode a stearoyl-ACP desaturase (S-ACP DES). S-ACP DES is an archetypical member of a family of soluble fatty acid (FA) desaturases; these enzymes play an important role in regulating the overall level of desaturated FAs in the cell. The activity of mutant S-ACP DES enzyme was reduced 10-fold, resulting in elevation of the 18:0 FA content in
ssi2
plants. Because reduced S-ACP DES activity leads to the induction of certain defense responses and the inhibition of others, we propose that a FA-derived signal modulates crosstalk between different defense signaling pathways.
PNA interference mapping demonstrates functional domains in the noncoding RNA
Xist
Authors: Beletskii Anton , Hong Young-Kwon , Pehrson John , Egholm Michael , Strauss William M.
Published: July 2002

The noncoding RNA
Xist
has been shown to be essential for X-chromosome inactivation and to coat the inactive X-chromosome (Xi). Thus, an important question in understanding the formation of Xi is whether the binding reaction of
Xist
is necessary for X-chromosome inactivation. In this article, we demonstrate the failure of X-chromosome silencing if the association of
Xist
with the X-chromosome is inhibited. The chromatin-binding region was functionally mapped and evaluated by using an approach for studying noncoding RNA function in living cells that we call peptide nucleic acid (PNA) interference mapping. In the reported experiments, a single 19-bp antisense cell-permeating PNA targeted against a particular region of
Xist
RNA caused the disruption of the Xi. The association of the Xi with macro-histone H2A is also disturbed by PNA interference mapping.
AML1-ETO expression is directly involved in the development of acute myeloid leukemia in the presence of additional mutations
Authors: Yuan Youzhong , Zhou Liming , Miyamoto Toshihiro , Iwasaki Hiromi , Harakawa Nari , Hetherington Christopher J. , Burel Sebastien A. , Lagasse Eric , Weissman Irving L. , Akashi Koichi , Zhang Dong-Er
Published: July 2002

The t(8;21) is one of the most frequent chromosomal abnormalities associated with acute myeloid leukemia (AML). The translocation, which involves the
AML1
gene on chromosome 21 and the
ETO
gene on chromosome 8, generates an AML1-ETO fusion transcription factor. To examine the effect of the AML1-ETO fusion protein on leukemogenesis, we made transgenic mice in which expression of AML1-ETO is under the control of the human
MRP8
promoter (hMRP8-AML1-ETO). AML1-ETO is specifically expressed in myeloid cells, including common myeloid progenitors of hMRP8-AML1-ETO transgenic mice. The transgenic mice were healthy during their life spans, suggesting that AML1-ETO alone is not sufficient for leukemogenesis. However, after treatment of newborn hMRP8-AML1-ETO transgenic mice and their wild-type littermates with a strong DNA-alkylating mutagen,
N-
ethyl-
N
-nitrosourea, 55% of transgenic mice developed AML and the other 45% of transgenic mice and all of the wild-type littermates developed acute T lymphoblastic leukemia. Our results provide direct evidence that AML1-ETO is critical for causing myeloid leukemia, but one or more additional mutations are required for leukemogenesis. The hMRP8-AML1-ETO-transgenic mice provide an excellent model that can be used to isolate additional genetic events and to further understand the molecular pathogenesis of AML1-ETO-related leukemia.
Phase identity of the maize leaf is determined after leaf initiation
Authors: Orkwiszewski Joseph A. J. , Poethig R. Scott
Published: July 2002

