bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct mechanisms underlie activation of hypothalamic neurosecretory neurons and their medullary catecholaminergic afferents in categorically different stress paradigms.
Authors: Li H Y , Ericsson A , Sawchenko P E
Published: July 2002

Intermittent electrical footshock induces c-fos expression in parvocellular neurosecretory neurons expressing corticotropin-releasing factor and in other visceromotor cell types of the paraventricular hypothalamic nucleus (PVH). Since catecholaminergic neurons of the nucleus of the solitary tract and ventrolateral medulla make up the dominant loci of footshock-responsive cells that project to the PVH, these were evaluated as candidate afferent mediators of hypothalamic neuroendocrine responses. Rats bearing discrete unilateral transections of this projection system were exposed to a single 30-min footshock session and sacrificed 2 hr later. Despite depletion of the aminergic innervation on the ipsilateral side, shock-induced up-regulation of Fos protein and corticotropin-releasing factor mRNA were comparable in strength and distribution in the PVH on both sides of the brain. This lesion did, however, result in a substantial reduction of Fos expression in medullary aminergic neurons on the ipsilateral side. These results contrast diametrically with those obtained in a systemic cytokine (interleukin 1) challenge paradigm, where similar cuts ablated the Fos response in the ipsilateral PVH but left intact the induction seen in the ipsilateral medulla. We conclude that (i) footshock-induced activation of medullary aminergic neurons is a secondary consequence of stress, mediated via a descending projection transected by our ablation, (ii) stress-induced activation of medullary aminergic neurons is not necessarily predictive of an involvement of these cell groups in driving hypothalamic visceromotor responses to a given stressor, and (iii) despite striking similarities in the complement of hypothalamic effector neurons and their afferents that may be activated by stresses of different types, distinct mechanisms may underlie adaptive hypothalamic responses in each.
The yeast ZRT1 gene encodes the zinc transporter protein of a high-affinity uptake system induced by zinc limitation.
Authors: Zhao H , Eide D
Published: July 2002

The yeast Saccharomyces cerevisiae has two separate systems for zinc uptake. One system has high affinity for substrate and is induced in zinc-deficient cells. The second system has lower affinity and is not highly regulated by zinc status. The ZRT1 gene encodes the transporter for the high-affinity system, called Zrt1p. The predicted amino acid sequence of Zrt1p is similar to that of Irt1p, a probable Fe(II) transporter from Arabidopsis thaliana. Like Irt1p, Zrt1p contains eight potential transmembrane domains and a possible metal-binding domain. Consistent with the proposed role of ZRT1 in zinc uptake, overexpressing this gene increased high-affinity uptake activity, whereas disrupting it eliminated that activity and resulted in poor growth of the mutant in zinc-limited media. Furthermore, ZRT1 mRNA levels and uptake activity were closely correlated, as was zinc-limited induction of a ZRT1-lacZ fusion. These results suggest that ZRT1 is regulated at the transcriptional level by the intracellular concentration of zinc. ZRT1 is an additional member of a growing family of metal transport proteins.
High yield conversion of doxorubicin to 2-pyrrolinodoxorubicin, an analog 500-1000 times more potent: structure-activity relationship of daunosamine-modified derivatives of doxorubicin.
Authors: Nagy A , Armatis P , Schally A V
Published: July 2002

A convenient, high yield conversion of doxorubicin to 3'-deamino-3'-(2''-pyrroline-1''-yl)doxorubicin is described. This daunosamine-modified analog of doxorubicin is 500-1000 times more active in vitro than doxorubicin. The conversion is effected by using a 30-fold excess of 4-iodobutyraldehyde in anhydrous dimethylformamide. The yield is higher than 85%. A homolog of this compound, 3'-deamino-3'-(1'',3''-tetrahydropyridine-1''-yl)doxorubicin, was also synthesized by using 5-iodovaleraldehyde. In this homolog, the daunosamine nitrogen is incorporated into a six- instead of a five-membered ring. This analog was 30-50 times less active than its counterpart with a five-membered ring. A similar structure-activity relationship was found when 3'-deamino-3'-(3''-pyrrolidone-1''-yl)doxorubicin (containing a five-membered ring) and 3'-deamino-3'-(3''-piperidone-1''-yl)doxorubicin (with a six-membered ring) were tested in vitro, the former being 5 times more potent than the latter. To further elucidate structure-activity relationships, 3'-deamino-3'-(pyrrolidine-1''-yl)doxorubicin, 3'-deamino-3'-(isoindoline-2''-yl)doxorubicin, 3'-deamino-3'-(2''-methyl-2''-pyrroline-1''-yl)doxorubicin, and 3'-deamino-3'-(3''-pyrroline-1''-yl)doxorubicin were also synthesized and tested. All the analogs were prepared by using reactive halogen compounds for incorporating the daunosamine nitrogen of doxorubicin into a five- or six-membered ring. These highly active antineoplastic agents can be used for incorporation into targeted cytotoxic analogs of luteinizing hormone-releasing hormone intended for cancer therapy.
Enhanced pathologic properties of Dutch-type mutant amyloid beta-protein.
Authors: Davis J , Van Nostrand W E
Published: July 2002

