bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutrophils employ the myeloperoxidase system to generate antimicrobial brominating and chlorinating oxidants during sepsis
Authors: Gaut Joseph P. , Yeh George C. , Tran Hung Duy , Byun Jaeman , Henderson Jeffrey P. , Richter Grace M. , Brennan Marie-Luise , Lusis Aldons J. , Belaaouaj Abderrazzaq , Hotchkiss Richard S. , Heinecke Jay W.
Published: July 2002

The myeloperoxidase system of neutrophils uses hydrogen peroxide and chloride to generate hypochlorous acid, a potent bactericidal oxidant
in vitro
. In a mouse model of polymicrobial sepsis, we observed that mice deficient in myeloperoxidase were more likely than wild-type mice to die from infection. Mass spectrometric analysis of peritoneal inflammatory fluid from septic wild-type mice detected elevated concentrations of 3-chlorotyrosine, a characteristic end product of the myeloperoxidase system. Levels of 3-chlorotyrosine did not rise in the septic myeloperoxidase-deficient mice. Thus, myeloperoxidase seems to protect against sepsis
in vivo
by producing halogenating species. Surprisingly, levels of 3-bromotyrosine also were elevated in peritoneal fluid from septic wild-type mice and were markedly reduced in peritoneal fluid from septic myeloperoxidase-deficient mice. Furthermore, physiologic concentrations of bromide modulated the bactericidal effects of myeloperoxidase
in vitro
. It seems, therefore, that myeloperoxidase can use bromide as well as chloride to produce oxidants
in vivo
, even though the extracellular concentration of bromide is at least 1,000-fold lower than that of chloride. Thus, myeloperoxidase plays an important role in host defense against bacterial pathogens, and bromide might be a previously unsuspected component of this system.
The maternal effect gene,
abnormal oocyte
(
abo
), of
Drosophila melanogaster
encodes a specific negative regulator of histones
Authors: Berloco Maria , Fanti Laura , Breiling Achim , Orlando Valerio , Pimpinelli Sergio
Published: July 2002

The
abnormal oocyte
(
abo
) gene of
Drosophila melanogaster
is a peculiar maternal effect gene whose mutations cause a maternal-effect lethality that can be rescued by specific regions of heterochromatin during early embryogenesis. Here we show that
abo
encodes an evolutionary conserved chromosomal protein that localizes exclusively to the histone gene cluster and binds to the regulatory regions of such genes. We also show a significant increase of histone transcripts in eggs of
abo
mutant mothers and a partial rescue of the
abo
maternal-effect defect by deficiencies of the histone gene cluster. On the basis of these results, we suggest that the Abo protein functions specifically as a negative regulator of histone transcription and propose a molecular model to account for the ability of heterochromatin to partially rescue the
abo
maternal-effect defect. Our model proposes that increased doses of specific heterochromatic regions titrate out abnormally high levels of histones present in embryos from mutant
abo
mothers and that a balanced pool of histones is critical for normal embryogenesis in
Drosophila
.
Role of p42/p44 mitogen-activated-protein kinase and p21
waf1/cip1
in the regulation of vascular smooth muscle cell proliferation by nitric oxide
Authors: Bauer Philip M. , Buga Georgette M. , Ignarro Louis J.
Published: July 2002

The purpose of this study was to determine the involvement of the p42/p44 mitogen-activated protein kinase (MAPK) pathway and induction of p21
waf1/cip1
in the antiproliferative effects of nitric oxide (NO) on rat aortic smooth muscle cells (RASMC). NO, like α-difluoromethylornithine (DFMO), interferes with cell proliferation by inhibiting ornithine decarboxylase (ODC) and, therefore, polyamine synthesis.
S
-nitroso-
N
-acetylpenicillamine or (
Z
)-1-[
N
-(2-aminoethyl)-
N
-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate inhibited RASMC growth at concentrations as low as 3 μM, and DFMO elicited effects at concentrations of 100 μM or greater. The cytostatic effect of NO and DFMO was prevented by the MAPK kinase 1/2 inhibitors PD 098,059 or U0126. This finding suggests that the p42/p44 MAPK pathway is involved in the inhibition of RASMC proliferation by NO. Western blot analysis revealed that treatment of RASMC with NO or DFMO leads to activation of p42/p44 MAPK and induction of p21
waf1/cip1
. This effect was prevented by MAPK kinase 1/2 inhibitors, suggesting that induction of p21
waf1/cip1
depended on activation of p42/p44. Moreover, activation of p42/p44 and induction of p21
waf1/cip1
were prevented by exogenous putrescine but not ornithine, suggesting this effect was due to the inhibition of ODC by NO or DFMO. Finally, activation of p42/p44 MAPK and induction of p21
waf1/cip1
were cGMP-independent. Neither 1H-(1,2,4)oxadiazolo[4,3-α]quinoxalin-1-one nor zaprinast influenced the cytostatic effect of NO or DFMO or their ability to activate these signal transduction pathways. These observations suggest that inhibition of ODC and accompanying putrescine production are the underlying mechanisms by which NO and DFMO activate the MAPK pathway to promote induction of p21
waf1/cip1
and consequent inhibition of cell proliferation.
High-pressure transformations in xenon hydrates
Authors: Sanloup Chrystèle , Mao Ho-kwang , Hemley Russell J.
Published: July 2002

