bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytotoxic analogs of luteinizing hormone-releasing hormone containing doxorubicin or 2-pyrrolinodoxorubicin, a derivative 500-1000 times more potent.
Authors: Nagy A , Schally A V , Armatis P , Szepeshazi K , Halmos G , Kovacs M , Zarandi M , Groot K , Miyazaki M , Jungwirth A , Horvath J
Published: July 2002

Doxorubicin (DOX) and its daunosamine-modified derivative, 2-pyrrolino-DOX, which is 500-1000 times more active than DOX, were incorporated into agonistic and antagonistic analogs of luteinizing hormone-releasing hormone (LH-RH). The conjugation of DOX with LH-RH analogs was performed by using N-(9-fluorenylmethoxycarbonyl)-DOX-14-O-hemiglutarate, a dicarboxylic acid ester derivative of DOX. Coupling this derivative covalently to the epsilon-amino group of the D-Lys side chain of agonist [D-Lys6]LH-RH or antagonistic analog AC-D-Nal(2)-D-Phe(4Cl)-D-Pal(3)-Ser-Tyr-D-Lys-Leu-Arg-Pro-D-Ala-NH 2 [where Nal(2) = 3-(2-naphthyl)alanine, Pal(3) = 3-(3-pyridyl)alanine, and Phe(4CI) = 4-chlorophenylalanine] was followed by the removal of the 9-fluorenylmethoxycarbonyl protective group to yield cytotoxic derivatives of LH-RH analogs containing DOX. From these DOX containing LH-RH hybrids, intensely potent analogs with daunosamine-modified derivatives of DOX can be readily formed. Thus, cytotoxic LH-RH agonist containing DOX (AN-152) can be converted in a 66% yield by a reaction with a 30-fold excess of 4-iodobutyraldehyde in N,N-dimethylformamide into a derivative having 2-pyrrolino-DOX (AN-207). Hybrid molecules AN-152 and AN-207 fully preserve the cytotoxic activity of their radicals, DOX or 2-pyrrolino-DOX, respectively, in vitro, and also retain the high binding affinity of the peptide hormone portion of the conjugates to rat pituitary receptors for LH-RH. These highly potent cytotoxic analogs of LH-RH were designed as targeted anti-cancer agents for the treatment of various tumors that possess receptors for the carrier peptide. Initial in vivo studies show that the hybrid molecules are much less toxic than the respective cytotoxic radicals incorporated and significantly more active in inhibiting tumor growth.
A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes.
Authors: Huber R , Hof P , Duarte R O , Moura J J , Moura I , Liu M Y , LeGall J , Hille R , Archer M , Romão M J
Published: July 2002

The crystal structure of the xanthine oxidase-related molybdenum-iron protein aldehyde oxido-reductase from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas (Mop) was analyzed in its desulfo-, sulfo-, oxidized, reduced, and alcohol-bound forms at 1.8-A resolution. In the sulfo-form the molybdenum molybdopterin cytosine dinucleotide cofactor has a dithiolene-bound fac-[Mo, = O, = S, ---(OH2)] substructure. Bound inhibitory isopropanol in the inner compartment of the substrate binding tunnel is a model for the Michaelis complex of the reaction with aldehydes (H-C = O,-R). The reaction is proposed to proceed by transfer of the molybdenum-bound water molecule as OH- after proton transfer to Glu-869 to the carbonyl carbon of the substrate in concert with hydride transfer to the sulfido group to generate [MoIV, = O, -SH, ---(O-C = O, -R)). Dissociation of the carboxylic acid product may be facilitated by transient binding of Glu-869 to the molybdenum. The metal-bound water is replenished from a chain of internal water molecules. A second alcohol binding site in the spacious outer compartment may cause the strong substrate inhibition observed. This compartment is the putative binding site of large inhibitors of xanthine oxidase.
Identification of a new calcitonin gene in the salmon Oncorhynchus gorbuscha.
Authors: Jansz H , Martial K , Zandberg J , Milhaud G , Benson A A , Julienne A , Moukhtar M S , Cressent M
Published: July 2002

Three isoforms of calcitonin (CT) exist in salmonids. Isohormones I and II are expressed in the pink salmon Oncorhynchus gorbuscha. We report here the existence in this species of a CT gene and of its transcripts, which encode for a fourth isohormone, the salmon CT (sCT) IV. This new CT gene was identified by PCR from genomic DNA and by sequencing the amplified DNA. The expression of this CT gene was established in ultimobranchial body and brain, by reverse transcription-PCR, hybridization and sequencing. The sCT IV gene, like the sCT I gene, is a complex transcription unit, containing exons encoding for a CT as a calcitonin gene-related peptide (CGRP) molecule. The predicted peptide, sCT IV, has a greater homology with the eel CT and the sCT II than with the sCT I. Alignment of the sCT IV with other fish and chicken CT showed amino acid modifications in similar positions as those found during evolution. The predicted salmon CGRP IV peptide is highly homologous to the known CGRP molecules in other species, confirming the high conservation of the molecule during evolution. This identification of a new salmon CT gene is interesting both for the therapeutic potential represented by the new molecules encoded by this gene and for phylogenetic studies.
The carboxyl terminus of the bacteriophage T4 DNA polymerase is required for holoenzyme complex formation
Authors: Berdis Anthony J. , Soumillion Patrice , Benkovic Stephen J.
Published: July 2002

