bluuebunny/crossref_metadata_2025 huggingface dataset:
Introduction of human apolipoprotein E4 “domain interaction” into mouse apolipoprotein E
Authors: Raffaï Robert L. , Dong Li-Ming , Farese Robert V. , Weisgraber Karl H.
Published: July 2002

Human apolipoprotein E4 (apoE4) binds preferentially to lower density lipoproteins, including very low density lipoproteins, and is associated with increased risk of atherosclerosis and neurodegenerative disorders, including Alzheimer's disease. This binding preference is the result of the presence of Arg-112, which causes Arg-61 in the amino-terminal domain to interact with Glu-255 in the carboxyl-terminal domain. ApoE2 and apoE3, which have Cys-112, bind preferentially to high density lipoproteins (HDL) and do not display apoE4 domain interaction. Mouse apoE, like apoE4, contains the equivalent of Arg-112 and Glu-255, but lacks the critical Arg-61 equivalent (it contains Thr-61). Thus, mouse apoE does not display apoE4 domain interaction and, as a result, behaves like human apoE3, including preferential binding to HDL. To assess the potential role of apoE4 domain interaction in atherosclerosis and neurodegeneration, we sought to introduce apoE4 domain interaction into mouse apoE. Replacing Thr-61 in mouse apoE with arginine converted the binding preference from HDL to very low density lipoproteinsin vitro, suggesting that apoE4 domain interaction could be introduced into mouse apoEin vivo. Using gene targeting in embryonic stem cells, we created mice expressing Arg-61 apoE. Heterozygous Arg-61/wild-type apoE mice displayed two phenotypes found in human apoE4/E3 heterozygotes: preferential binding to lower density lipoproteins and reduced abundance of Arg-61 apoE in the plasma, reflecting its more rapid catabolism. These findings demonstrate the successful introduction of apoE4 domain interaction into mouse apoEin vivo. The Arg-61 apoE mouse model will allow the effects of apoE4 domain interaction in lipoprotein metabolism, atherosclerosis, and neurodegeneration to be determined.
The
in vivo
role of p38 MAP kinases in cardiac remodeling and restrictive cardiomyopathy
Authors: Liao Pu , Georgakopoulos Dimitrios , Kovacs Attila , Zheng Meizi , Lerner Deborah , Pu Haiying , Saffitz Jeffrey , Chien Kenneth , Xiao Rui-Ping , Kass David A. , Wang Yibin
Published: July 2002

Stress-induced mitogen-activated protein kinase (MAP) p38 is activated in various forms of heart failure, yet its effects on the intact heart remain to be established. Targeted activation of p38 MAP kinase in ventricular myocytes was achieved
in vivo
by using a gene-switch transgenic strategy with activated mutants of upstream kinases MKK3bE and MKK6bE. Transgene expression resulted in significant induction of p38 kinase activity and premature death at 7–9 weeks. Both groups of transgenic hearts exhibited marked interstitial fibrosis and expression of fetal marker genes characteristic of cardiac failure, but no significant hypertrophy at the organ level. Echocardiographic and pressure-volume analyses revealed a similar extent of systolic contractile depression and restrictive diastolic abnormalities related to markedly increased passive chamber stiffness. However, MKK3bE-expressing hearts had increased end-systolic chamber volumes and a thinned ventricular wall, associated with heterogeneous myocyte atrophy, whereas MKK6bE hearts had reduced end-diastolic ventricular cavity size, a modest increase in myocyte size, and no significant myocyte atrophy. These data provide
in vivo
evidence for a negative inotropic and restrictive diastolic effect from p38 MAP kinase activation in ventricular myocytes and reveal specific roles of p38 pathway in the development of ventricular end-systolic remodeling.
Functional genomics reveals a family of eukaryotic oxidation protection genes
Authors: Volkert Michael R. , Elliott Nathan A. , Housman David E.
Published: July 2002

Reactive oxygen species (ROS) are toxic compounds produced by
normal metabolic processes. Their reactivity with cellular components
is a major stress for aerobic cells that results in lipid, protein, and
DNA damage. ROS-mediated DNA damage contributes to spontaneous
mutagenesis, and cells deficient in repair and protective mechanisms
have elevated levels of spontaneous mutations. In
Escherichia
coli
a large number of genes are involved in the repair of
oxidative DNA damage and its prevention by detoxification of ROS. In
humans, the genes required for these processes are not well defined. In
this report we describe the human
OXR1
(o
x
idation resistance) gene discovered in a search for
human genes that function in protection against oxidative damage.
OXR1
is a member of a conserved family of genes found in
eukaryotes but not in prokaryotes. We also outline the procedures
developed to identify human genes involved in the prevention and repair
of oxidative damage that were used to identify the human
OXR1
gene. This procedure makes use of the spontaneous
mutator phenotype of
E. coli
oxidative repair-deficient
mutants and identifies genes of interest by screening for antimutator
activity resulting from cDNA expression.
Bazooka and atypical protein kinase C are required to regulate oocyte differentiation in the
Drosophila
ovary
Authors: Cox Daniel N. , Seyfried Salim Abdelilah , Jan Lily Yeh , Jan Yuh Nung
Published: July 2002