A high-pressure investigation of the Xe⋅H
2
O
chemical system was conducted by using diamond-anvil cell techniques
combined with
in situ
Raman spectroscopy, synchrotron
x-ray diffraction, and laser heating. Structure I xenon
clathrate was observed to be stable up to 1.8 GPa, at which pressure it
transforms to a new Xe clathrate phase stable up to 2.5 GPa before
breaking down to ice VII plus solid xenon. The bulk modulus and
structure of both phases were determined: 9 ± 1 GPa for Xe
clathrate A with structure I (cubic,
a
=
11.595 ± 0.003 Å,
V
= 1,558.9 ± 1.2
Å
3
at 1.1 GPa) and 45 ± 5 GPa for Xe clathrate B
(tetragonal,
a
= 8.320 ± 0.004 Å,
c
= 10.287 ± 0.007 Å,
V
= 712.1 ± 1.2 Å
3
at 2.2 GPa). The extended pressure
stability field of Xe clathrate structure I (A) and the discovery of a
second Xe clathrate (B) above 1.8 GPa have implications for xenon in
terrestrial and planetary interiors.
A PP2C-type phosphatase dephosphorylates the PII signaling protein in the cyanobacterium
Synechocystis
PCC 6803
Authors: Irmler Angelika , Forchhammer Karl
Published: July 2002

The family of the PII signal transduction proteins contains the most highly conserved signaling proteins in nature. The cyanobacterial PII-homologue transmits signals of the cellular nitrogen status and carbon status through phosphorylation of a seryl-residue. To identify the enzyme responsible for dephosphorylation of the phosphorylated PII protein in
Synechocystis
PCC 6803, prospective phosphatase encoding genes were inactivated by targeted insertion of kanamycin resistance cassettes. Disruption of ORF sll1771 generates a mutant unable to dephosphorylate PII under various experimental conditions. On the basis of conserved signature motifs, the sll1771 product (termed PphA) is a member of the protein phosphatase 2C (PP2C) superfamily, which is characterized by Mg
2+
/Mn
2+
-dependent catalytic activity. Biochemical analysis of overexpressed and purified PphA confirms its PP2C-type enzymatic properties and demonstrated its reactivity toward the phosphorylated PII protein. Thus, PphA is the first protein phosphatase in
Synechocystis
PCC 6803 for which the physiological substrate and function is known.
Association with capsid proteins promotes nuclear targeting of simian virus 40 DNA.
Authors: Nakanishi A , Clever J , Yamada M , Li P P , Kasamatsu H
Published: July 2002

All animal DNA viruses except pox virus utilize the cell nucleus as the site for virus reproduction. Yet, a critical viral infection process, nuclear targeting of the viral genome, is poorly understood. The role of capsid proteins in nuclear targeting of simian virus 40 (SV40) DNA, which is assessed by the nuclear accumulation of large tumor (T) antigen, the initial sign of the infectious process, was tested by two independent approaches: antibody interception experiments and reconstitution experiments. When antibody against viral capsid protein Vp1 or Vp3 was introduced into the cytoplasm, the nuclear accumulation of T antigen was not observed in cells either infected or cytoplasmically injected with virion. Nuclearly introduced anti-Vp3 IgG also showed the inhibitory effect. In the reconstitution experiments, SV40 DNA was allowed to interact with protein components of the virus, either empty particles or histones, and the resulting complexes were tested for the capability of protein components to target the DNA to the nucleus from cytoplasm as effectively as the targeting of DNA in the mature virion. In cells injected with empty particle-DNA, but not in minichromosome-injected cells, T antigen was observed as effectively as in SV40-injected cells. These results demonstrate that SV40 capsid proteins can facilitate transport of SV40 DNA into the nucleus and indicate that Vp3, one of the capsid proteins, accompanies SV40 DNA as it enters the nucleus during virus infection.
The platelet-derived growth factor alpha-receptor is encoded by a growth-arrest-specific (gas) gene.
Authors: Lih C J , Cohen S N , Wang C , Lin-Chao S
Published: July 2002

Using the Escherichia coli lacZ gene to identify chromosomal loci that are transcriptionally active during growth arrest of NIH 3T3 fibroblasts, we found that an mRNA expressed preferentially in serum-deprived cells specifies the previously characterized alpha-receptor (alphaR) for platelet-derived growth factor (PDGF), which mediates mitogenic responsiveness to all PDGF isoforms. Both PDGFalphaR mRNA, which was shown to include a 111-nt segment encoded by a DNA region thought to contain only intron sequences, and PDGFalphaR protein accumulated in serum-starved cells and decreased as cells resumed cycling. Elevated PDGFalphaR gene expression during serum starvation was not observed in cells that had been transformed with oncogenes erbB2, src, or raf, which prevent starvation-induced growth arrest. Our results support the view that products of certain genes expressed during growth arrest function to promote, rather than restrict, cell cycling. We suggest that accumulation of the PDGFalphaR gene product may facilitate the exiting of cells from growth arrest upon mitogenic stimulation by PDGF, leading to the state of "competence" required for cell cycling.
Two Arabidopsis cyclin promoters mediate distinctive transcriptional oscillation in synchronized tobacco BY-2 cells.
Authors: Shaul O , Mironov V , Burssens S , Van Montagu M , Inze D
Published: July 2002

Cyclins are cell cycle regulators whose proteins oscillate dramatically during the cell cycle. Cyclin steady-state mRNA levels also fluctuate, and there are indications that both their rate of transcription and mRNA stability are under cell cycle control. Here, we demonstrate the transcriptional regulation of higher eukaryote cyclins throughout the whole cell cycle with a high temporal resolution. The promoters of two Arabidopsis cyclins, cyc3aAt and cyc1At, mediated transcriptional oscillation of the beta-glucuronidase (gus) reporter gene in stably transformed tobacco BY-2 cell lines. The rate of transcription driven by the cyc3aAt promoter was very low during G1, slowly increased during the S phase, peaked at the G2 phase and G2-to-M transition, and was down-regulated before early metaphase. In contrast, the rate of the cyc1At-related transcription increased upon exit of the S phase, peaked at the G2-to-M transition and during mitosis, and decreased upon exit from the M phase. This study indicates that transcription mechanisms that seem to be conserved among species play a significant role in regulating the mRNA abundance of the plant cyclins. Furthermore, the transcription patterns of cyc3aAt and cyc1At were coherent with their slightly higher sequence similarity to the A and B groups of animal cyclins, respectively, suggesting that they may fulfill comparable roles during the cell cycle.
Immunotargeting of antioxidant enzyme to the pulmonary endothelium.
Authors: Muzykantov V R , Atochina E N , Ischiropoulos H , Danilov S M , Fisher A B
Published: July 2002