To further elucidate the mechanism and dynamics of bacteriophage T4
holoenzyme formation, a mutant polymerase in which the last six
carboxyl-terminal amino acids are deleted, was constructed,
overexpressed, and purified to homogeneity. The mutant polymerase,
designated ΔC6 exo
−
, is identical to wild-type
exo
−
polymerase with respect to
k
cat
,
k
pol
, and
dissociation constants for nucleotide and DNA substrate. However,
unlike wild-type exo
−
polymerase, the ΔC6
exo
−
polymerase is unable to interact with the 45 protein
to form the stable holoenzyme. A synthetic polypeptide corresponding to
the carboxyl terminus of the wild-type exo
−
polymerase was
tested as an
in vitro
inhibitor of bacteriophage T4 DNA
replication. Surprisingly, the peptide does not directly inhibit
holoenzyme complex formation by disrupting the interaction of the
polymerase with the 45 protein. On the contrary, the peptide appears to
disrupt the interaction of the 44/62 protein with the 45 protein,
suggesting that the 44/62 protein and the polymerase use the same
site on the 45 protein for functional interactions. Data presented are
discussed in terms of a model correlating the functionality of the
carboxyl terminus of the polymerase for productive interactions with
the 45 protein as well as in terms of the 45 protein concomitantly
interacting with the 44/62 protein and polymerase.
NADPH-oxidase and a hydrogen peroxide-sensitive K
+
channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines
Authors: Wang Dashou , Youngson Charlotte , Wong Veronica , Yeger Herman , Dinauer Mary C. , de Miera Eleazar Vega-Saenz , Rudy Bernardo , Cutz Ernest
Published: July 2002

Pulmonary neuroepithelial bodies (NEB) are widely distributed
throughout the airway mucosa of human and animal lungs. Based on the
observation that NEB cells have a candidate oxygen sensor enzyme
complex (NADPH oxidase) and an oxygen-sensitive K
+
current,
it has been suggested that NEB may function as airway chemoreceptors.
Here we report that mRNAs for both the hydrogen peroxide sensitive
voltage gated potassium channel subunit (KH
2
O
2
)
KV3.3a and membrane components of NADPH oxidase (gp91
phox
and p22
phox
) are coexpressed in the NEB cells of fetal
rabbit and neonatal human lungs. Using a microfluorometry and
dihydrorhodamine 123 as a probe to assess H
2
O
2
generation, NEB cells exhibited oxidase activity under basal
conditions. The oxidase in NEB cells was significantly stimulated by
exposure to phorbol esther (0.1 μM) and inhibited by diphenyliodonium
(5 μM). Studies using whole-cell voltage clamp showed that the
K
+
current of cultured fetal rabbit NEB cells exhibited
inactivating properties similar to KV3.3a transcripts expressed in
Xenopus
oocyte model. Exposure of NEB cells to hydrogen
peroxide (H
2
O
2
, the dismuted by-product of the
oxidase) under normoxia resulted in an increase of the outward
K
+
current indicating that H
2
O
2
could be the transmitter modulating the O
2
-sensitive
K
+
channel. Expressed mRNAs or orresponding protein
products for the NADPH oxidase membrane cytochrome b as well as mRNA
encoding KV3.3a were identified in small cell lung carcinoma cell
lines. The studies presented here provide strong evidence for an
oxidase-O
2
sensitive potassium channel molecular complex
operating as an O
2
sensor in NEB cells, which function as
chemoreceptors in airways and in NEB related tumors. Such a complex may
represent an evolutionary conserved biochemical link for a membrane
bound O
2
-signaling mechanism proposed for other cells and
life forms.
RETRACTED: Tumor necrosis factor receptor-associated factor (TRAF)-1, TRAF-2, and TRAF-3 interact
in vivo
with the CD30 cytoplasmic domain; TRAF-2 mediates CD30-induced nuclear factor kappa B activation
Authors: Ansieau Stéphane , Scheffrahn Inka , Mosialos George , Brand Heike , Duyster Justus , Kaye Kenneth , Harada Josephine , Dougall Bill , Hübinger Gabi , Kieff Elliott , Herrmann Friedhelm , Leutz Achim , Gruss Hans-Jürgen
Published: July 2002

CD30 is a member of the tumor necrosis factor receptor
superfamily, which can transduce signals for proliferation, death, or
nuclear factor kappa B (NF-κB) activation. Investigation of CD30
signaling pathways using a yeast two-hybrid interaction system trapped
a cDNA encoding the tumor necrosis factor receptor-associated factor
(TRAF)-2 TRAF homology domain. TRAF-1 and TRAF-3 also interacted with
CD30, and >90% of
in vitro
-translated TRAF-1 or -2, or
50% of TRAF-3, bound to the CD30 cytoplasmic domain. TRAF-1, -2, and
-3 bound mostly, but not exclusively, to the carboxyl-terminal 36
residues of CD30. The binding was strongly inhibited by a CD30
oligopeptide centered around a PXQXT (where X is any amino acid) motif
shared with CD40 and the Epstein–Barr virus transforming protein LMP1,
indicating that this motif in CD30 is an important determinant of
TRAF-1, -2 or -3 interaction. At least 15% of TRAF-1, -2, or -3
associated with CD30 when coexpressed in 293 cells. The association was
not affected by CD30 cross-linking. However, cross-linking of CD30
activated NF-κB. NF-κB activation was dependent on the
carboxyl-terminal 36 amino acids of CD30 that mediate TRAF association.
TRAF-2 has been previously shown to have a unique role in TRAF-mediated
NF-κB activation, and NF-κB activation following CD30 cross-linking
was blocked by a dominant negative TRAF-2 mutant. These data indicate
that CD30 cross-linking-induced NF-κB activation is predominantly
TRAF-2-mediated.
Intrastriatally injected
c-fos
antisense oligonucleotide interferes with striatonigral but not striatopallidal γ-aminobutyric acid transmission in the conscious rat
Authors: Sommer W. , Rimondini R. , O’Connor W. , Hansson A. C. , Ungerstedt U. , Fuxe K.
Published: July 2002