The
par
genes, identified by their role in the
establishment of anterior-posterior polarity in the
Caenorhabditis elegans
zygote,
subsequently have been shown to regulate cellular polarity in diverse
cell types by means of an evolutionarily conserved protein complex
including PAR-3, PAR-6, and atypical protein kinase C (aPKC).
The
Drosophila
homologs of
par-1, par-3 (bazooka,
baz), par-6
(
DmPar-6),
and
pkc-3
(Drosophila a
PKC,
DaPKC)
each are known to play
conserved roles in the generation of cell polarity in the germ line as
well as in epithelial and neural precursor cells within the embryo. In
light of this functional conservation, we examined the potential role
of
baz
and
DaPKC
in the regulation of
oocyte polarity. Our analyses reveal germ-line autonomous roles for
baz
and
DaPKC
in the establishment of
initial anterior-posterior polarity within germ-line cysts and
maintenance of oocyte cell fate. Germ-line clonal analyses indicate
both proteins are essential for two key aspects of oocyte
determination: the posterior translocation of oocyte specification
factors and the posterior establishment of the microtubule organizing
center within the presumptive oocyte. We demonstrate BAZ and DaPKC
colocalize to belt-like structures between germarial cyst cells.
However, in contrast to their regulatory relationship in the
Drosophila
and
C. elegans
embryos, these
proteins are not mutually dependent for their germ-line localization,
nor is either protein specifically required for PAR-1 localization to
the fusome. Therefore, whereas BAZ, DaPKC, and PAR-1 are functionally
conserved in establishing oocyte polarity, the regulatory relationships
among these genes are not well conserved, indicating these molecules
function differently in different cellular contexts.
Structure and function in rhodopsin: correct folding and misfolding in two point mutants in the intradiscal domain of rhodopsin identified in retinitis pigmentosa.
Authors: Liu X , Garriga P , Khorana H G
Published: July 2002

The rhodopsin mutants P23H and G188R, identified in autosomal dominant retinitis pigmentosa (ADRP), and the site-specific mutants D190A and DeltaY191-Y192 were expressed in COS cells from synthetic mutant opsin genes containing these mutations. The proteins expressed from P23H and D190A partially regenerated the rhodopsin chromophore with 11-cis-retinal and were mixtures of the correctly folded (retinal-binding) and misfolded (non-retinal-binding) opsins. The mixtures were separated into pure, correctly folded mutant rhodopsins and misfolded opsins. The proteins expressed from the ADRP mutant G188R and the mutant DeltaY191-Y192 were composed of totally misfolded non-retinal-binding opsins. Far-UV CD spectra showed that the correctly folded mutant rhodopsins had helical content similar to that of the wild-type rhodopsin, whereas the misfolded opsins had helical content 50-70% of the wild type. The near-UV CD spectra of the misfolded mutant proteins lack the characteristic band pattern seen in the wild-type opsin, indicative of a different tertiary structure. Further, whereas the folded mutant rhodopsins were essentially resistant to trypsin digestion, the misfolded opsins were degraded to small fragments under the same conditions. Therefore, the misfolded opsins appear to be less compact in their structures than the correctly folded forms. We suggest that most, if not all, of the point mutations in the intradiscal domain identified in ADRP cause partial or complete misfolding of rhodopsin.
Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment.
Authors: Li S C , Goto N K , Williams K A , Deber C M
Published: July 2002

Proline is established as a potent breaker of both alpha-helical and beta-sheet structures in soluble (globular) proteins. Thus, the frequent occurrence of the Pro residue in the putative transmembrane helices of integral membrane proteins, particularly transport proteins, presents a structural dilemma. We propose that this phenomenon results from the fact that the structural propensity of a given amino acid may be altered to conform to changes imposed by molecular environment. To test this hypothesis on proline, we synthesized model peptides of generic sequence H2N-(Ser-LyS)2-Ala- Leu-Z-Ala-Leu-Z-Trp-Ala-Leu-Z-(Lys-Ser)3-OH (Z = Ala and/or Pro). Peptide conformations were analyzed by circular dichroism spectroscopy in aqueous buffer, SDS, lysophosphatidylglycerol micelles, and organic solvents (methanol, trifluoroethanol, and 2-propanol). The helical propensity of Pro was found to be greatly enhanced in the membrane-mimetic environments of both lipid micelles and organic solvents. Proline was found to stabilize the alpha-helical conformation relative to Ala at elevated temperatures in 2-propanol, an observation that argues against the doctrine that Pro is the most potent alpha-helix breaker as established in aqueous media. Parallel studies in deoxycholate micelles of the temperature-induced conformational transitions of the single-spanning membrane bacteriophage IKe major coat protein, in which the Pro-containing wild type was compared with Pro30 --> Ala mutant, Pro was found to protect the helix, but disrupt the beta-sheet structure as effectively as it does to model peptides in water. The intrinsic capacity of Pro to disrupt beta-sheets was further reflected in a survey of porins where Pro was found to be selectively excluded from the core of membrane-spanning beta-sheet barrels. The overall data provide a rationale for predicting and understanding the structural consequences when Pro occurs in the context of a membrane.
Nucleus-associated pools of Rna1p, the Saccharomyces cerevisiae Ran/TC4 GTPse activating protein involved in nucleus/cytosol transit.
Authors: Traglia H M , O'Connor J P , Tung K S , Dallabrida S , Shen W C , Hopper A K
Published: July 2002

Rna1p is the GTPase activating enzyme for Ran/TC4, a Ras-like GTPase necessary for nuclear/cytosolic exchange. Although most wild-type Rna1p is located in the cytosol, we found that the vast majority of the mutant Rna1-1p and, under appropriate physiological conditions, a small portion of the wild-type Rna1p cofractionate with yeast nuclei. Subnuclear fractionation studies show that most of the Rna1p is tightly associated with nuclear components, and that a portion of the active protein can be solubilized by treatments that fail to solubilize inactive Rna1-1p. To learn the precise nuclear locations of the Rna1 proteins, we studied their subcellular distributions in HeLa cells. By indirect immuno-fluorescence we show that wild-type Rna1p has three subcellular locations. The majority of the protein is distributed throughout the cytosol, but a portion of the protein is nucleus-associated, located at both the cytosolic surface and within the nucleoplasm. Mutant Rna1-1p is found at the outer nuclear surface and in the cytosol. We propose that a small pool of the wild-type Rna1p is located in the nuclear interior, supporting the model that the same components of the Ran/TC4 GTPase cycle exist on both sides of the nuclear membrane.
Action spectra for phytochrome A- and B-specific photoinduction of seed germination in Arabidopsis thaliana.
Authors: Shinomura T , Nagatani A , Hanzawa H , Kubota M , Watanabe M , Furuya M
Published: July 2002