Oxidative injury to the pulmonary endothelium has pathological significance for a spectrum of diseases. Administration of antioxidant enzymes, superoxide dismutase (SOD) and catalase (Cat), has been proposed as a method to protect endothelium. However, neither these enzymes nor their derivatives possess specific affinity to endothelium and do not accumulate in the lung. Previously we have described a monoclonal antibody to angiotensin-converting enzyme (ACE) that accumulates selectively in the lung after systemic injection in rats, hamsters, cats, monkeys, and humans. In the present work we describe a system for selective intrapulmonary delivery of CuZn-SOD and Cat conjugated with biotinylated anti-ACE antibody mAb 9B9 (b-mAb 9B9) by a streptavidin (SA)-biotin bridge. Both enzymes biotinylated with biotin ester at biotin/enzyme ratio 20 retain enzymatic activity and bind SA without loss of activity. We have constructed tri-molecular heteropolymer complexes consisting of b-mAb 9B9, SA, and biotinylated SOD or biotinylated Cat and have studied biodistribution and pulmonary uptake of these complexes in the rat after i.v. injection. Biodistribution of biotinylated enzymes was similar to that of nonmodified enzymes. Binding of SA markedly prolonged lifetime of biotinylated enzymes in the circulation. In contrast to enzymes conjugated with nonspecific IgG, other enzyme derivatives, and nonmodified enzymes, biotinylated enzymes conjugated with b-mAb 9B9 accumulated specifically in the rat lung (9% of injected SOD/g of lung tissue and 7.5% of injected Cat/g of lung tissue). Pulmonary uptake of nonmodified enzymes or derivatives with nonspecific IgG did not exceed 0.5% of injected dose/g. Both SOD and Cat conjugated with b-mAb 9B9 were retained in the rat lung for at least several hours. Trichloracetic acid-precipitable radiolabeled Cat was associated with microsomal and plasma membrane fractions of the lung tissue homogenate. Thus, modification of antioxidant enzymes with biotin and SA-mediated conjugation with b-mAb 9B9 prolongs the circulation of enzymes resulting in selective accumulation in the lung and intracellular delivery of enzymes to the pulmonary endothelium. These results provide the background for an approach to provide protection of pulmonary endothelium against oxidative insults.
Long-term culture of lymphohematopoietic stem cells.
Authors: Palacios R , Bucana C , Xie X
Published: July 2002

Pluripotent hematopoietic stem cells (PHSCs) show self-renewal and give rise to all blood cell types. The extremely low number of these cells in primary hematopoietic organs and the lack of culture systems that support proliferation of undifferentiated PHSCs have precluded the study of both the biology of these cells and their clinical application. We describe here cell lines and clones derived from PHSCs that were established from hematopoietic cells from the fetal liver or bone marrow of normal and p53-deficient mice with a combination of four growth factors. Most cell lines were Sca-1+, c-Kit+, PgP-1+, HSA+, and Lin- (B-220-, Joro 75-, 8C5-, F4/80-, CD4-, CD8-, CD3-, IgM-, and TER 119-negative) and expressed three new surface markers: Joro 177, Joro 184, and Joro 96. They did not synthesize RNA transcripts for several genes expressed at early stages of lymphocyte and myeloid/erythroid cell development. The clones were able to generate lymphoid, myeloid, and erythroid hematopoietic cells and to reconstitute the hematopoietic system of irradiated mice for a long time. The availability of lymphohematopoietic stem cell lines should facilitate the analysis of the molecular mechanisms that control self-renewal and differentiation and the development of efficient protocols for somatic gene therapy.
Membrane healing and restoration of contractility after mechanical injury in isolated skeletal muscle fibers of the frog.
Authors: Gonzalez-Serratos H , Rozycka M , Cordoba-Rodriguez R , Ortega A
Published: July 2002

In single isolated skeletal muscle fibers of the frog, we studied (i) the recovery from large sarcolemmal mechanical injuries of the response to electric stimulation and (ii) the integrity of the sarcolemma under the light microscope. In Ringer's solution, the damaged cells stopped contracting and deteriorated completely within 1 hr. In the presence of phosphatidylcholine (0.025 g/ml in Ringer's solution), the injured cells initially responded with local twitches. Within 0.5 hr, contractility and membrane integrity started to recover and both were back to control levels within 3 hr. When these cells were placed back in normal Ringer's solution, they remained viable and active for several hours. Our results suggest that phosphatidylcholine can protect muscle fibers from the effects of sarcolemmal injury.
Surface-epitope masking and expression cloning identifies the human prostate carcinoma tumor antigen gene PCTA-1 a member of the galectin gene family.
Authors: Su Z Z , Lin J , Shen R , Fisher P E , Goldstein N I , Fisher P B
Published: July 2002