The vegetative development of the maize shoot can be divided into juvenile and adult phases based on the types of leaves produced at different times in shoot development. Models for the regulation of phase change make explicit predictions about when the identity of these types of leaves is determined. To test these models, we examined the timing of leaf type determination in maize. Clones induced in transition leaf primordia demonstrated that the juvenile and adult regions of these leaves do not become clonally distinct until after the primordium is 700 μm in length, implying that these cell fates were undetermined at this stage of leaf development. Adult shoot apices were cultured
in vitro
to induce rejuvenation. We found that leaf primordia as large as 3 mm in length can be at least partially rejuvenated by this treatment, and the location of rejuvenated tissue is correlated with the maturation pattern of the leaf. The amount and distribution of juvenile tissue in rejuvenated leaves suggests that rejuvenation occurs nearly simultaneously in all leaf primordia.
In vitro
culture rejuvenated existing leaf primordia and the P0 primordium, but did not change the identity of subsequent primordia or the total number of leaves produced by the shoot. This result suggests that leaf identity can be regulated independently of the identity of the shoot apical meristem, and it implies that vegetative phase change is not initiated by a change in the identity of the shoot apical meristem.
Two tandem verprolin homology domains are necessary for a strong activation of Arp2/3 complex-induced actin polymerization and induction of microspike formation by N-WASP
Authors: Yamaguchi Hideki , Miki Hiroaki , Suetsugu Shiro , Ma Le , Kirschner Marc W. , Takenawa Tadaomi
Published: July 2002

All WASP family proteins share a common C terminus that consists of
the verprolin homology domain (V), cofilin homology domain (C), and
acidic region (A), through which they activate Arp2/3 complex-induced
actin polymerization. In this study, we characterized the Arp2/3
complex-mediated actin polymerization activity of VCA fragments of all
of the WASP family proteins: WASP, N-WASP, WAVE1, WAVE2, and WAVE3. All
of the VCA fragments stimulated the nucleating activity of Arp2/3
complex. Among them, N-WASP VCA, which possesses two tandem V motifs,
had a more potent activity than other VCA proteins. The chimeric
protein experiments revealed that the V motif was more important to the
activation potency than the CA region; two V motifs were required for
full activity of N-WASP. COS7 cells overexpressing N-WASP form
microspikes in response to epidermal growth factor. However, when a
chimeric protein in which the VCA region of N-WASP is replaced with
WAVE1 VCA was overexpressed, microspike formation was suppressed.
Interestingly, when the N-WASP VCA region was replaced with WAVE1 VCA,
having two V motifs, this chimeric protein could induce microspike
formation. These results indicate that strong activation of Arp2/3
complex by N-WASP is mainly caused by its two tandem V motifs, which
are essential for actin microspike formation.
Inhibition of both paracrine and autocrine VEGF/ VEGFR-2 signaling pathways is essential to induce long-term remission of xenotransplanted human leukemias
Authors: Dias Sergio , Hattori Koichi , Heissig Beate , Zhu Zhenping , Wu Yan , Witte Larry , Hicklin Daniel J. , Tateno Masatoshi , Bohlen Peter , Moore Malcolm A. S. , Rafii Shahin
Published: July 2002

Antiangiogenic agents block the effects of tumor-derived angiogenic factors (paracrine factors), such as vascular endothelial growth factor (VEGF), on endothelial cells (EC), inhibiting the growth of solid tumors. However, whether inhibition of angiogenesis also may play a role in liquid tumors is not well established. We recently have shown that certain leukemias not only produce VEGF but also selectively express functional VEGF receptors (VEGFRs), such as VEGFR-2 (Flk-1, KDR) and VEGFR1 (Flt1), resulting in the generation of an autocrine loop. Here, we examined the relative contribution of paracrine (EC-dependent) and autocrine (EC-independent) VEGF/VEGFR signaling pathways, by using a human leukemia model, where autocrine and paracrine VEGF/VEGFR loops could be selectively inhibited by neutralizing mAbs specific for murine EC (paracrine pathway) or human tumor (autocrine) VEGFRs. Blocking either the paracrine or the autocrine VEGF/VEGFR-2 pathway delayed leukemic growth and engraftment
in vivo
, but failed to cure inoculated mice. Long-term remission with no evidence of disease was achieved only if mice were treated with mAbs against both murine and human VEGFR-2, whereas mAbs against human or murine VEGFR-1 had no effect on mice survival. Therefore, effective antiangiogenic therapies to treat VEGF-producing, VEGFR-expressing leukemias may require blocking both paracrine and autocrine VEGF/VEGFR-2 angiogenic loops to achieve remission and long-term cure.
Structure of linkage disequilibrium and phenotypic associations in the maize genome
Authors: Remington David L. , Thornsberry Jeffry M. , Matsuoka Yoshihiro , Wilson Larissa M. , Whitt Sherry R. , Doebley John , Kresovich Stephen , Goodman Major M. , Buckler Edward S.
Published: July 2002