We have examined the seed germination in Arabidopsis thaliana of wild type (wt), and phytochrome A (PhyA)- and B (PhyB)-mutants in terms of incubation time and environmental light effects. Seed germination of the wt and PhyA-null mutant (phyA) was photoreversibly regulated by red and far-red lights of 10-1,000 micromol m-2 when incubated in darkness for 1-14 hr, but no germination occurred in PhyB-null mutant (phyB). When wt seeds and the phyB mutant seeds were incubated in darkness for 48 hr, they synthesized PhyA during dark incubation and germinated upon exposure to red light of 1-100 nmol m-2 and far-red light of 0.5-10 micromol m-2, whereas the phyA mutant showed no such response. The results indicate that the seed germination is regulated by PhyA and PhyB but not by other phytochromes, and the effects of PhyA and PhyB are separable in this assay. We determined action spectra separately for PhyA- and PhyB-specific induction of seed germination at Okazaki large spectrograph. Action spectra for the PhyA response show that monochromatic 300-780 nm lights of very low fluence induced the germination, and this induction was not photoreversible in the range examined. Action spectra for the PhyB response show that germination was photoreversibly regulated by alternate irradiations with light of 0.01-1 mmol m-2 at wavelengths of 540-690 nm and 695-780 nm. The present work clearly demonstrated that PhyA photoirreversibly triggers the germination upon irradiations with ultraviolet, visible and far-red light of very low fluence, while PhyB controls the photoreversible effects of low fluence.
Meiosis: how could it work?
Authors: Kleckner N
Published: July 2002

A physical connection between homologs is required for reductional segregation at the first division of meiosis. This connection is usually provided by one or a few well-spaced crossovers. A speculative overview of processes leading to formation of these crossovers is presented.
Chromosomal localization of the proteasome Z subunit gene reveals an ancient chromosomal duplication involving the major histocompatibility complex.
Authors: Kasahara M , Hayashi M , Tanaka K , Inoko H , Sugaya K , Ikemura T , Ishibashi T
Published: July 2002

Proteasomes are the multi-subunit protease thought to play a key role in the generation of peptides presented by major histocompatibility complex (MHC) class I molecules. When cells are stimulated with interferon gamma, two MHC-encoded subunits, low molecular mass polypeptide (LMP) 2 and LMP7, and the MECL1 subunit encoded outside the MHC are incorporated into the proteasomal complex, presumably by displacing the housekeeping subunits designated Y, X, and Z, respectively. These changes in the subunit composition appear to facilitate class I-mediated antigen presentation, presumably by altering the cleavage specificities of the proteasome. Here we show that the mouse gene encoding the Z subunit (Psmb7) maps to the paracentromeric region of chromosome 2. Inspection of the mouse loci adjacent to the Psmb7 locus provides evidence that the paracentromeric region of chromosome 2 and the MHC region on chromosome 17 most likely arose as a result of a duplication that took place at an early stage of vertebrate evolution. The traces of this duplication are also evident in the homologous human chromosome regions (6p21.3 and 9q33-q34). These observations have implications in understanding the genomic organization of the present-day MHC and offer insights into the origin of the MHC.
TRAF5, a novel tumor necrosis factor receptor-associated factor family protein, mediates CD40 signaling.
Authors: Ishida T K , Tojo T , Aoki T , Kobayashi N , Ohishi T , Watanabe T , Yamamoto T , Inoue J
Published: July 2002

Signals emanating from CD40 play crucial roles in B-cell function. To identify molecules that transduce CD40 signalings, we have used the yeast two-hybrid system to done cDNAs encoding proteins that bind the cytoplasmic tail of CD40. A cDNA encoding a putative signal transducer protein, designated TRAF5, has been molecularly cloned. TRAF5 has a tumor necrosis factor receptor-associated factor (TRAF) domain in its carboxyl terminus and is most homologous to TRAF3, also known as CRAF1, CD40bp, or LAP-1, a previously identified CD40-associated factor. The amino terminus has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other members of the TRAF family protein except for TRAF1. In vitro binding assays revealed that TRAF5 associates with the cytoplasmic tail of CD40, but not with the cytoplasmic tail of tumor receptor factor receptor type 2, which associates with TRAF2. Based on analysis of the association between TRAF5 and various CD40 mutants, residues 230-269 of CD40 are required for the association with TRAF5. In contrast to TRAF3, overexpression of TRAF5 activates transcription factor nuclear factor kappa B. Furthermore, amino-terminally truncated forms of TRAF5 suppress the CD40-mediated induction of CD23 expression, as is the case with TRAF3. These results suggest that TRAF5 and TRAF3 could be involved in both common and distinct signaling pathways emanating from CD40.
Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice.
Authors: Rockman H A , Choi D J , Rahman N U , Akhter S A , Lefkowitz R J , Koch W J
Published: July 2002

Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase-5 (GRK5), a serine/threonine kinase most abundantly expressed in the heart compared with other tissues. Animals overexpressing GRK5 showed marked beta-adrenergic receptor desensitization in both the anesthetized and conscious state compared with nontransgenic control mice, while the contractile response to angiotensin II receptor stimulation was unchanged. In contrast, the angiotensin II-induced rise in contractility was significantly attenuated in transgenic mice overexpressing the beta-adrenergic receptor kinase-1, another member of the GRK family. These data suggest that myocardial overexpression of GRK5 results in selective uncoupling of G protein-coupled receptors and demonstrate that receptor specificity of the GRKs may be important in determining the physiological phenotype.
Newly identified stress-responsive protein kinases, Krs-1 and Krs-2.
Authors: Taylor L K , Wang H C , Erikson R L
Published: July 2002

The activation of protein kinases is a frequent response of cells to treatment with growth factors, chemicals, heat shock, or apoptosis-inducing agents. However, when several agents result in the activation of the same enzymes, it is unclear how specific biological responses are generated. We describe here two protein kinases that are activated by a subset of stress conditions or apoptotic agents but are not activated by commonly used mitogenic stimuli. Purification and cloning demonstrate that these protein kinases are members of a subfamily of kinases related to Ste20p, a serine/threonine kinase that functions early in a pheromone responsive signal transduction cascade in yeast. The specificity of Krs-1 and Krs-2 activation and their similarity to Ste20p suggest that they may function at an early step in phosphorylation events that are specific responses to some forms of chemical stress or extreme heat shock.
Substitution rate comparisons between grasses and palms: synonymous rate differences at the nuclear gene Adh parallel rate differences at the plastid gene rbcL.
Authors: Gaut B S , Morton B R , McCaig B C , Clegg M T
Published: July 2002

A number of studies have noted that nucleotide substitution rates at the chloroplast-encoded rbcL locus violate the molecular clock principle. Substitution rate variation at this plastid gene is particularly pronounced between palms and grasses; for example, a previous study estimated that substitution rates in rbcL sequences are approximately 5-fold faster in grasses than in palms. To determine whether a proportionate change in substitution rates also occurs in plant nuclear genes, we characterized nucleotide substitution rates in palm and grass sequences for the nuclear gene Adh. In this article, we report that palm sequences evolve at a rate of 2.61 x 10(-9) substitution per synonymous site per year, a rate which is slower than most plant nuclear genes. Grass Adh sequences evolve approximately 2.5-fold faster than palms at synonymous sites. Thus, synonymous rates in nuclear Adh genes show a marked decrease in palms relative to grasses, paralleling the pattern found at the plastid rbcL locus. This shared pattern indicates that synonymous rates are correlated between a nuclear and a plastid gene. Remarkably, nonsynonymous rates do not show this correlation. Nonsynonymous rates vary between two duplicated grass Adh loci, and nonsynonymous rates at the palm Adh locus are not markedly reduced relative to grasses.
Substructure of the inner core of the Earth.
Authors: Herndon J M
Published: July 2002

The rationale is disclosed for a substructure within the Earth's inner core, consisting of an actinide subcore at the center of the Earth, surrounded by a subshell composed of the products of nuclear fission and radioactive decay. Estimates are made as to possible densities, physical dimensions, and chemical compositions. The feasibility for self-sustaining nuclear fission within the subcore is demonstrated, and implications bearing on the structure and geodynamic activity of the inner core are discussed.
Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death.
Authors: Ohshima T , Ward J M , Huh C G , Longenecker G , Veeranna  , Pant H C , Brady R O , Martin L J , Kulkarni A B
Published: July 2002

Although cyclin-dependent kinase 5 (Cdk5) is closely related to other cyclin-dependent kinases, its kinase activity is detected only in the postmitotic neurons. Cdk5 expression and kinase activity are correlated with the extent of differentiation of neuronal cells in developing brain. Cdk5 purified from nervous tissue phosphorylates neuronal cytoskeletal proteins including neurofilament proteins and microtubule-associated protein tau in vitro. These findings indicate that Cdk5 may have unique functions in neuronal cells, especially in the regulation of phosphorylation of cytoskeletal molecules. We report here generation of Cdk5(-/-) mice through gene targeting and their phenotypic analysis. Cdk5(-/-) mice exhibit unique lesions in the central nervous system associated with perinatal mortality. The brains of Cdk5(-/-) mice lack cortical laminar structure and cerebellar foliation. In addition, the large neurons in the brain stem and in the spinal cord show chromatolytic changes with accumulation of neurofilament immunoreactivity. These findings indicate that Cdk5 is an important molecule for brain development and neuronal differentiation and also suggest that Cdk5 may play critical roles in neuronal cytoskeleton structure and organization.
Interplay of structure and disorder in cochaperonin mobile loops.
Authors: Landry S J , Taher A , Georgopoulos C , van der Vies S M
Published: July 2002

Protein-protein interactions typically are characterized by highly specific interfaces that mediate binding with precisely tuned affinities. Binding of the Escherichia coli cochaperonin GroES to chaperonin GroEL is mediated, at least in part, by a mobile polypeptide loop in GroES that becomes immobilized in the GroEL/GroES/nucleotide complex. The bacteriophage T4 cochaperonin Gp31 possesses a similar highly flexible polypeptide loop in a region of the protein that shows low, but significant, amino acid similarity with GroES and other cochaperonins. When bound to GroEL, a synthetic peptide representing the mobile loop of either GroES or Gp31 adopts a characteristic bulged hairpin conformation as determined by transferred nuclear Overhauser effects in NMR spectra. Thermodynamic considerations suggest that flexible disorder in the cochaperonin mobile loops moderates their affinity for GroEL to facilitate cycles of chaperonin-mediated protein folding.
Temporal mapping of gene expression levels during the differentiation of individual primary hematopoietic cells
Authors: Cheng Tao , Shen Hongmei , Giokas Dena , Gere John , Tenen Daniel G. , Scadden David T.
Published: July 2002