The selective production of monoclonal antibodies (mAbs) reacting with defined cell surface-expressed molecules is now readily accomplished with an immunological subtraction approach, surface-epitope masking (SEM). Using SEM, prostate carcinoma (Pro 1.5) mAbs have been developed that react with tumor-associated antigens expressed on human prostate cancer cell lines and patient-derived carcinomas. Screening a human LNCaP prostate cancer cDNA expression library with the Pro 1.5 mAb identifies a gene, prostate carcinoma tumor antigen-1 (PCTA-1). PCTA-1 encodes a secreted protein of approximately 35 kDa that shares approximately 40% sequence homology with the N-amino terminal region of members of the S-type galactose-binding lectin (galectin) gene family. Specific galectins are found on the surface of human and marine neoplastic cells and have been implicated in tumorigenesis and metastasis. Primer pairs within the 3' untranslated region of PCTA-1 and reverse transcription-PCR demonstrate selective expression of PCTA-1 by prostate carcinomas versus normal prostate and benign prostatic hypertrophy. These findings document the use of the SEM procedure for generating mAbs reacting with tumor-associated antigens expressed on human prostate cancers. The SEM-derived mAbs have been used for expression cloning the gene encoding this human tumor antigen. The approaches described in this paper, SEM combined with expression cloning, should prove of wide utility for developing immunological reagents specific for and identifying genes relevant to human cancer.
A chromosomal locus required for copper resistance, competitive fitness, and cytochrome c biogenesis in Pseudomonas fluorescens.
Authors: Yang C H , Azad H R , Cooksey D A
Published: July 2002

A chromosomal locus required for copper resistance and competitive fitness was cloned from a strain of Pseudomonas fluorescens isolated from copper-contaminated agricultural soil. Sequence analysis of this locus revealed six open reading frames with homology to genes involved in cytochrome c biogenesis in other bacteria, helC, cycJ, cycK, tipB, cycL, and cycH, with the closest similarity being to the aeg-46.5(yej) region of the Escherichia coli chromosome. The proposed functions of these genes in other bacteria include the binding, transport, and coupling of heme to apocytochrome c in the periplasm of these Gram-negative bacteria. Putative heme-binding motifs were present in the predicted products of cycK and cycL, and TipB contained a putative disulfide oxidoreductase active site proposed to maintain the heme-binding site of the apocytochrome in a reduced state for ligation of heme. Tn3-gus mutagenesis showed that expression of the genes was constitutive but enhanced by copper, and confirmed that the genes function both in copper resistance and production of active cytochrome c. However, two mutants in cycH were copper-sensitive and oxidase-positive, suggesting that the functions of these genes, rather than cytochrome c oxidase itself, were required for resistance to copper.
A knock-out model of paroxysmal nocturnal hemoglobinuria: Pig-a(-) hematopoiesis is reconstituted following intercellular transfer of GPI-anchored proteins.
Authors: Dunn D E , Yu J , Nagarajan S , Devetten M , Weichold F F , Medof M E , Young N S , Liu J M
Published: July 2002

We created a "knockout" embryonic stem cell via targeted disruption of the phosphatidylinositol glycan class A (Pig-a) gene, resulting in loss of expression of cell surface glycosyl phosphatidylinositol-anchored proteins and reproducing the mutant phenotype of the human disease paroxysmal nocturnal hemoglobinuria. Morphogenesis of Pig-a- embryoid bodies (EB) in vitro was grossly aberrant and, unlike EB derived from normal embryonic stem cells, Pig-A EB produced no secondary hematopoietic colonies. Chimeric EB composed of control plus Pig-A- cells, however, appeared normal, and hematopoiesis from knock-out cells was reconstituted. Transfer in situ of glycosyl phosphatidylinositol-anchored proteins from normal to knock-out cells was demonstrated by two-color fluorescent analysis, suggesting a possible mechanism for these functional effects. Hematopoietic cells with mutated PIG-A genes in humans with paroxysmal nocturnal hemoglobinuria may be subject to comparable pathophysiologic processes and amenable to similar therapeutic protein transfer.
Antibody-induced engagement of beta 2 integrins on adherent human neutrophils triggers activation of p21ras through tyrosine phosphorylation of the protooncogene product Vav.
Authors: Zheng L , Sjölander A , Eckerdal J , Andersson T
Published: July 2002

It is known that beta 2 integrins are crucial for leukocyte cell-cell and cell-matrix interactions, and accumulating evidence now suggests that integrins serve not only as a structural link but also as a signal-transducing unit that controls adhesion-induced changes in cell functions. In the present study, we plated human neutrophils on surface-bound anti-beta 2 (CD18) antibodies and found that the small GTP-binding protein p21ras is activated by beta 2 integrins. Pretreatment of the cells with genistein, a tyrosine kinase inhibitor, led to a complete block of p21ras activation, an effect that was not achieved with either U73122, which abolishes the beta 2 integrin-induced Ca2+ signal, or wortmannin, which totally inhibits the phosphatidylinositol 3-kinase activity. Western blot analysis revealed that antibody-induced engagement of beta 2 integrins causes tyrosine phosphorylation of several proteins in the cells. One of these tyrosine-phosphorylated proteins had an apparent molecular mass of 95 kDa and was identified as the protooncogene product Vav, a p21ras guanine nucleotide exchange factor that is specifically expressed in cells of hematopoietic lineage. A role for Vav in the activation of p21ras is supported by the observations that antibody-induced engagement of beta 2 integrins causes an association of Vav with p21ras and that the effect of genistein on p21ras activation coincided with its ability to inhibit both the tyrosine phosphorylation of Vav and the Vav-p21ras association. Taken together, these results indicate that antibody-induced engagement of beta 2 integrins on neutrophils triggers tyrosine phosphorylation of Vav and, possibly through its association, a downstream activation of p21ras.
A molecular mechanism for the effect of lithium on development.
Authors: Klein P S , Melton D A
Published: July 2002