Association studies based on linkage disequilibrium (LD) can
provide high resolution for identifying genes that may contribute to
phenotypic variation. We report patterns of local and genome-wide LD in
102 maize inbred lines representing much of the worldwide genetic
diversity used in maize breeding, and address its implications for
association studies in maize. In a survey of six genes, we found that
intragenic LD generally declined rapidly with distance
(
r
2
< 0.1 within 1500 bp), but rates
of decline were highly variable among genes. This rapid decline
probably reflects large effective population sizes in maize during its
evolution and high levels of recombination within genes. A set of 47
simple sequence repeat (SSR) loci showed stronger evidence of
genome-wide LD than did single-nucleotide polymorphisms (SNPs) in
candidate genes. LD was greatly reduced but not eliminated by grouping
lines into three empirically determined subpopulations. SSR data also
supplied evidence that divergent artificial selection on flowering time
may have played a role in generating population structure. Provided the
effects of population structure are effectively controlled, this
research suggests that association studies show great promise for
identifying the genetic basis of important traits in maize with very
high resolution.
FLU: A negative regulator of chlorophyll biosynthesis in
Arabidopsis
thaliana
Authors: Meskauskiene Rasa , Nater Mena , Goslings David , Kessler Felix , op den Camp Roel , Apel Klaus
Published: July 2002

Tetrapyrroles such as chlorophylls and bacteriochlorophylls play a fundamental role in the energy absorption and transduction activities of photosynthetic organisms. Because of these molecules, however, photosynthetic organisms are also prone to photooxidative damage. They had to evolve highly efficient strategies to control tetrapyrrole biosynthesis and to prevent the accumulation of free intermediates that potentially are extremely destructive when illuminated. In higher plants, the metabolic flow of tetrapyrrole biosynthesis is regulated at the step of δ-aminolevulinic acid synthesis. This regulation previously has been attributed to feedback control of Glu tRNA reductase, the first enzyme committed to tetrapyrrole biosynthesis, by heme. With the recent discovery of chlorophyll intermediates acting as signals that control both nuclear gene activities and tetrapyrrole biosynthesis, it seems likely that heme is not the only regulator of this pathway. A genetic approach was used to identify additional factors involved in the control of tetrapyrrole biosynthesis. In
Arabidopsis thaliana
, we have found a negative regulator of tetrapyrrole biosynthesis, FLU, which operates independently of heme and seems to selectively affect only the Mg
2+
branch of tetrapyrrole biosynthesis. The identity of this protein was established by map-based cloning and sequencing the
FLU
gene. FLU is a nuclear-encoded plastid protein that, after import and processing, becomes tightly associated with plastid membranes. It is unrelated to any of the enzymes known to be involved in tetrapyrrole biosynthesis. Its predicted features suggest that FLU mediates its regulatory effect through interaction with enzymes involved in chlorophyll synthesis.
Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action
Authors: Lauer Thomas , Preik Michael , Rassaf Tienush , Strauer Bodo E. , Deussen Andreas , Feelisch Martin , Kelm Malte
Published: July 2002