A hierarchical order of gene expression has been proposed to
control developmental events in hematopoiesis, but direct demonstration
of the temporal relationships between regulatory gene expression and
differentiation has been difficult to achieve. We modified a
single-cell PCR method to detect 2-fold changes in mRNA copies per cell
(dynamic range, 250–250,000 copies/cell) and used it to sequentially
quantitate gene expression levels as single primitive
(CD34
+
,CD38
−
) progenitor cells underwent
differentiation to become erythrocytes, granulocytes, or
monocyte/macrophages. Markers of differentiation such as CD34 or
cytokine receptor mRNAs and transcription factors associated with their
regulation were assessed. All transcription factors tested were
expressed in multipotent progenitors. During lineage-specific
differentiation, however, distinct patterns of expression emerged. SCL,
GATA-2, and GATA-1 expression sequentially extinguished during
erythroid differentiation. PU.1, AML1B, and C/EBPα expression
profiles and their relationship to cytokine receptor expression in
maturing granulocytes could be distinguished from similar profiles in
monocytic cells. These data characterize the dynamics of gene
expression accompanying blood cell development and define a signature
gene expression pattern for specific stages of hematopoietic
differentiation.
Multiple A-type cyclins express sequentially during the cell cycle in
Nicotiana tabacum
BY2 cells
Authors: Reichheld Jean-Philippe , Chaubet Nicole , Shen Wen Hui , Renaudin Jean-Pierre , Gigot Claude
Published: July 2002

Four full-length and one partial cDNA clones encoding four
different A-type cyclins were isolated from a tobacco S-phase-specific
library. The corresponding mRNAs displayed sequential appearance and
disappearance during the cell cycle of highly synchronized
suspension-cultured tobacco cells. Sequence analysis showed that the
plant A-type cyclins can be subdivided into three distinct structural
groups that are likely to be represented in every plant species. Two of
the isolated tobacco cyclins belonging to the same group were highly
expressed throughout S and G
2
phases but showed different
kinetics of induction at the G
1
/S transition. Another one
belonging to a second group was induced at mid-S phase and expressed
until mid-M phase. A similar expression pattern was previously reported
for a tobacco cyclin belonging to the third group. This sequential
expression of multiple A-type cyclins in one type of plant cells makes
a clear distinction from the situation in animal cells in which only
one A-type cyclin exists in a given species. Furthermore, the
expression of the different A-type cyclin genes responded differently
upon a block at mid-S phase by DNA synthesis inhibitors. These results
suggest that the multiple A-type cyclins act at different steps of the
plant cell cycle and, therefore, exert distinct functions. In contrast,
the expression of B-type cyclins was restricted to a narrow window
corresponding to the M phase.
The purified
Bacillus subtilis
tetracycline efflux protein TetA(L) reconstitutes both tetracycline–cobalt/H
+
and Na
+
(K
+
)/H
+
exchange
Authors: Cheng Jianbo , Hicks David B. , Krulwich Terry A.
Published: July 2002

Recent work has suggested that the chromosomally encoded TetA(L)
transporter of
Bacillus subtilis
, for which no
physiological function had been shown earlier, not only confers
resistance to low concentrations of tetracycline but is also a
multifunctional antiporter protein that has dominant roles in both
Na
+
- and K
+
-dependent pH homeostasis and in
Na
+
resistance during growth at alkaline pH. To rigorously
test this hypothesis, TetA(L) has been purified with a hexahistidine
tag at its C terminus and reconstituted into proteoliposomes. The
TetA(L)–hexahistidine proteoliposomes exhibit high activities of
tetracycline–cobalt/H
+
,
Na
+
/H
+
, and K
+
/H
+
antiport in an assay in which an outwardly directed proton gradient is
artificially imposed and solute uptake is monitored. Tetracycline
uptake depends on the presence of cobalt and
vice versa
,
with the cosubstrates being transported in a 1:1 ratio. Evidence for
the electrogenicity of both tetracycline–cobalt/H
+
and
Na
+
/H
+
antiports is presented. K
+
and Li
+
inhibit Na
+
uptake, but there is little
cross-inhibition between Na
+
and tetracycline–cobalt
uptake activities. The results strongly support the conclusion that
TetA(L) is a multifunctional antiporter. They expand the roster of such
porters to encompass one with a complex organic substrate and
monovalent cation substrates that may have distinct binding domains,
and provide the first functional reconstitution of a member of the
14-transmembrane segment transporter family.
31P magnetic resonance spectroscopy of the Sherpa heart: a phosphocreatine/adenosine triphosphate signature of metabolic defense against hypobaric hypoxia.
Authors: Hochachka P W , Clark C M , Holden J E , Stanley C , Ugurbil K , Menon R S
Published: July 2002