Lithium, one of the most effective drugs for the treatment of bipolar (manic-depressive) disorder, also has dramatic effects on morphogenesis in the early development of numerous organisms. How lithium exerts these diverse effects is unclear, but the favored hypothesis is that lithium acts through inhibition of inositol monophosphatase (IMPase). We show here that complete inhibition of IMPase has no effect on the morphogenesis of Xenopus embryos and present a different hypothesis to explain the broad action of lithium. Our results suggest that lithium acts through inhibition of glycogen synthase kinase-3 beta (GSK-3 beta), which regulates cell fate determination in diverse organisms including Dictyostelium, Drosophila, and Xenopus. Lithium potently inhibits GSK-3 beta activity (Ki = 2 mM), but is not a general inhibitor of other protein kinases. In support of this hypothesis, lithium treatment phenocopies loss of GSK-3 beta function in Xenopus and Dictyostelium. These observations help explain the effect of lithium on cell-fate determination and could provide insights into the pathogenesis and treatment of bipolar disorder.
The entry and clearance of Ca2+ at individual presynaptic active zones of hair cells from the bullfrog's sacculus.
Authors: Issa N P , Hudspeth A J
Published: July 2002

Neurotransmitter is released when Ca2+ triggers the fusion of synaptic vesicles with the plasmalemma. To study factors that regulate Ca2+ concentration at the presynaptic active zones of hair cells, we used laser-scanning confocal microscopy with the fluorescent Ca2+ indicator fluo 3. The experimental results were compared with the predictions of a model of presynaptic Ca2+ concentration in which Ca2+ enters a cell through a point source, diffuses from the entry site, and binds to fixed or mobile Ca2+ buffers. The observed time course and magnitude of fluorescence changes under a variety of conditions were well fit when the model included mobile molecules as the only Ca2+ buffer. The results confirm the localized entry of Ca2+ underlying neurotransmitter release and suggest that Ca2+ is cleared from an active zone almost exclusively by mobile buffer.
Estrogen reduces atherosclerotic lesion development in apolipoprotein E-deficient mice.
Authors: Bourassa P A , Milos P M , Gaynor B J , Breslow J L , Aiello R J
Published: July 2002

We have studied the effects of endogenous and exogenous estrogen on atherosclerotic lesions in apolipoprotein E-deficient mice. Female mice ovariectomized (OVX) at weaning displayed increases (P < 0.01) in fatty streak lesions in the proximal aorta and aortic sinus compared with female mice with intact ovarian function. These differences between the OVX and sham controls were apparent in both chow- and "Western-type" diet-fed mice. Moreover, increases in lesion size following OVX occurred without changes in plasma cholesterol. Hormone replacement with subdermal 17-beta-estradiol pellets releasing either 6, 14, or 28 micrograms/day significantly decreased (P < 0.001) atherosclerotic lesion area in both male and OVX female mice. In contrast, neither 17-alpha-estradiol (28 micrograms/day) or tamoxifen (85 micrograms/day) affected lesion progression in OVX female mice. In the Western diet-fed group, exogenous estradiol markedly reduced plasma cholesterol and triglycerides, whereas, in animals fed the chow diet, exogenous estrogen and tamoxifen treatment only decreased plasma and very low density lipoprotein triglycerides. However, lesion area was only weakly correlated with plasma cholesterol and triglycerides, 0.35 and 0.44 tau values, respectively (P < 0.01). In summary, in the apolipoprotein E-deficient mouse 17-beta-estradiol protects against atherosclerotic lesion formation, and this can only be partially explained through effects on plasma lipoprotein levels.
Cell fate determination in the vertebrate retina.
Authors: Cepko C L , Austin C P , Yang X , Alexiades M , Ezzeddine D
Published: July 2002

In the vertebrate central nervous system, the retina has been a useful model for studies of cell fate determination. Recent results from studies conducted in vitro and in vivo suggest a model of retinal development in which both the progenitor cells and the environment change over time. The model is based upon the notion that the mitotic cells within the retina change in their response properties, or "competence", during development. These changes presage the ordered appearance of distinct cell types during development and appear to be necessary for the production of the distinct cell types. As the response properties of the cells change, so too do the environmental signals that the cells encounter. Together, intrinsic properties and extrinsic cues direct the choice of cell fate.
Mimicry of the calcium-induced conformational state of troponin C by low temperature under pressure.
Authors: Foguel D , Suarez M C , Barbosa C , Rodrigues J J , Sorenson M M , Smillie L B , Silva J L
Published: July 2002

Calcium binding to the N-domain of troponin C initiates a series of conformational changes that lead to muscle contraction. Calcium binding provides the free energy for a hydrophobic region in the core of N-domain to assume a more open configuration. Fluorescence measurements on a tryptophan mutant (F29W) show that a similar conformational change occurs in the absence of Ca2+ when the temperature is lowered under pressure. The conformation induced by subzero temperatures binds the hydrophobic probe bis-aminonaphthalene sulfonate, and the tryptophan has the same fluorescence lifetime (7 ns) as in the Ca2+-bound form. The decrease in volume (delta V = -25.4 ml/mol) corresponds to an increase in surface area. Thermodynamic measurements suggest an enthalpy-driven conformational change that leads to an intermediate with an exposed N-domain core and a high affinity for Ca2+.
A zinc finger directory for high-affinity DNA recognition
Authors: Jamieson Andrew C. , Wang Hongming , Kim Sung-Hou
Published: July 2002