Antisense
c-fos
oligonucleotides injected into the
neostriatum of conscious rats selectively inhibited
c-fos
expression associated with compensatory increases
in striatal
c-fos
mRNA levels and also with increased
expression of
junB
and
NGFI-A
mRNA,
probably as a result of regulatory phenomena. Dual probe
in
vivo
microdialysis was used to investigate γ-aminobutyric
acid (GABA) release in the substantia nigra and the globus pallidus,
which represent the terminal sites of the dopamine D
1
receptor regulated striatonigral and the dopamine D
2
receptor regulated striatopallidal GABA pathways, respectively.
Intrastriatal infusion of the
c-fos
antisense
oligonucleotide profoundly decreased dialysate GABA levels in the
ipsilateral substantia nigra within 60 min but did not influence the
dialysate GABA levels in the globus pallidus compared with the sham and
control oligonucleotide treated groups. The site of action of the
antisense oligonucleotides was mainly restricted to striatal neurons as
shown by the distribution of locally injected fluoresceine
isothiocyanate and radiolabeled oligonucleotides. The findings
demonstrate a facilitatory role for
c-fos
mediated gene
regulation in striatonigral GABA transmission and strengthen the
evidence that the regulation of neurotransmission is different in the
striatonigral and striatopallidal GABA pathways.
Cloning of a gene (RIG-G) associated with retinoic acid-induced differentiation of acute promyelocytic leukemia cells and representing a new member of a family of interferon-stimulated genes
Authors: Yu Man , Tong Jian-Hua , Mao Mao , Kan Li-Xin , Liu Meng-Min , Sun Yi-Wu , Fu Gang , Jing Yong-Kui , Yu Long , Lepaslier Denis , Lanotte Michel , Wang Zhen-Yi , Chen Zhu , Waxman Samuel , Wang Ya-Xin , Tan Jia-Zhen , Chen Sai-Juan
Published: July 2002

In a cell line (NB4) derived from a patient with acute promyelocytic leukemia, all-trans-retinoic acid (ATRA) and interferon (IFN) induce the expression of a novel gene we callRIG-G(for retinoic acid-induced gene G). This gene codes for a 58-kDa protein containing 490 amino acids with several potential sites for post-translational modification. In untreated NB4 cells, the expression ofRIG-Gis undetectable. ATRA treatment induces the transcriptional expression ofRIG-Grelatively late (12–24 hr) in a protein synthesis-dependent manner, whereas IFN-α induces its expression early (30 min to 3 hr). Database search has revealed a high-level homology betweenRIG-Gand several IFN-stimulated genes in human (ISG54K,ISG56K, and IFN-inducible and retinoic acid-inducible 58K gene) and some other species, defining a well conserved gene family. The gene is composed of two exons and has been mapped by fluorescencein situhybridization to chromosome 10q24, where two other human IFN-stimulated gene members are localized. A synergistic induction ofRIG-Gexpression in NB4 cells by combined treatment with ATRA and IFNs suggests that a collaboration exists between their respective signaling pathways.
Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53
Authors: Sugrue Mary M. , Shin Deug Y. , Lee Sam W. , Aaronson Stuart A.
Published: July 2002

The p53 tumor suppressor gene has been shown to play an important role in determining cell fate. Overexpression of wild-type p53 in tumor cells has been shown to lead to growth arrest or apoptosis. Previous studies in fibroblasts have provided indirect evidence for a link between p53 and senescence. Here we show, using an inducible p53 expression system, that wild-type p53 overexpression in EJ bladder carcinoma cells, which have lost functional p53, triggers the rapid onset of G
1
and G
2
/M growth arrest associated with p21 up-regulation and repression of mitotic cyclins (cyclin A and B) and cdc2. Growth arrest in response to p53 induction became irreversible within 48-72 h, with cells exhibiting morphological features as well as specific biochemical and ultrastructural markers of the senescent phenotype. These findings provide direct evidence that p53 overexpression can activate the rapid onset of senescence in tumor cells.
The epitopes for some antiphospholipid antibodies are adducts of oxidized phospholipid and β2glycoprotein 1 (and other proteins)
Authors: Hörkkö Sohvi , Miller Elizabeth , Branch D. Ware , Palinski Wulf , Witztum Joseph L.
Published: July 2002