The plasma level of NO
x
, i.e., the sum of NO
2
−
and NO
3
−
, is frequently used to assess NO bioavailability
in vivo
. However, little is known about the kinetics of NO conversion to these metabolites under physiological conditions. Moreover, plasma nitrite recently has been proposed to represent a delivery source for intravascular NO. We therefore sought to investigate in humans whether changes in NO
x
concentration are a reliable marker for endothelial NO production and whether physiological concentrations of nitrite are vasoactive. NO
2
−
and NO
3
−
concentrations were measured in blood sampled from the antecubital vein and brachial artery of 24 healthy volunteers. No significant arterial-venous gradient was observed for either NO
2
−
or NO
3
−
. Endothelial NO synthase (eNOS) stimulation with acetylcholine (1–10 μg/min) dose-dependently augmented venous NO
2
−
levels by maximally 71%. This effect was paralleled by an almost 4-fold increase in forearm blood flow (FBF), whereas an equieffective dose of papaverine produced no change in venous NO
2
−
. Intraarterial infusion of NO
2
−
had no effect on FBF. NOS inhibition (N
G
-monomethyl-
l
-arginine; 4–12 μmol/min) dose-dependently reduced basal NO
2
−
and FBF and blunted acetylcholine-induced vasodilation and NO release by more than 80% and 90%, respectively. In contrast, venous NO
3
−
and total NO
x
remained unchanged as did systemic arterial NO
2
−
and NO
3
−
levels during all these interventions. FBF and NO release showed a positive association (
r
= 0.85;
P
< 0.001). These results contradict the current paradigm that plasma NO
3
−
and/or total NO
x
are generally useful markers of endogenous NO production and demonstrate that only NO
2
−
reflects acute changes in regional eNOS activity. Our results further demonstrate that physiological levels of nitrite are vasodilator-inactive.
Caspase 3-cleaved N-terminal fragments of wild-type and mutant huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent proteolysis
Authors: Kim Yun J. , Yi Yong , Sapp Ellen , Wang Yumei , Cuiffo Ben , Kegel Kimberly B. , Qin Zheng-Hong , Aronin Neil , DiFiglia Marian
Published: July 2002

The Huntington's disease (HD) mutation is a polyglutamine expansion in the N-terminal region of huntingtin (N-htt). How neurons die in HD is unclear. Mutant N-htt aggregates in neurons in the HD brain; expression of mutant N-htt
in vitro
causes cell death. Other
in vitro
studies show that proteolysis by caspase 3 could be important in regulating mutant N-htt function, but there has been no direct evidence for caspase 3-cleaved N-htt fragments in brain. Here, we show that N-htt fragments consistent with the size produced by caspase 3 cleavage
in vitro
are resident in the cortex, striatum, and cerebellum of normal and adult onset HD brain and are similar in size to the fragments seen after exogenous expression of human huntingtin in mouse clonal striatal neurons. HD brain extracts treated with active caspase 3 had increased levels of N-htt fragments. Compared with the full-length huntingtin, the caspase 3-cleaved N-htt fragments, especially the mutant fragment, preferentially segregated with the membrane fraction. Partial proteolysis of the human caspase 3-cleaved N-htt fragment by calpain occurred
in vitro
and resulted in smaller N-terminal products; products of similar size appeared when mouse brain protein extracts were treated with calpain. Results support the idea that sequential proteolysis by caspase 3 and calpain may regulate huntingtin function at membranes and produce N-terminal mutant fragments that aggregate and cause cellular dysfunction in HD.
Functional magnetic resonance imaging of reorganization in rat brain after stroke
Authors: Dijkhuizen Rick M. , Ren JingMei , Mandeville Joseph B. , Wu Ona , Ozdag Fatih M. , Moskowitz Michael A. , Rosen Bruce R. , Finklestein Seth P.
Published: July 2002

Functional recovery after stroke has been associated with brain plasticity; however, the exact relationship is unknown. We performed behavioral tests, functional MRI, and histology in a rat stroke model to assess the correlation between temporal changes in sensorimotor function, brain activation patterns, cerebral ischemic damage, and cerebrovascular reactivity. Unilateral stroke induced a large ipsilateral infarct and acute dysfunction of the contralateral forelimb, which significantly recovered at later stages. Forelimb impairment was accompanied by loss of stimulus-induced activation in the ipsilesional sensorimotor cortex; however, local tissue and perfusion were only moderately affected and cerebrovascular reactivity was preserved in this area. At 3 days after stroke, extensive activation-induced responses were detected in the contralesional hemisphere. After 14 days, we found reduced involvement of the contralesional hemisphere, and significant responses in the infarction periphery. Our data suggest that limb dysfunction is related to loss of brain activation in the ipsilesional sensorimotor cortex and that restoration of function is associated with biphasic recruitment of peri- and contralesional functional fields in the brain.
Parkin functions as an E2-dependent ubiquitin– protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1
Authors: Zhang Yi , Gao Jun , Chung Kenny K. K. , Huang Hui , Dawson Valina L. , Dawson Ted M.
Published: July 2002