We have used two monovalent phage display libraries containing
variants of the Zif268 DNA-binding domain to obtain families of zinc
fingers that bind to alterations in the last 4 bp of the DNA sequence
of the Zif268 consensus operator, GCG TG
GGCG
. Affinity
selection was performed by altering the Zif268 operator three base
pairs at a time, and simultaneously selecting for sets of 16 related
DNA sequences. In this way, only four experiments were required to
select for all possible 64 combinations of DNA triplet sequences. The
results show that (
i
) for high-affinity DNA binding in
the range observed for the Zif268 wild-type complex
(
K
d
= 0.5–5 nM), finger 1 specifically
requires the arginine at the carboxy terminus of its recognition helix
that forms a bidentate hydrogen-bond with the guanine base
(
G
) in the crystal structure of Zif268 complexed to its DNA
operator sequence GCG TGG
G
CG; (
ii
) when the
guanine base (G) is replaced by A, C, or T, a lower-affinity family
(
K
d
⩾ 50 nM) can be detected that shows an
overall tendency to bind G-rich DNA; (
iii
) the residues
at position 2 on the finger 2 recognition helix do not appear to
interact strongly with the complementary 5′ base in the finger 1
binding site; and (
iv
) unexpected substitutions at the
amino terminus of finger 1 can occasionally result in specificity for
the 3′ base in the finger 1 binding site. A DNA recognition directory
was constructed for high-affinity zinc fingers that recognize all three
bases in a DNA triplet for seven sequences of the type GNN. Similar
approaches may be applied to other zinc fingers to broaden the scope of
the directory.
Energy transfer to a proton-transfer fluorescence probe: Tryptophan to a flavonol in human serum albumin
Authors: Sytnik Alexander , Litvinyuk Igor
Published: July 2002

A protein fluorescence probe system, coupling excited-state intermolecular Förster energy transfer and intramolecular proton transfer (PT), is presented. As an energy donor for this system, we used tryptophan, which transfers its excitation energy to 3-hydroxyflavone (3-HF) as a flavonol prototype, an acceptor exhibiting excited-state intramolecular PT. We demonstrate such a coupling in human serum albumin–3-HF complexes, excited via the single intrinsic tryptophan (Trp-214). Besides the PT tautomer fluorescence (λmax= 526 nm), these protein–probe complexes exhibit a 3-HF anion emission (λmax= 500 nm). Analysis of spectroscopic data leads to the conclusion that two binding sites are involved in the human serum albumin–3-HF interaction. The 3-HF molecule bound in the higher affinity binding site, located in the IIIA subdomain, has the association constant (k1) of 7.2 × 105M−1and predominantly exists as an anion. The lower affinity site (k2= 2.5 × 105M−1), situated in the IIA subdomain, is occupied by the neutral form of 3-HF (normal tautomer). Since Trp-214 is situated in the immediate vicinity of the 3-HF normal tautomer bound in the IIA subdomain, the intermolecular energy transfer for this donor/acceptor pair has a 100% efficiency and is followed by the PT tautomer fluorescence. Intermolecular energy transfer from the Trp-214 to the 3-HF anion bound in the IIIA subdomain is less efficient and has the rate of 1.61 × 108s−1, thus giving for the donor/acceptor distance a value of 25.5 Å.
A cytokine mRNA-destabilizing element that is structurally and functionally distinct from A+U-rich elements
Authors: Brown Cheryl Y. , Lagnado Cathy A. , Goodall Gregory J.
Published: July 2002

The control of mRNA stability is crucial to the regulation of cytokine expression. We describe here a novel, potent destabilizing element found in the 3′ untranslated region of granulocyte colony-stimulating factor mRNA. This element, which appears to require at least one stem–loop structure, we term the stem–loop destabilizing element (SLDE). Functionally equivalent elements appear to also exist in the interleukin 2 and interleukin 6 mRNAs. The SLDE is functionally distinct from the A+U-rich elements, which are also present in these and other cytokine mRNAs, because it destabilizes a chimeric mRNA in a tumor cell line in which A+U-rich elements do not function. In addition, the effect of the SLDE is insensitive to calcium ionophore and is therefore regulated independently of A+U destabilizing elements. The existence of two distinct mRNA-destabilizing elements provides an additional mechanism for the differential regulation of cytokine expression.
Symmetries in bacterial motility
Authors: Berg Howard C.
Published: July 2002

Descriptions are given of three kinds of symmetries encountered in
studies of bacterial locomotion, and of the ways in which they are
circumvented or broken. A bacterium swims at very low Reynolds number:
it cannot propel itself using reciprocal motion (by moving through a
sequence of shapes, first forward and then in reverse); cyclic motion
is required. A common solution is rotation of a helical filament,
either right- or left-handed. The flagellar rotary motor that drives
each filament generates the same torque whether spinning clockwise or
counterclockwise. This symmetry is broken by coupling to the filament.
Finally, bacterial populations, grown in a nutrient medium from an
inoculum placed at a single point, usually move outward in symmetric
circular rings. Under certain conditions, the cells excrete a
chemoattractant, and the rings break up into discrete aggregates that
can display remarkable geometric order.
NMR characterization and solution structure determination of the oxidized cytochrome
c
7
from
Desulfuromonas acetoxidans
Authors: Banci Lucia , Bertini Ivano , Bruschi Mireille , Sompornpisut Pornthep , Turano Paola
Published: July 2002