Circulating autoantibodies to phospholipids (aPLs), such as cardiolipin (CL), are found in patients with antiphospholipid antibody syndrome (APS). We recently demonstrated that many aPLs bound to CL only after it had been oxidized (OxCL), but not to a reduced CL analogue that could not undergo oxidation. We now show that the neoepitopes recognized by some aPLs consist of adducts formed between breakdown products of oxidized phospholipid and associated proteins, such as β2glycoprotein 1 (β2GP1). Addition of human β2GP1, polylysine, native low-density lipoprotein, or apolipoprotein AI to OxCL-coated wells increased the anticardiolipin antibody (aCL) binding from APS sera that first had been diluted so that no aCL binding to OxCL could be detected. No increase in aCL binding was observed when these proteins were added to wells coated with reduced CL. The ability of β2GP1, polylysine, or low-density lipoprotein to be a “cofactor” for aCL binding to OxCL was greatly reduced when the proteins were methylated. Incubation of β2GP1 with oxidized 1-palmitoyl-2-linoleyl-[1-14C]-phosphatidylcholine (PC), but not with dipalmitoyl-[1-14C]-PC, led to formation of covalent adducts with β2GP1 recognized by APS sera. These data suggest that the reactive groups of OxCL, such as aldehydes generated during the decomposition of oxidized polyunsaturated fatty acids, form covalent adducts with β2GP1 (and other proteins) and that these are epitopes for aCLs. Knowledge that the epitopes recognized by many aPLs are adducts of oxidized phospholipid and associated proteins, including β2GP1, may give new insights into the pathogenic events underlying the clinical manifestations of APS.
Inactivation of FtsI inhibits constriction of the FtsZ cytokinetic ring and delays the assembly of FtsZ rings at potential division sites
Authors: Pogliano Joe , Pogliano Kit , Weiss David S. , Losick Richard , Beckwith Jon
Published: July 2002

A universally conserved event in cell division is the formation of a cytokinetic ring at the future site of division. In the bacterium
Escherichia coli
, this ring is formed by the essential cell division protein FtsZ. We have used immunofluorescence microscopy to show that FtsZ assembles early in the division cycle, suggesting that constriction of the FtsZ ring is regulated and supporting the view that FtsZ serves as a bacterial cytoskeleton. Assembly of FtsZ rings was heterogeneously affected in an
ftsI
temperature-sensitive mutant grown at the nonpermissive temperature, some filaments displaying a striking defect in FtsZ assembly and others displaying little or no defect. By using low concentrations of the β-lactams cephalexin and piperacillin to specifically inhibit FtsI (PBP3), an enzyme that synthesizes peptidoglycan at the division septum, we show that FtsZ ring constriction requires the transpeptidase activity of FtsI. Unconstricted FtsZ rings are stably trapped at the midpoint of the cell for several generations after inactivation of FtsI, whereas partially constricted FtsZ rings are less effectively trapped. In addition, FtsZ rings are able to assemble in newborn cells in the presence of cephalexin, suggesting that newborn cells contain a site at which FtsZ can assemble (the nascent division site) and that the transpeptidase activity of FtsI is not required for assembly of FtsZ at these sites. However, aside from this first round of FtsZ ring assembly, very few additional FtsZ rings assemble in the presence of cephalexin, even after several generations of growth. One interpretation of these results is that the transpeptidase activity of FtsI is required, directly or indirectly, for the assembly of nascent division sites and thereby for future assembly of FtsZ rings.
Characterization of recombinant phytochrome from the cyanobacterium
Synechocystis
Authors: Lamparter Tilman , Mittmann Franz , Gärtner Wolfgang , Börner Thomas , Hartmann Elmar , Hughes Jon
Published: July 2002

The complete sequence of the
Synechocystis
chromosome has revealed a phytochrome-like sequence that yielded an authentic phytochrome when overexpressed in
Escherichia coli
. In this paper we describe this recombinant
Synechocystis
phytochrome in more detail. Islands of strong similarity to plant phytochromes were found throughout the cyanobacterial sequence whereas C-terminal homologies identify it as a likely sensory histidine kinase, a family to which plant phytochromes are related. An
≈
300 residue portion that is important for plant phytochrome function is missing from the
Synechocystis
sequence, immediately in front of the putative kinase region. The recombinant apoprotein is soluble and can easily be purified to homogeneity by affinity chromatography. Phycocyanobilin and similar tetrapyrroles are covalently attached within seconds, an autocatalytic process followed by slow conformational changes culminating in red-absorbing phytochrome formation. Spectral absorbance characteristics are remarkably similar to those of plant phytochromes, although the conformation of the chromophore is likely to be more helical in the
Synechocystis
phytochrome. According to size-exclusion chromatography the native recombinant apoproteins and holoproteins elute predominantly as 115- and 170-kDa species, respectively. Both tend to form dimers
in vitro
and aggregate under low salt conditions. Nevertheless, the purity and solubility of the recombinant gene product make it a most attractive model for molecular studies of phytochrome, including x-ray crystallography.
Xenopus Zic
3, a primary regulator both in neural and neural crest development
Authors: Nakata Katsunori , Nagai Takeharu , Aruga Jun , Mikoshiba Katsuhiko
Published: July 2002

Xenopus Zic3
is a
Xenopus
homologue of mouse
Zic
and
Drosophila
pair-rule gene,
odd-paired
. We show here that
Zic3
has significant roles both in neural and neural crest development in
Xenopus
embryo. Expression of
Zic3
is first detected in prospective neural plate region at gastrulation. Onset of the expression was earlier than most proneural genes and followed
chordin
expression. The expression was induced by blockade of BMP4 signal. Overexpression of
Zic3
resulted in hyperplastic neural and neural crest derived tissue. In animal cap explant, the overexpression of
Zic3
induced expression of all the proneural genes and neural crest marker genes. These findings suggest that
Zic3
can determine the ectodermal cell fate and promote the earliest step of neural and neural crest development.
Correct protein folding in glycerol
Authors: Rariy Roman V. , Klibanov Alexander M.
Published: July 2002