Of all humans thus far studied, Sherpas are considered by many high-altitude biomedical scientists as most exquisitely adapted for life under continuous hypobaric hypoxia. However, little is known about how the heart is protected in hypoxia. Hypoxia defense mechanisms in the Sherpa heart were explored by in vivo, noninvasive 31P magnetic resonance spectroscopy. Six Sherpas were examined under two experimental conditions [normoxic (21% FiO2) and hypoxic (11% FiO2) and in two adaptational states--the acclimated state (on arrival at low-altitude study sites) and the deacclimating state (4 weeks of ongoing exposure to low altitude). Four lowland subjects were used for comparison. We found that the concentration ratios of phosphocreatine (PCr)/adenosine triphosphate (ATP) were maintained at steady-state normoxic values (0.96, SEM = 0.22) that were about half those found in normoxic lowlanders (1.76, SEM = 0.03) monitored the same way at the same time. These differences in heart energetic status between Sherpas and lowlanders compared under normoxic conditions remained highly significant (P < 0.02) even after 4 weeks of deacclimation at low altitudes. In Sherpas under acute hypoxia, the heart rate increased by 20 beats per min from resting values of about 70 beats per min, and the percent saturation of hemoglobin decreased to about 75%. However, these perturbations did not alter the PCr/ATP concentration ratios, which remained at about 50% of the values expected in healthy lowlanders. Because the creatine phosphokinase reaction functions close to equilibrium, these steady-state PCr/ATP ratios presumably coincided with about 3-fold higher free adenosine diphosphate (ADP) concentrations. Higher ADP concentrations (i.e., lower [PCr]/[ATP] ratios) were interpreted to correlate with the Km values for ADP-requiring kinases of glycolysis and to reflect elevated carbohydrate contributions to heart energy needs. This metabolic organization is postulated as advantageous in hypobaria because the ATP yield per O2 molecule is 25-60% higher with glucose than with free fatty acids (the usual fuels utilized in the human heart in postfasting conditions).
Increased dopamine turnover in the prefrontal cortex impairs spatial working memory performance in rats and monkeys.
Authors: Murphy B L , Arnsten A F , Goldman-Rakic P S , Roth R H
Published: July 2002

The selective activation of the prefrontal cortical dopamine system by mild stress can be mimicked by anxiogenic beta-carbolines such as FG7142. To investigate the functional relevance of elevated levels of dopamine turnover in the prefrontal cortex, the current study examined the effects of FG7142 on the performance of spatial working memory tasks in the rat and monkey. FG7142 selectively increased prefrontal cortical dopamine turnover in rats and significantly impaired performance on spatial working memory tasks in both rats and monkeys. Spatial discrimination, a task with similar motor and motivational demands (rats), or delayed response performance following zero-second delays (monkeys) was unaffected by FG7142. Further, biochemical analysis in rats revealed a significant positive correlation between dopamine turnover in the prefrontal cortex and cognitive impairment on the delayed alternation task. The cognitive deficits in both rats and monkeys were prevented by pretreatment with the benzodiazepine receptor antagonist, RO15-1788, which blocked the increase in dopamine turnover and by the dopamine receptor antagonists, haloperidol, clozapine, and SCH23390. These findings indicate that excessive dopamine activity in the prefrontal cortex is detrimental to cognitive functions mediated by the prefrontal cortex.
Peroxynitrite disables the tyrosine phosphorylation regulatory mechanism: Lymphocyte-specific tyrosine kinase fails to phosphorylate nitrated cdc2(6-20)NH2 peptide.
Authors: Kong S K , Yim M B , Stadtman E R , Chock P B
Published: July 2002

To determine if nitration of tyrosine residues by peroxynitrite (PN), which can be generated endogenously, can disrupt the phosphorylation of tyrosine residues in proteins involved in cell signaling networks, we studied the effect of PN-promoted nitration of tyrosine residues in a pentadecameric peptide, cdc2(6-20)NH2, on the ability of the peptide to be phosphorylated. cdc2(6-20)NH2 corresponds to the tyrosine phosphorylation site of p34cdc2 kinase, which is phosphorylated by lck kinase (lymphocyte-specific tyrosine kinase, p56lck). PN nitrates both Tyr-15 and Tyr-19 of the peptide in phosphate buffer (pH 7.5) at 37 degrees C. Nitration of Tyr-15. which is the phosphorylated amino acid residue, inhibits completely the phosphorylation of the peptide. The nitration reaction is enhanced by either Fe(III)EDTA or Cu(II)-Zn(II)-superoxide dismutase (Cu,Zn-SOD). The kinetic data are consistent with the view that reactions of Fe(111)EDTA or Cu,Zn-SOD with the cis form of PN yield complexes in which PN decomposes more slowly to form N02+, the nitrating agent. Thus, the nitration efficiency of PN is enhanced. These results are discussed from the point of view that PN-promoted nitration will result in permanent impairment of cyclic cascades that control signal transduction processes and regulate cell cycles.
Parkinson disease: A new link between monoamine oxidase and mitochondrial electron flow
Authors: Cohen Gerald , Farooqui Rafay , Kesler Natasa
Published: July 2002

Two factors that contribute to the progression of Parkinson disease
are a brain defect in mitochondrial respiration and the generation of
hydrogen peroxide (H
2
O
2
) by monoamine oxidase
(MAO). Here we show that the two are linked. Metabolism of the
neurotransmitter dopamine, or other monoamines (benzylamine, tyramine),
by intact rat brain mitochondria suppresses pyruvate- and
succinate-dependent electron transport. MAO inhibitors prevent this
action. Mitochondrial damage is also reversed during electron flow. A
probable explanation is that MAO-generated H
2
O
2
oxidizes glutathione to glutathione disulfide (GSSG), which undergoes
thiol-disulfide interchange to form protein mixed disulfides, thereby
interfering reversibly with thiol-dependent enzymatic function. In
agreement with this premise, direct addition of GSSG to mitochondria
resulted in similar reversible inhibition of electron transport. In
addition, the monoamines induced an elevation in protein mixed
disulfides within mitochondria. These observations imply that
(
i
) heightened activity and metabolism of
neurotransmitter by monoamine neurons may affect neuronal function, and
(
ii
) apparent defects in mitochondrial respiration
associated with Parkinson disease may reflect, in part, an established
increase in dopamine turnover. The experimental results also target
mitochondrial repair mechanisms for further investigation and may, in
time, lead to newer forms of therapy.
Activation of mitogen-activating protein kinase by glucose is not required for insulin secretion
Authors: Khoo Shih , Cobb Melanie H.
Published: July 2002