The solution structure of the three-heme electron transfer protein
cytochrome
c
7
from
Desulfuromonas
acetoxidans
is reported. The determination of the structure is
obtained through NMR spectroscopy on the fully oxidized, paramagnetic
form. The richness of structural motifs and the presence of three
prosthetic groups in a protein of 68 residues is discussed in
comparison with the four-heme cytochromes
c
3
already characterized through x-ray crystallography. In particular, the
orientation of the three hemes present in cytochrome
c
7
is similar to that of three out of four
hemes of cytochromes
c
3
. The reduction
potentials of the individual hemes, which have been obtained through
the sequence-specific assignment of the heme resonances, are discussed
with respect to the properties of the protein matrix. This information
is relevant for any attempt to understand the electron transfer
pathway.
Phenotype of arylsulfatase A-deficient mice: Relationship to human metachromatic leukodystrophy
Authors: Hess Barbara , Saftig Paul , Hartmann Dieter , Coenen Ruth , Lüllmann-Rauch Renate , Goebel Hans H. , Evers Meike , von Figura Kurt , D’Hooge Rudi , Nagels Guy , De Deyn Peter , Peters Christoph , Gieselmann Volkmar
Published: July 2002

Metachromatic leukodystrophy is a lysosomal sphingolipid storage
disorder caused by the deficiency of arylsulfatase A. The disease is
characterized by progressive demyelination, causing various neurologic
symptoms. Since no naturally occurring animal model of the disease is
available, we have generated arylsulfatase A-deficient mice. Deficient
animals store the sphingolipid cerebroside-3-sulfate in various
neuronal and nonneuronal tissues. The storage pattern is comparable to
that of affected humans, but gross defects of white matter were not
observed up to the age of 2 years. A reduction of axonal
cross-sectional area and an astrogliosis were observed in 1-year-old
mice; activation of microglia started at 1 year and was generalized at
2 years. Purkinje cell dendrites show an altered morphology. In the
acoustic ganglion numbers of neurons and myelinated fibers are severely
decreased, which is accompanied by a loss of brainstem
auditory-evoked potentials. Neurologic examination reveals significant
impairment of neuromotor coordination.
The ATM homologue
MEC1
is required for phosphorylation of replication protein A in yeast
Authors: Brush George S. , Morrow Dwight M. , Hieter Philip , Kelly Thomas J.
Published: July 2002

Replication protein A (RPA) is a highly conserved single-stranded
DNA-binding protein, required for cellular DNA replication, repair, and
recombination. In human cells, RPA is phosphorylated during the S and
G
2
phases of the cell cycle and also in response to
ionizing or ultraviolet radiation.
Saccharomyces
cerevisiae
exhibits a similar pattern of cell cycle-regulated
RPA phosphorylation, and our studies indicate that the
radiation-induced reactions occur in yeast as well. We have examined
yeast RPA phosphorylation during the normal cell cycle and in response
to environmental insult, and have demonstrated that the checkpoint gene
MEC1
is required for the reaction under all conditions
tested. Through examination of several checkpoint mutants, we have
placed RPA phosphorylation in a novel pathway of the DNA damage
response.
MEC1
is similar in sequence to human ATM, the
gene mutated in patients with ataxia-telangiectasia (A-T). A-T cells
are deficient in multiple checkpoint pathways and are hypersensitive to
killing by ionizing radiation. Because A-T cells exhibit a delay in
ionizing radiation-induced RPA phosphorylation, our results indicate a
functional similarity between
MEC1
and ATM, and suggest
that RPA phosphorylation is involved in a conserved eukaryotic DNA
damage-response pathway defective in A-T.
Template-nucleated alanine-lysine helices are stabilized by position-dependent interactions between the lysine side chain and the helix barrel.
Authors: Groebke K , Renold P , Tsang K Y , Allen T J , McClure K F , Kemp D S
Published: July 2002

The helicity in water has been determined for several series of alanine-rich peptides that contain single lysine residues and that are N-terminally linked to a helix-inducing and reporting template termed Ac-Hel1. The helix-propagating constant for alanine (sAla value) that best fits the properties of these peptides lies in the range of 1.01-1.02, close to the value reported by Scheraga and coworkers [Wojcik, J., Altmann, K.-H. & Scheraga, H.A. (1990) Biopolymers 30, 121-134], but significantly lower than the value assigned by Baldwin and coworkers [Chakrabartty, A., Kortemme, T. & Baldwin, R.L. (1994) Protein Sci. 3,843-852]. From a study of conjugates Ac-Hel1-Ala(n)-Lys-Ala(m)-NH2 and analogs in which the methylene portion of the lysine side chain is truncated, we find that the unusual helical stability of Ala(n)Lys peptides is controlled primarily by interactions of the lysine side chain with the helix barrel, and only passively by the alanine matrix. Using 1H NMR spectroscopy, we observe nuclear Overhauser effect crosspeaks consistent with proton-proton contacts expected for these interactions.
Aromatic amino acid transamination and methionine recycling in trypanosomatids.
Authors: Berger B J , Dai W W , Wang H , Stark R E , Cerami A
Published: July 2002

Although trypanosomatids are known to rapidly transaminate exogenous aromatic amino acids in vitro and in vivo, the physiological significance of this reaction is not understood. In postmitochondrial supernatants prepared from Trypanosoma brucei brucei and Crithidia fasciculata, we have found that aromatic amino acids were the preferred amino donors for the transamination of alpha-ketomethiobutyrate to methionine. Intact C. fasciculata grown in the presence of [15N]tyrosine were found to contain detectable [15N]methionine, demonstrating that this reaction occurs in situ in viable cells. This process is the final step in the recycling of methionine from methylthioadenosine, a product of decarboxylated S-adenosylmethionine from the polyamine synthetic pathway. Mammalian liver, in contrast, preferentially used glutamine for this reaction and utilized a narrower range of amino donors than seen with the trypanosomatids. Studies with methylthioadenosine showed that this compound was readily converted to methionine, demonstrating a fully functional methionine-recycling pathway in trypanosomatids.
Major histocompatibility complex class II-transfected tumor cells present endogenous antigen and are potent inducers of tumor-specific immunity
Authors: Armstrong Todd D. , Clements Virginia K. , Martin Brian K. , Ting Jenny P.-Y. , Ostrand-Rosenberg Suzanne
Published: July 2002