Water is the natural medium for protein folding, which is also used in all
in vitro
studies. In the present work, we posed, and answered affirmatively, a question of whether it is possible to fold correctly a typical protein in a nonaqueous solvent. To this end, unfolded and reduced hen egg-white lysozyme was refolded and reoxidized in glycerol containing varying amounts of water. The unfolded/reduced enzyme was found to regain spontaneously substantial catalytic activity even in the nearly anhydrous solvent; for example, the refolding yield in 99% glycerol was still some one-third of that in pure water, and one-half of that was regained even in 99.8% glycerol. The less than full recovery of the enzymatic activity in glycerol is, as in water, because of competing protein aggregation during the refolding. Lysozyme reoxidation in glycerol was successfully mediated by two dissimilar oxidizing systems, and the refolding yield was markedly affected by the pH of the last aqueous solution before the transfer into glycerol. No recovery of the lysozyme activity was observed when the refolding/reoxidation reaction was carried out in the denaturing solvent dimethyl sulfoxide. This study paves the way for a systematic investigation of the solvent effect on protein folding and demonstrates that water is not a unique milieu for this process.
Identification of a prion protein epitope modulating transmission of bovine spongiform encephalopathy prions to transgenic mice
Authors: Scott Michael R. , Safar Jiri , Telling Glenn , Nguyen Oanh , Groth Darlene , Torchia Marilyn , Koehler Ruth , Tremblay Patrick , Walther Dirk , Cohen Fred E. , DeArmond Stephen J. , Prusiner Stanley B.
Published: July 2002

There is considerable concern that bovine prions from cattle with bovine spongiform encephalopathy (BSE) may have been passed to humans (Hu), resulting in a new form of Creutzfeldt–Jakob disease (CJD). We report here the transmission of bovine (Bo) prions to transgenic (Tg) mice expressing BoPrP; one Tg line exhibited incubation times of ≈200 days. Like most cattle with BSE, vacuolation and astrocytic gliosis were confined in the brainstems of these Tg mice. Unexpectedly, mice expressing a chimeric Bo/Mo PrP transgene were resistant to BSE prions whereas mice expressing Hu or Hu/Mo PrP transgenes were susceptible to Hu prions. A comparison of differences in Mo, Bo, and Hu residues within the C terminus of PrP defines an epitope that modulates conversion of PrP
C
into PrP
Sc
and, as such, controls prion transmission across species. Development of susceptible Tg(BoPrP) mice provides a means of measuring bovine prions that may prove critical in minimizing future human exposure.
Glutathione deficiency is associated with impaired survival in HIV disease
Authors: Herzenberg Leonore A. , De Rosa Stephen C. , Dubs J. Gregson , Roederer Mario , Anderson Michael T. , Ela Stephen W. , Deresinski Stanley C. , Herzenberg Leonard A.
Published: July 2002

Glutathione (GSH), a cysteine-containing tripeptide, is essential for the viability and function of virtually all cells.In vitrostudies showing that low GSH levels both promote HIV expression and impair T cell function suggested a link between GSH depletion and HIV disease progression. Clinical studies presented here directly demonstrate that low GSH levels predict poor survival in otherwise indistinguishable HIV-infected subjects. Specifically, we show that GSH deficiency in CD4 T cells from such subjects is associated with markedly decreased survival 2–3 years after baseline data collection (Kaplan–Meier and logistic regression analyses,P< 0.0001 for both analyses). This finding, supported by evidence demonstrating that oral administration of the GSH prodrugN-acetylcysteine replenishes GSH in these subjects and suggesting thatN-acetylcysteine administration can improve their survival, establishes GSH deficiency as a key determinant of survival in HIV disease. Further, it argues strongly that the unnecessary or excessive use of acetaminophen, alcohol, or other drugs known to deplete GSH should be avoided by HIV-infected individuals.
Noninvasive assessment of the developing
Xenopus
cardiovascular system using optical coherence tomography
Authors: Boppart Stephen A. , Tearney Gary J. , Bouma Brett E. , Southern James F. , Brezinski Mark E. , Fujimoto James G.
Published: July 2002

Studies investigating normal and abnormal cardiac development are frequently limited by an inability to assess cardiovascular function within the intact organism. In this work, optical coherence tomography (OCT), a new method of micron-scale, noninvasive imaging based on the measurement of backscattered infrared light, was introduced for the high resolution assessment of structure and function in the developing
Xenopus laevis
cardiovascular system. Microstructural details, such as ventricular size and wall positions, were delineated with OCT at 16-μm resolution and correlated with histology. Three-dimensional representation of the cardiovascular system also was achieved by repeated cross-sectional imaging at intervals of 25 μm. In addition to structural information, OCT provides high speed
in vivo
axial ranging and imaging, allowing quantitative dynamic activity, such as ventricular ejection fraction, to be assessed. The sensitivity of OCT for dynamic assessment was demonstrated with an inotropic agent that altered cardiac function and dimensions. Optical coherence tomography is an attractive new technology for assessing cardiovascular development because of its high resolution, its ability to image through nontransparent structures, and its inexpensive portable design.
In vivo
and
in vitro
imaging are performed at a resolution approaching that of histopathology without the need for animal killing.
Rescue of cardiac α-actin-deficient mice by enteric smooth muscle γ-actin
Authors: Kumar A. , Crawford K. , Close L. , Madison M. , Lorenz J. , Doetschman T. , Pawlowski S. , Duffy J. , Neumann J. , Robbins J. , Boivin G. P. , O’Toole B. A. , Lessard J. L.
Published: July 2002