Parkinson's disease is a common neurodegenerative disorder in
which familial-linked genes have provided novel insights into the
pathogenesis of this disorder. Mutations in Parkin, a
ring-finger-containing protein of unknown function, are implicated in
the pathogenesis of autosomal recessive familial Parkinson's disease.
Here, we show that Parkin binds to the E2 ubiquitin-conjugating human
enzyme 8 (UbcH8) through its C-terminal ring-finger. Parkin has
ubiquitin–protein ligase activity in the presence of UbcH8. Parkin
also ubiquitinates itself and promotes its own degradation. We also
identify and show that the synaptic vesicle-associated protein,
CDCrel-1, interacts with Parkin through its ring-finger domains.
Furthermore, Parkin ubiquitinates and promotes the degradation of
CDCrel-1. Familial-linked mutations disrupt the ubiquitin–protein
ligase function of Parkin and impair Parkin and CDCrel-1 degradation.
These results suggest that Parkin functions as an E3 ubiquitin–protein
ligase through its ring domains and that it may control protein levels
via ubiquitination. The loss of Parkin's ubiquitin–protein ligase
function in familial-linked mutations suggests that this may be the
cause of familial autosomal recessive Parkinson's disease.
Computational study of protein specificity: The molecular basis of HIV-1 protease drug resistance
Authors: Wang Wei , Kollman Peter A.
Published: July 2002

Drug resistance has sharply limited the effectiveness of
HIV-1 protease inhibitors in AIDS therapy. It is critically important
to understand the basis of this resistance for designing new drugs. We
have evaluated the free energy contribution of each residue in the HIV
protease in binding to one of its substrates and to the five
FDA-approved protease drugs. Analysis of these free energy profiles and
the variability at each sequence position suggests: (
i
)
single drug resistance mutations are likely to occur at not well
conserved residues if they interact more favorably with drugs than with
the substrate; and (
ii
) resistance-evading drugs should
have a free energy profile similar to the substrate and interact most
favorably with well conserved residues. We also propose an empirical
parameter, called the free energy/variability value, which combines
free energy calculation and sequence analysis to suggest possible drug
resistance mutations on the protease. The free energy/variability
value is defined as the product of one residue's contribution to the
binding free energy and the variability of that residue. This parameter
can assist in designing resistance-evading drugs for any target.
A vertebrate fatty acid desaturase with Δ5 and Δ6 activities
Authors: Hastings Nicola , Agaba Morris , Tocher Douglas R. , Leaver Michael J. , Dick James R. , Sargent John R. , Teale Alan J.
Published: July 2002

Δ5 and Δ6 fatty acid desaturases are critical enzymes in
the pathways for the biosynthesis of the polyunsaturated fatty acids
arachidonic, eicosapentaenoic, and docosahexaenoic acids. They are
encoded by distinct genes in mammals and
Caenorhabditis elegans
. This paper
describes a cDNA isolated from zebrafish (
Danio rerio
)
with high similarity to mammalian Δ6 desaturase genes. The 1,590-bp
sequence specifies a protein that, in common with other fatty acid
desaturases, contains an N-terminal cytochrome
b
5
domain and three histidine boxes,
believed to be involved in catalysis. When the zebrafish cDNA was
expressed in
Saccharomyces cerevisiae
it conferred the
ability to convert linoleic acid (18:2
n
-6) and
α-linolenic acid (18:3
n
-3) to their corresponding Δ6
desaturated products, 18:3
n
-6 and
18:4
n
-3. However, in addition it conferred on the yeast
the ability to convert di-homo-γ-linoleic acid
(20:3
n
-6) and eicosatetraenoic acid
(20:4
n
-3) to arachidonic acid (20:4
n
-6)
and eicosapentaenoic acid (20:5
n
-3), respectively,
indicating that the zebrafish gene encodes an enzyme having both Δ5
and Δ6 desaturase activity. The zebrafish Δ5/Δ6
desaturase may represent a component of a prototypic vertebrate
polyunsaturated fatty acids biosynthesis pathway.
Activation of single whisker barrel in rat brain localized by functional magnetic resonance imaging.
Authors: Yang X , Hyder F , Shulman R G
Published: July 2002