In the insulinoma cell line INS-1, a model system for glucose-regulated insulin secretion, the mitogen-activating protein (MAP) kinases/extracellular signal-regulated protein kinases, ERK1 and ERK2 are activated up to 15-fold by physiological concentrations of glucose, in the range of 3–12 mM. The related MAP kinase family members, the c-Jun–N-terminal kinases/stress-activated protein kinases are insensitive to glucose, while the p38 MAP kinase is slightly glucose responsive (1.5-fold). ERK activation is dependent on glucose metabolism and the subsequent increase in calcium influx. Inhibiting activation of ERK1 and ERK2 with the MEK1/2 inhibitor PD98059 has no effect on insulin secretion, indicating that ERK activity is not necessary for secretion under these conditions. Glucose activates ERK1 and ERK2 in cytosolic and purified nuclear fractions of INS-1 cells and more of each is found in nuclei from glucose-treated cells. These findings suggest that some of the glucose-dependent actions of ERKs will be exerted in the nucleus.
Transient aggregates in protein folding are easily mistaken for folding intermediates
Authors: Silow Maria , Oliveberg Mikael
Published: July 2002

It has been questioned recently whether populated intermediates are important for the protein folding process or are artefacts trapped in nonproductive pathways. We report here that the rapidly formed intermediate of the spliceosomal protein U1A is an off-pathway artefact caused by transient aggregation of denatured protein under native conditions. Transient aggregates are easily mistaken for structured monomers and could be a general problem in time-resolved folding studies.
c-Myc transactivation of
LDH-A
: Implications for tumor metabolism and growth
Authors: Shim Hyunsuk , Dolde Christine , Lewis Brian C. , Wu Chyi-Sun , Dang Gerard , Jungmann Richard A. , Dalla-Favera Riccardo , Dang Chi V.
Published: July 2002

Cancer cells are able to overproduce lactic acid aerobically, whereas normal cells undergo anaerobic glycolysis only when deprived of oxygen. Tumor aerobic glycolysis was recognized about seven decades ago; however, its molecular basis has remained elusive. The lactate dehydrogenase-A gene (
LDH-A
), whose product participates in normal anaerobic glycolysis and is frequently increased in human cancers, was identified as a c-Myc-responsive gene. Stably transfected Rat1a fibroblasts that overexpress
LDH-A
alone or those transformed by c-Myc overproduce lactic acid.
LDH-A
overexpression is required for c-Myc-mediated transformation because lowering its level through antisense
LDH-A
expression reduces soft agar clonogenicity of c-Myc-transformed Rat1a fibroblasts, c-Myc-transformed human lymphoblastoid cells, and Burkitt lymphoma cells. Although antisense expression of LDH-A did not affect the growth of c-Myc-transformed fibroblasts adherent to culture dishes under normoxic conditions, the growth of these adherent cells in hypoxia was reduced. These observations suggest that an increased LDH-A level is required for the growth of a transformed spheroid cell mass, which has a hypoxic internal microenvironment. Our studies have linked c-Myc to the induction of
LDH-A
, whose expression increases lactate production and is necessary for c-Myc-mediated transformation.
Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase
Authors: Rosenquist Thomas A. , Zharkov Dmitry O. , Grollman Arthur P.
Published: July 2002

Oxidative DNA damage is generated by reactive oxygen species. The mutagenic base, 8-oxoguanine, formed by this process, is removed from oxidatively damaged DNA by base excision repair. Genes coding for DNA repair enzymes that recognize 8-oxoguanine have been reported in bacteria and yeast. We have identified and characterized mouse and human cDNAs encoding homologs of the 8-oxoguanine DNA glycosylase (
ogg1
) gene of
Saccharomyces cerevisiae. Escherichia coli
doubly mutant for mutM and mutY have a mutator phenotype and are deficient in 8-oxoguanine repair. The recombinant mouse gene (mOgg1) suppresses the mutator phenotype of
mutY/mutM E. coli
. Extracts prepared from
mutY/mutM E. coli
expressing mOgg1 contain an activity that excises 8-oxoguanine from DNA and a β-lyase activity that nicks DNA 3′ to the lesion. The mouse
ogg1
gene product acts efficiently on DNA duplexes in which 7,8-dihydroxy-8-oxo-2′-deoxyguanosine (8-oxodG) is paired with dC, acts weakly on duplexes in which 8-oxodG is paired with dT or dG, and is inactive against duplexes in which 8-oxodG is paired with dA. Mouse and human
ogg1
genes contain a helix–hairpin–helix structural motif with conserved residues characteristic of a recently defined family of DNA glycosylases.
Ogg1
mRNA is expressed in several mouse tissues; highest levels were detected in testes. Isolation of the mouse
ogg1
gene makes it possible to modulate its expression in mice and to explore the involvement of oxidative DNA damage and associated repair processes in aging and cancer.
Proteasome regulation of activation-induced T cell death
Authors: Cui Haili , Matsui Ken , Omura Satoshi , Schauer Stephanie L. , Matulka Ray A. , Sonenshein Gail E. , Ju Shyr-Te
Published: July 2002