The muscle actins in higher vertebrates display highly conserved amino acid sequences, yet they show distinct expression patterns. Thus, cardiac α-actin, skeletal α-actin, vascular smooth muscle α-actin, and enteric smooth muscle γ-actin comprise the major actins in their respective tissues. To assess the functional and developmental significance of cardiac α-actin, the murine (129/SvJ) cardiac α-actin gene was disrupted by homologous recombination. The majority (≈56%) of the mice lacking cardiac α-actin do not survive to term, and the remainder generally die within 2 weeks of birth. Increased expression of vascular smooth muscle and skeletal α-actins is observed in the hearts of newborn homozygous mutants and also heterozygotes but apparently is insufficient to maintain myofibrillar integrity in the homozygous mutants. Mice lacking cardiac α-actin can be rescued to adulthood by the ectopic expression of enteric smooth muscle γ-actin using the cardiac α-myosin heavy chain promoter. However, the hearts of such rescued cardiac α-actin-deficient mice are extremely hypodynamic, considerably enlarged, and hypertrophied. Furthermore, the transgenically expressed enteric smooth muscle γ-actin reduces cardiac contractility in wild-type and heterozygous mice. These results demonstrate that alterations in actin composition in the fetal and adult heart are associated with severe structural and functional perturbations.
Biodiversity at the edge: A test of the importance of spatial “mass effects” in the Rothamsted Park Grass experiments
Authors: Kunin William E.
Published: July 2002

The coexistence of many plant species competing for a few resources is one of the central puzzles of community ecology. One explanation is that different species may be competitively superior in different microhabitats. Many species could then coexist within each piece of a mosaic landscape by what has been termed “mass effects,” because subpopulations in areas with negative growth rates would be supplemented by propagules from areas with reproductive surpluses. If mass effects are important, plant species diversity should increase near habitat boundaries, especially where habitat differences are moderate. In the first experimental test of this prediction, plants were censused on 54 transects within the long-established Rothamsted Park Grass plots. Very few showed significant declines in species richness with distance from subplot boundaries. Nonetheless, the regression coefficients were negative much more often than expected by chance, suggesting that weak mass effects operated. The effect was strongest where neighboring subplots differed greatly, with no evidence of the predicted decline where differences were extreme. Detailed analyses of transects with apparent mass effects revealed few species that behaved as predicted. This study serves both to provide evidence of the existence of mass effects and to question their importance in the maintenance of local plant diversity in this system.
Patterns of variance in stage-structured populations: Evolutionary predictions and ecological implications
Authors: Pfister Catherine A.
Published: July 2002

Variability in population growth rate is thought to have negative consequences for organism fitness. Theory for matrix population models predicts that variance in population growth rate should be the sum of the variance in each matrix entry times the squared sensitivity term for that matrix entry. I analyzed the stage-specific demography of 30 field populations from 17 published studies for pattern between the variance of a demographic term and its contribution to population growth. There were no instances in which a matrix entry both was highly variable and had a large effect on population growth rate; instead, correlations between estimates of temporal variance in a term and contribution to population growth (sensitivity or elasticity) were overwhelmingly negative. In addition, survivorship or growth sensitivities or elasticities always exceeded those of fecundity, implying that the former two terms always contributed more to population growth rate. These results suggest that variable life history stages tend to contribute relatively little to population growth rates because natural selection may alter life histories to minimize stages with both high sensitivity and high variation.
Coupling the folding of homologous proteins
Authors: Keasar Chen , Tobi Dror , Elber Ron , Skolnick Jeff
Published: July 2002

The empirical observation that homologous proteins fold to similar
structures is used to enhance the capabilities of an
ab
initio
algorithm to predict protein conformations. A penalty
function that forces homologous proteins to look alike is added to the
potential and is employed in the coupled energy optimization of several
homologous proteins. Significant improvement in the quality of the
computed structures (as compared with the computational folding of a
single protein) is demonstrated and discussed.
Crystal structure of chemically synthesized [N33A] stromal cell-derived factor 1α, a potent ligand for the HIV-1 “fusin” coreceptor
Authors: Dealwis Chris , Fernandez Elias J. , Thompson Darren A. , Simon Reyna J. , Siani Michael A. , Lolis Elias
Published: July 2002

Stromal cell-derived factor-1α (SDF-1α ) is a member of the chemokine superfamily and functions as a growth factor and chemoattractant through activation of CXCR4/LESTR/Fusin, a G protein-coupled receptor. This receptor also functions as a coreceptor for T-tropic syncytium-inducing strains of HIV-1. SDF-1α antagonizes infectivity of these strains by competing with gp120 for binding to the receptor. The crystal structure of a variant SDF-1α ([N33A]SDF-1α ) prepared by total chemical synthesis has been refined to 2.2-Å resolution. Although SDF-1α adopts a typical chemokine β-β-β-α topology, the packing of the α-helix against the β-sheet is strikingly different. Comparison of SDF-1α with other chemokine structures confirms the hypothesis that SDF-1α may be either an ancestral protein from which all other chemokines evolved or the chemokine that is the least divergent from a primordial chemokine. The structure of SDF-1α reveals a positively charged surface ideal for binding to the negatively charged extracellular loops of the CXCR4 HIV-1 coreceptor. This ionic complementarity is likely to promote the interaction of the mobile N-terminal segment of SDF-1α with interhelical sites of the receptor, resulting in a biological response.
NF-κB activation and interleukin 6 production in fibroblasts by estrogen receptor-negative breast cancer cell-derived interleukin 1α
Authors: Bhat-Nakshatri Poornima , Newton Thomas R. , Goulet Robert , Nakshatri Harikrishna
Published: July 2002