The previously established cortical representation of rat whiskers in layer IV of the cortex contains distinct cylindrical columns of cellular aggregates, which are termed barrels and correlate in a one-to-one relation to whiskers on the contralateral rat face. In the present study, functional magnetic resonance imaging (fMRI) of the rat brain was used to map whisker barrel activation during mechanical up-down movement (+/- 2.5 mm amplitude at 8 Hz) of single/multiple whisker(s). Multislice gradient echo fMRI experiments were performed at 7 T with in-plane image resolution of 220 x 220 microns, slice thickness of 1 mm, and echo time of 16 ms. Highly significant (P < 0.001) and localized contralateral regions of activation were observed upon stimulation of single/multiple whisker(s). In all experiments (n = 10), the locations of activation relative to bregma and midline were highly correlated with the neuroanatomical position of the corresponding whisker barrels, and the results were reproducible intra- and interanimal. Our results indicate that fMRI based on blood oxygenation level-dependent image contrast has the sensitivity to depict activation of a single whisker barrel in the rat brain. This noninvasive technique will supplement existing methods in the study of rat barrel cortex and should be particularly useful for the long-term investigations of central nervous system in the same animal.
Targeted disruption of the mZP3 gene results in production of eggs lacking a zona pellucida and infertility in female mice.
Authors: Liu C , Litscher E S , Mortillo S , Sakai Y , Kinloch R A , Stewart C L , Wassarman P M
Published: July 2002

Mammalian eggs are surrounded by a thick extracellular coat, the zona pellucida, that plays important roles during early development. The mouse egg zona pellucida is constructed of three glycoproteins, called mZP1, mZP2, and mZP3. The gene encoding mZP3 is expressed only by growing oocytes during a 2- to 3-week period of oogenesis. Here, the mZP3 gene was disrupted by targeted mutagenesis using homologous recombination in mouse embryonic stem cells. Viable female mice homozygous for the mutated mZP3 allele (mZP3-/-) were obtained. These mice are indistinguishable in appearance from wild-type (mZP3+/+) and heterozygous (mZP3+/-) littermates. However, although ovaries of juvenile and adult mZP3-/- females possess growing and fully grown oocytes, the oocytes completely lack a zona pellucida. Consistent with this observation, eggs recovered from oviducts of superovulated, adult mZP3-/- females also lack a zona pellucida. Thus far, mZP3-/- females mated with wild-type males have failed to become pregnant.
Quantal release at a neuronal nicotinic synapse from rat adrenal gland.
Authors: Barbara J G , Takeda K
Published: July 2002

The neuronal nicotinic synapse in tissue slices of the adrenal medulla was studied with whole-cell patch-clamp. Excitatory postsynaptic currents (EPSCs) were evoked by local field stimulation or occurred spontaneously especially when external [K+] was increased. EPSCs were carried by channels sharing biophysical and pharmacological properties of neuronal-type nicotinic receptors (nAChRs). A single-channel conductance (gamma) of 43-45 pS was found from nonstationary variance analysis of EPSCs. Spontaneous EPSCs were tetrodotoxin-insensitive and Ca(2+)-dependent and occurred in burst-like clusters. Quantal analysis of spontaneous EPSCs gave a quantal size of 20 pA and amplitude histograms were well described by binomial models with low values of quantal content, consistent with a small number of spontaneously active release sites. However, rare large amplitude EPSCs suggest that the total number of sites is higher and that extrajunctional receptors are involved. Our estimates of quantal content and size at the chromaffin cell neuronal nicotinic synapse may be useful in characterizing central neuronal-type nicotinic receptor-mediated cholinergic synaptic transmission.
Comparison of aquatic food chains using nitrogen isotopes.
Authors: Cabana G , Rasmussen J B
Published: July 2002