Lactacystin, a microbial metabolite that inhibits protease activity only in the proteasome, was used to study the role of the proteasome in the activation-induced cell death (AICD) of T cells. Lactacystin induces DNA fragmentation and apoptosis in a T cell hybridoma (DO.11.10) in a dose-dependent manner. Between 1 and 10 μM, the mildly cytotoxic lactacystin inhibited the AICD of DO.11.10 cells cultured in anti-CD3-coated wells. Degradation of IκBβ and the translocation of the NF-κB (p50/RelA) into the nucleus, which occurred at 1.5 hr after anti-CD3 activation, were inhibited by lactacystin. Lactacystin did not inhibit the expression of nuclear transcription factor Oct-1. The activation-induced expression of the immediate–early gene, Nur77, and the T cell death genes, CD95 (Fas) and CD95 ligand (FasL), were inhibited. Functional expression of FasL cytotoxicity and the increase of cell surface Fas were also inhibited. Lactacystin must be added within 2 hr of activation to efficiently block AICD. In addition, lactacystin failed to inhibit the killing of DO.11.10 by FasL-expressing allo-specific cytotoxic effector cells. These observations strongly suggest a direct link between the proteasome-dependent degradation of IκBβ and the AICD that occurs through activation of the FasL gene and up-regulation of the Fas gene.
Pig but not human interferon-γ initiates human cell-mediated rejection of pig tissue in vivo
Authors: Sultan Parvez , Murray Allan G. , McNiff Jennifer M. , Lorber Marc I. , Askenase Philip W. , Bothwell Alfred L. M. , Pober Jordan S.
Published: July 2002

Split-thickness pig skin was transplanted on severe combined immunodeficient mice so that pig dermal microvessels spontaneously inosculated with mouse microvessels and functioned to perfuse the grafts. Pig endothelial cells in the healed grafts constitutively expressed class I and class II major histocompatibility complex molecules. Major histocompatibility complex molecule expression could be further increased by intradermal injection of pig interferon-γ (IFN-γ) but not human IFN-γ or tumor necrosis factor. Grafts injected with pig IFN-γ also developed a sparse infiltrate of mouse neutrophils and eosinophils without evidence of injury. Introduction of human peripheral blood mononuclear cells into the animals by intraperitoneal inoculation resulted in sparse perivascular mononuclear cell infiltrates in the grafts confined to the pig dermis. Injection of pig skin grafts on mice that received human peripheral blood mononuclear cells with pig IFN-γ (but not human IFN-γ or heat-inactivated pig IFN-γ) induced human CD4+and CD8+T cells and macrophages to more extensivley infiltrate the pig skin grafts and injure pig dermal microvessels. These findings suggest that human T cell-mediated rejection of xenotransplanted pig organs may be prevented if cellular sources of pig interferon (e.g., passenger lymphocytes) are eliminated from the graft.
Typing of urinary JC virus DNA offers a novel means of tracing human migrations
Authors: Sugimoto Chie , Kitamura Tadaichi , Guo Jing , Al-Ahdal Mohammed N. , Shchelkunov Sergei N. , Otova Berta , Ondrejka Paul , Chollet Jean-Yves , El-Safi Sayda , Ettayebi Mohamed , Grésenguet Gérard , Kocagöz Tanil , Chaiyarasamee Sanong , Thant Kyaw Zin , Thein Soe , Moe Kyaw , Kobayashi Nobuyoshi , Taguchi Fumiaki , Yogo Yoshiaki
Published: July 2002

Although polyomavirus JC (JCV) is the proven pathogen of
progressive multifocal leukoencephalopathy, the fatal demyelinating
disease, this virus is ubiquitous as a usually harmless symbiote among
human beings. JCV propagates in the adult kidney and excretes its
progeny in urine, from which JCV DNA can readily be recovered. The main
mode of transmission of JCV is from parents to children through long
cohabitation. In this study, we collected a substantial number of urine
samples from native inhabitants of 34 countries in Europe, Africa, and
Asia. A 610-bp segment of JCV DNA was amplified from each urine sample,
and its DNA sequence was determined. A worldwide phylogenetic tree
subsequently constructed revealed the presence of nine subtypes
including minor ones. Five subtypes (EU, Af2, B1, SC, and CY) occupied
rather large territories that overlapped with each other at their
boundaries. The entire Europe, northern Africa, and western Asia were
the domain of EU, whereas the domain of Af2 included nearly all of
Africa and southwestern Asia all the way to the northeastern edge of
India. Partially overlapping domains in Asia were occupied by subtypes
B1, SC, and CY. Of particular interest was the recovery of JCV subtypes
in a pocket or pockets that were separated by great geographic
distances from the main domains of those subtypes. Certain of these
pockets can readily be explained by recent migrations of human
populations carrying these subtypes. Overall, it appears that JCV
genotyping promises to reveal previously unknown human migration
routes: ancient as well as recent.
The gene for albicidin detoxification from
Pantoea dispersa
encodes an esterase and attenuates pathogenicity of
Xanthomonas albilineans
to sugarcane
Authors: Zhang Lianhui , Birch Robert G.
Published: July 2002

Albicidin phytotoxins are pathogenicity factors in a devastating disease of sugarcane known as leaf scald, caused by
Xanthomonas albilineans
. A gene (
albD
) from
Pantoea dispersa
has been cloned and sequenced and been shown to code for a peptide of 235 amino acids that detoxifies albicidin. The gene shows no significant homology at the DNA or protein level to any known sequence, but the gene product contains a GxSxG motif that is conserved in serine hydrolases. The AlbD protein, purified to homogeneity by means of a glutathione
S
-transferase gene fusion system, showed strong esterase activity on
p-
nitrophenyl butyrate and released hydrophilic products during detoxification of albicidins. AlbD hydrolysis of
p-
nitrophenyl butyrate and detoxification of albicidins required no complex cofactors. Both processes were strongly inhibited by phenylmethylsulfonyl fluoride, a serine enzyme inhibitor. These data strongly suggest that AlbD is an albicidin hydrolase. The enzyme detoxifies albicidins efficiently over a pH range from 5.8 to 8.0, with a broad temperature optimum from 15 to 35°C. Expression of
albD
in transformed
X. albilineans
strains abolished the capacity to release albicidin toxins and to incite disease symptoms in sugarcane. The gene is a promising candidate for transfer into sugarcane to confer a form of disease resistance.