Several angiogenic factors and extracellular matrix-degrading enzymes that promote invasion and metastasis of cancer are produced by stromal fibroblasts that surround cancer cells. The expression of genes that code for some of these proteins is regulated by the transcription factor NF-κB. In this report, we demonstrate that conditioned medium (CM) from estrogen receptor (ER)-negative but not ER-positive breast cancer cells induces NF-κB in fibroblasts. In contrast, CM from both ER-positive and ER-negative breast cancer cells induces NF-κB in macrophages and endothelial cells. NF-κB activation in fibroblasts was accompanied by induction of interleukin 6 (IL-6) and urokinase plasminogen activator (uPA), both of which promote angiogenesis and metastasis. A survey of cytokines known for their ability to induce NF-κB identified IL-1α as the factor responsible for NF-κB activation in fibroblasts. Analysis of primary breast carcinomas revealed the presence of IL-1α transcripts in majority of lymph node-positive breast cancers. These results along with the known role of IL-1α and IL-6 in osteoclast formation provide insight into the mechanism of metastasis and hypercalcemia in advanced breast cancers.
Agrobacterium
VirD2 protein interacts with plant host cyclophilins
Authors: Deng Wanyin , Chen Lishan , Wood Derek W. , Metcalfe Tracee , Liang Xiaoyou , Gordon Milton P. , Comai Luca , Nester Eugene W.
Published: July 2002

Agrobacterium tumefaciens
induces crown gall tumors on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing plasmid, VirD2, an endonuclease covalently bound to the 5′ end of the T-DNA, and perhaps VirE2, a single-stranded DNA binding protein. The yeast two-hybrid system was used to screen for proteins interacting with VirD2 and VirE2 to identify components in
Arabidopsis thaliana
that interact with the T-complex. Three VirD2- and two VirE2-interacting proteins were identified. Here we characterize the interactions of VirD2 with two isoforms of
Arabidopsis
cyclophilins identified by using this analysis. The VirD2 domain interacting with the cyclophilins is distinct from the endonuclease, omega, and the nuclear localization signal domains. The VirD2–cyclophilin interaction is disrupted
in vitro
by cyclosporin A, which also inhibits
Agrobacterium
-mediated transformation of
Arabidopsis
and tobacco. These data strongly suggest that host cyclophilins play a role in T-DNA transfer.
Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: Involvement of MEK upstream of Bad phosphorylation
Authors: Scheid Michael P. , Duronio Vincent
Published: July 2002

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. We have shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and we now show that when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. Interleukin 3 (IL-3), on the other hand, requires PI3K for survival, and blocking PI3K partially inhibited Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras–mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it did not stimulate Bad phosphorylation. Finally, although our data suggest that the MAPK pathway is not required for inhibition of apoptosis, we provide evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. Our data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect.
A way of following individual cells in the migrating slugs of
Dictyostelium discoideum
Authors: Bonner J. T.
Published: July 2002

In the development of the cellular slime mold
Dictyostelium discoideum
there is a stage in which the aggregated amoebae form a migrating slug that moves forward in a polar fashion, showing sensitive orientation to environmental cues, as well as early signs of differentiation into anterior prestalk and posterior prespore cells. Heretofore it has been difficult to follow the movement of the individual cells within the slug, but a new method is described in which small, flat (one cell thick) slugs are produced in a glass-mineral oil interface where one can follow the movement of all the cells. Observations of time-lapse videos reveal the following facts about slug migration: (
i
) While the posterior cells move straight forward, the anterior cells swirl about rapidly in a chaotic fashion. (
ii
) Turning involves shifting the high point of these hyperactive cells. (
iii
) Both the anterior and the posterior cells move forward on their own power as the slug moves forward. (
iv
) There are no visible regular oscillations within the slug. (
v
) The number of prestalk and prespore cells is proportional for a range of sizes of these mini-slugs. All of these observations on thin slugs are consistent with what one finds in normal, three-dimensional slugs.
Effects of transition metals on nitric oxide synthase catalysis
Authors: Perry Jason M. , Marletta Michael A.
Published: July 2002

The biosynthesis of nitric oxide (NO) by the enzyme NO synthase (NOS) proceeds by the hydroxylation of
l
-arginine to form
N
G
-hydroxy-
l
-arginine followed by the conversion of
N
G
-hydroxy-
l
-arginine to
l
-citrulline and NO. The previously identified requirements of this relatively complicated reaction include several protein-bound cofactors: cytochrome P450-type heme, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and tetrahydrobiopterin (H
4
B). In addition to
l
-arginine, NOS also requires the substrates NADPH and molecular oxygen. The role of H
4
B in NOS catalysis has long been a subject of debate and uncertainty fueled, in part, by the failure to detect any dependence of the NOS reaction on nonheme iron, a cofactor integral to catalysis in every other H
4
B-dependent enzyme. Here we report the ability of NOS to bind transition metals stoichiometrically, and demonstrate that the rate of catalysis is enhanced by nonheme iron. We also show that other divalent transition metals, including Cu, Zn, Co, and Ni, inhibit NOS catalysis. Also, the addition of Cu
2+
to NOS inhibits heme reduction, whereas the addition of Fe
2+
does not. Overall, the results appear to connect NOS to the known H
4
B/nonheme iron-dependent hydroxylases, and suggest a similar, if not identical, step in the NOS reaction mechanism.
ATP hydrolysis catalyzed by human replication factor C requires participation of multiple subunits
Authors: Cai Jinsong , Yao Nina , Gibbs Emma , Finkelstein Jeff , Phillips Barbara , O’Donnell Michael , Hurwitz Jerard
Published: July 2002