Recent studies have shown the utility of delta(15)N to model trophic structure and contaminant bioaccumulation in aquatic food webs. However, cross-system comparisons in delta(15)N can be complicated by differences in delta(15)N at the base of the food chain. Such baseline variation in delta(15)N is difficult to resolve using plankton because of the large temporal variability in the delta(15)N of small organisms that have fast nitrogen turnover. Comparisons using large primary consumers, which have stable tissue isotopic signatures because of their slower nitrogen turnover, show that delta(15)N increases markedly with the human population density in the lake watershed. This shift in delta(15)N likely reflects the high delta(15)N of human sewage. Correcting for this baseline variation in delta(15)N, we report that, contrary to expectations based on previous food-web analysis, the food chains leading up to fish varied by about only one trophic level among the 40 lakes studied. Our results also suggest that the delta(15)N signatures of nitrogen at the base of the food chain will provide a useful tool in the assessment of anthropogenic nutrient inputs.
A human homolog of the
Schizosaccharomyces pombe rad9
+
checkpoint control gene
Authors: Lieberman Howard B. , Hopkins Kevin M. , Nass Monica , Demetrick Douglas , Davey Scott
Published: July 2002

The product of the
Schizosaccharomyces pombe
rad9
+
gene is required for cell cycle arrest at the
G
2
checkpoints in response to incompletely replicated or
damaged DNA. We have identified a human cDNA from an infant brain
library that is a structural homolog of
S. pombe
rad9
+
, by searching the dBest data base for
sequences similar to the fission yeast gene. The human gene encodes a
391-amino acid long, 42,520-Da protein that is ≈25% identical and
52% similar to the yeast protein. The human and yeast gene products
demonstrate partial conservation of function, as the human cDNA can
rescue to different degrees the sensitivity of
S. pombe
rad9
::
ura4
+
cells to the DNA
synthesis inhibitor hydroxyurea and gamma rays, as well as the
associated checkpoint controls. These results suggest an underlying
conservation of the molecular mechanisms of S and G
2
checkpoint control pathways in most if not all eukaryotes. Fluorescence
in situ
hybridization using a fragment of the
corresponding human genome as a probe, in conjunction with PCR
reactions employing DNA from human X rodent somatic cell hybrids, has
localized the gene to human chromosome 11q13.1-13.2. This region
contains a number of tumor suppressor loci, and based on the biology of
checkpoint control genes,
HRAD9
should be considered a
strong candidate for one of them.
Role of guanine nucleotide-binding proteins--ras-family or trimeric proteins or both--in Ca2+ sensitization of smooth muscle.
Authors: Gong M C , Iizuka K , Nixon G , Browne J P , Hall A , Eccleston J F , Sugai M , Kobayashi S , Somlyo A V , Somlyo A P
Published: July 2002

The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) involved in the agonist- and guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S])-induced increase in the Ca2+ sensitivity of 20-kDa myosin light chain (MLC20) phosphorylation and contraction in smooth muscle. A constitutively active, recombinant val14p21rhoA.GTP expressed in the baculovirus/Sf9 system, but not the protein expressed without posttranslational modification in Escherichia coli, induced at constant Ca2+ (pCa 6.4) a slow contraction associated with increased MLC20 phosphorylation from 19.8% to 29.5% (P < 0.05) in smooth muscle permeabilized with beta-esein. The effect of val14p21rhoA.GTP was inhibited by ADP-ribosylation of the protein and was absent in smooth muscle extensively permeabilized with Triton X-100. ADP-ribosylation of endogenous p21rho with epidermal cell differentiation inhibitor (EDIN) inhibited Ca2+ sensitization induced by GTP [in rabbit mesenteric artery (RMA) and rabbit ileum smooth muscles], by carbachol (in rabbit ileum), and by endothelin (in RMA), but not by phenylephrine (in RMA), and only slowed the rate without reducing the amplitude of contractions induced in RMA by 1 microM GTP[gamma-S] at constant Ca2+ concentrations. AlF(4-)-induced Ca2+ sensitization was inhibited by both guanosine 5'-[beta-thio]diphosphate (GDP[beta-S]) and by EDIN. EDIN also inhibited, to a lesser extent, contractions induced by Ca2+ alone (pCa 6.4) in both RMA and rabbit ileum. ADP-ribosylation of trimeric G proteins with pertussis toxin did not inhibit Ca2+ sensitization. We conclude that p21rho may play a role in physiological Ca2+ sensitization as a cofactor with other messengers, rather than as a sole direct inhibitor of smooth muscle MLC20 phosphatase.