We have developed an immunotherapy in which tumor cells transfected with syngeneic major histocompatibility complex (MHC) class II genes are cell-based vaccines for the treatment of established tumor and metastatic disease. If this strategy is to be used clinically, convenient methods for generating class II+tumor cells are necessary. Interferon-γ treatment or transduction of the class II transactivator (CIITA) gene induces class II expression but also up-regulates the class II-associated accessory molecules, invariant chain (Ii) and DM. To determine if interferon-γ treatment and CIITA transduction are potential immunotherapies, we assessed the tumorigenicity of sarcoma cells expressing combinations of class II, Ii, and DM. Since we hypothesized that class II-transfected tumor cells not coexpressing Ii and DM present endogenously encoded tumor peptides, we have assessed the transfectants for antigen presentation activity to MHC class II-restricted antigen-specific CD4+T cells. Tumor challenge studies demonstrate that tumor cells expressing class II without coexpression of Ii or Ii plus DM are highly immunogenic and preferentially present endogenous antigens, while tumors coexpressing class II with Ii or Ii plus DM are not effective immunogens. Because tumor rejection correlates with expression of class II without coexpression of Ii and DM, the most efficacious vaccines will express MHC class II without coexpression of Ii and DM and will preferentially present endogenous antigen.
Fas-induced apoptosis of T cells occurs independently of ceramide generation
Authors: Watts Julian D. , Gu Ming , Polverino Anthony J. , Patterson Scott D. , Aebersold Ruedi
Published: July 2002

The Fas receptor is one of a number of important physiological inducers of programmed cell death (apoptosis). Current models for regulation of this process involve rapid conversion of sphingomyelin to ceramide by cellular sphingomyelinases. Induced changes in cellular levels of such sphingosine-based ceramides are normally extrapolated from measurements of sphingomyelinase activity or following their conversion to ceramide phosphate by treatment of cellular lipid extracts with bacterial diacylglycerol kinase (DAGK). To allow direct study of cellular sphingosine- and sphinganine-based ceramide levels, we developed a mass spectrometric technique capable of determining inducible changes in both overall ceramide levels and species distribution in cellular lipid preparations. Contrary to current models, we detected no changes in cellular ceramide levels up to 2 hr poststimulation of Jurkat T cells with an anti-Fas IgM, although this treatment did induce apoptosis. We also determined in the same system that, when utilizing the DAGK assay, increased phosphorylation of substrates that comigrated with ceramide standards was apparent but that this effect was due to an enhancement of DAGK activity rather than increases in levels of cellular ceramides as substrates
per se
. Thus, the first direct measurement of ceramides present in cells undergoing apoptosis indicates that, insofar as it can be measured, the induction of apoptosis does not involve the generation of sphingosine-based ceramides, contrary to many published accounts.
Endocrine modulation of the neurotoxicity of gp120: Implications for AIDS-related dementia complex
Authors: Brooke S. , Chan R. , Howard S. , Sapolsky R.
Published: July 2002

HIV infection often involves the development of AIDS-related dementia complex, a variety of neurologic, neuropsychologic, and neuropathologic impairments. A possible contributor to AIDS-related dementia complex is the HIV envelope glycoprotein gp120, which damages neurons via a complex glutamate receptor- and calcium-dependent cascade. We demonstrate an endocrine modulation of the deleterious effects of gp120 in primary hippocampal and cortical cultures. Specifically, we observe that gp120-induced calcium mobilization and neurotoxicity are exacerbated by glucocorticoids, the adrenal steroids secreted during stress. Importantly, this deleterious synergy can occur between gp120 and synthetic glucocorticoids (such as prednisone or dexamethasone) that are used clinically in high concentrations to treat severe cases of the
Pneumocystis carinii
pneumonia typical of HIV infection. Conversely, we also observe that estradiol protects neurons from the deleterious actions of gp120, reducing toxicity and calcium mobilization.
Yersiniasignals macrophages to undergo apoptosis and YopJ is necessary for this cell death
Authors: Monack Denise M. , Mecsas Joan , Ghori Nafisa , Falkow Stanley
Published: July 2002

PathogenicYersiniaspp. carry a large common plasmid that encodes a number of essential virulence determinants. Included in these factors are theYersinia-secreted proteins called Yops. We analyzed the consequences of wild-type and mutant strains ofYersinia pseudotuberculosisinteractions with the macrophage cell line RAW264.7 and murine bone marrow-derived macrophages. Wild-typeY. pseudotuberculosiskills ≈70% of infected RAW264.7 macrophages and marrow-derived macrophages after an 8-h infection. We show that the cell death mediated byY. pseudotuberculosisis apoptosis. MutantY. pseudotuberculosisthat do not make any Yop proteins no longer cause host cell death. Attachment to host cells via invasin or YadA is necessary for the cell death phenotype. Several Yop mutant strains that fail to express one or more Yop proteins were engineered and then characterized for their ability to cause host cell death. A mutant with a polar insertion in YpkA Ser/Thr kinase that does not express YpkA or YopJ is no longer able to cause apoptosis. In contrast, a mutant no longer making YopE or YopH (a tyrosine phosphatase) induces apoptosis in macrophages similar to wild type. WhenyopJis added in trans to theypkAyopJmutant, the ability of this strain to signal programmed cell death in macrophages is restored. Thus, YopJ is necessary for inducing apoptosis. The ability ofY. pseudotuberculosisto promote apoptosis of macrophages in cell culture suggests that this process is important for the establishment of infection in the host and for evasion of the host immune response.