Human replication factor C (hRFC) is a five-subunit protein complex (p140, p40, p38, p37, and p36) that acts to catalytically load proliferating cell nuclear antigen onto DNA, where it recruits DNA polymerase δ or ɛ to the primer terminus at the expense of ATP, leading to processive DNA synthesis. We have previously shown that a subcomplex of hRFC consisting of three subunits (p40, p37, and p36) contained DNA-dependent ATPase activity. However, it is not clear which subunit(s) hydrolyzes ATP, as all five subunits include potential ATP binding sites. In this report, we introduced point mutations in the putative ATP-binding sequences of each hRFC subunit and examined the properties of the resulting mutant hRFC complex and the ATPase activity of the hRFC or the p40·p37·p36 complex. A mutation in any one of the ATP binding sites of the p36, p37, p40, or p140 subunits markedly reduced replication activity of the hRFC complex and the ATPase activity of the hRFC or the p40·p37·p36 complex. A mutation in the ATP binding site of the p38 subunit did not alter the replication activity of hRFC. These findings indicate that the replication activity of hRFC is dependent on efficient ATP hydrolysis contributed to by the action of four hRFC subunits.
De novo
adipogenesis in mice at the site of injection of basement membrane and basic fibroblast growth factor
Authors: Kawaguchi Nobuko , Toriyama Kazuhiro , Nicodemou-Lena Eleni , Inou Kazuhiko , Torii Shuhei , Kitagawa Yasuo
Published: July 2002

Autografting of fat pads has a long history in plastic and reconstructive surgery for augmentation of lost soft tissue. However, the results are disappointing because of absorption of the grafts with time. The fate of transplanted fat is linked to adipose precursor cells distributed widely in connective tissues. Adipocyte precursor cells can proliferate and mature into adipocytes even in the adult body depending on microenvironment. When reconstituted basement membrane, Matrigel, supplemented with more than 1 ng/ml bFGF was injected s.c. into 6-week-old mice, the neovascularization induced within 1 week was followed by migration of endogenous adipose precursor cells, and a clearly visible fat pad was formed. The pad grew until 3 weeks after the injection and persisted for at least 10 weeks. Such
de novo
adipogenesis was induced reproducibly by s.c. injection of Matrigel and bFGF over the chest, lateral abdomen, or head. Adipogenesis could be induced even in ear cartilage or in muscle. Thus, our results demonstrated that an abundant population of adipose precursor cells is distributed widely in connective tissues of the adult body and that they migrate into the neovascularized plug of Matrigel for proliferation and maturation. These results suggest a technique of augmenting lost soft tissue in plastic and reconstructive surgery.
Reconstitution of hyperacetylated, DNase I-sensitive chromatin characterized by high conformational flexibility of nucleosomal DNA
Authors: Krajewski Wladyslaw A. , Becker Peter B.
Published: July 2002

Increased acetylation at specific N-terminal lysines of core histones is a hallmark of active chromatin
in vivo
, yet the structural consequences of acetylation leading to increased gene activity are only poorly defined. We employed a new approach to characterize the effects of histone acetylation: A
Drosophila
embryo-derived cell-free system for chromatin reconstitution under physiological conditions was programmed with exogenous histones to assemble hyperacetylated or matching control chromatin of high complexity. Hyperacetylated chromatin resembled unmodified chromatin at similar nucleosome density with respect to its sensitivity toward microccal nuclease, its nucleosomal repeat length, and the incorporation of the linker histone H1. In contrast, DNA in acetylated chromatin showed an increased sensitivity toward DNase I and a surprisingly high degree of conformational flexibility upon temperature shift pointing to profound alterations of DNA/histone interactions. This successful reconstitution of accessible and flexible chromatin outside of a nucleus paves the way for a thorough analysis of the causal relationship between histone acetylation and gene function.
Detection of P450c17-independent pathways for dehydroepiandrosterone (DHEA) biosynthesis in brain glial tumor cells
Authors: Cascio C. , Prasad V. V. K. , Lin Y. Y. , Lieberman S. , Papadopoulos V.
Published: July 2002

Dehydroepiandrosterone (D) is biosynthesized in the brain by a pathway different from that existing in the adrenal cortex. C6 rat glioma tumor cells in culture biosynthesize both pregnenolone (P) and D. They possess the mRNA, protein, and side-chain cleavage activity of P450scc. On the other hand, P450c17 was not detected. Adding FeSO
4
to C6 cells increased the synthesis of both P and D. Even in the presence of aminoglutethimide, an inhibitor of P450scc, FeSO
4
increased the synthesis of both steroids, indicating that the Fe
2+
-sensitive process does not involve P450scc. Likewise, the FeSO
4
-induced formation of D was not blocked by the P450c17 inhibitor, SU-10603. These results suggest that the FeSO
4
-induced synthesis of D as well as of P in C6 cells may be due to the fragmentation of
in situ
-formed tertiary hydroperoxides. It is likely, however, that the effect of the Fe
2+
is not limited to this one reaction. When exogenous P was added to C6 microsomes, along with FeSO
4
, the amount of D formed was greater than control values, indicating that Fe
2+
facilitated the conversion of P to D. Unlike the constituents that are converted by Fe
2+
to P, the precursor of D in C6 cells is not soluble in a 1:1 mixture of ether and ethylacetate. Treatment of C6 cells with KI, NaBH
4
, or HIO
4
resulted in an increase in D synthesis. From this it seems clear that a precursor of the D produced in C6 cells is a steroid where both C-17 and C-20 are oxygenated.