Cerebrovascular amyloid beta-protein (Abeta) deposition is a pathological feature of several related disorders including Alzheimer disease and hereditary cerebral hemorrhage with amyloidosis Dutch-type (HCHWA-D). HCHWA-D is caused by a point mutation in the gene that encodes the Abeta precursor and results in a Glu --> Gln substitution at position 22 of Abeta. In comparison to Alzheimer disease, the cerebrovascular Abeta deposition in HCHWA-D is generally more severe, often resulting in intracerebral hemorrhage when patients reach 50 years of age. We recently reported that Abeta(1-42), but not the shorter Abeta(1-40) induces pathologic responses in cultured human leptomeningeal smooth muscle cells including cellular degeneration that is accompanied by a marked increase in the levels of cellular Abeta precursor and soluble Abeta peptide. In the present study, we show that the HCHWA-D mutation converts the normally nonpathologic Abeta(1-40) into a highly pathologic form of the peptide for cultured human leptomeningeal smooth muscle cells. These findings suggest that these altered functional properties of HCHWA-D mutated Abeta may contribute to the early and often severe cerebrovascular pathology that is the hallmark of this disorder.
Defective dimerization of von Willebrand factor subunits due to a Cys-> Arg mutation in type IID von Willebrand disease.
Authors: Schneppenheim R , Brassard J , Krey S , Budde U , Kunicki T J , Holmberg L , Ware J , Ruggeri Z M
Published: July 2002

The same heterozygous T -> C transition at nt 8567 of the von Willebrand factor (vWF) transcript was found in two unrelated patients with type III) von Willebrand disease, with no other apparent abnormality. In one family, both alleles were normal in the parents and one sister; thus, the mutation originated de novo in the proposita. The second patient also had asymptomatic parents who, however, were not available for study. The structural consequences of the identified mutation, resulting in the CyS2010 -> Arg substitution, were evaluated by expression of the vWF carboxyl-terminal domain containing residues 1366-2050. Insect cells infected with recombinant baculovirus expressing normal vWF sequence secreted a disulfide linked dimeric molecule with an apparent molecular mass of 150 kDa before reduction, yielding a single band of 80 kDa after disulfide bond reduction. In contrast, cells expressing the mutant fragment secreted a monomeric molecule of apparent molecular mass of 80 kDa, which remained unchanged after reduction. We conclude that CyS2010 is essential for normal dimerization of vWF subunits through disulfide bonding of carboxyl-terminal domains and that a heterozygous mutation in the corresponding codon is responsible for defective multimer formation in type III) von Willebrand disease.
β cell apoptosis in T cell-mediated autoimmune diabetes
Authors: Kurrer Michael O. , Pakala Syamasundar V. , Hanson Holly L. , Katz Jonathan D.
Published: July 2002

Insulin-dependent diabetes mellitus results from T cell-mediated destruction of insulin-producing, pancreatic islet β cells. How this destruction takes place has remained elusive—largely due to the slow kinetics of disease progression. By crossing a transgenic mouse carrying a β cell-specific T cell receptor onto the NOD.
scid
background, we produced a simplified but robust and accelerated model of diabetes. This mouse produces CD4
+
T cells bearing transgenic T cell receptor but is devoid of CD8
+
T cells and B cells. More importantly, this mouse develops a rapid diabetes, which has allowed us to record and quantify β cell death. We have determined that β cells within the inflamed islets die by apoptosis.
Targeted null-mutation in the
vascular endothelial–cadherin
gene impairs the organization of vascular-like structures in embryoid bodies
Authors: Vittet Daniel , Buchou Thierry , Schweitzer Annie , Dejana Elisabetta , Huber Philippe
Published: July 2002

Vascular endothelial–cadherin (VE–cadherin) is exclusively expressed in endothelial cells and is strictly located at cell-to-cell junctions. As the other members of the cadherin family, VE–cadherin is able to mediate a homotypic type of cellular interaction in a Ca
2+
-dependent manner. In the mouse embryo, VE–cadherin transcripts are detected at the earliest stages of vascular development. To ascertain if VE–cadherin expression is required for the assembly of endothelial cells into vascular structures, we generated
VE–cadherin
-negative mouse embryonic stem cells (
VE–cadherin
−/−
ES cells) by gene targeting and examined the consequences on vascular development of ES-derived embryoid bodies (EBs). In contrast to wild-type EBs, we observed that endothelial cells remained dispersed and failed to organize a vessel-like pattern in
VE–cadherin
−/−
ES-derived EBs. However, dispersed
VE–cadherin
−/−
ES-derived endothelial cells expressed a large range of other endothelial markers. Moreover, the targeted null-mutation in the
VE–cadherin
locus did not interfere with the hematopoietic differentiation potential of ES cells. These
in vitro
experiments are consistent with a pivotal role of VE–cadherin in vascular structure assembly.
A cluster of bacterial genes for anaerobic benzene ring biodegradation
Authors: Egland Paul G. , Pelletier Dale A. , Dispensa Marilyn , Gibson Jane , Harwood Caroline S.
Published: July 2002

A reductive benzoate pathway is the central conduit for the anaerobic biodegradation of aromatic pollutants and lignin monomers. Benzene ring reduction requires a large input of energy and this metabolic capability has, so far, been reported only in bacteria. To determine the molecular basis for this environmentally important process, we cloned and analyzed genes required for the anaerobic degradation of benzoate and related compounds from the phototrophic bacterium,
Rhodopseudomonas palustris
. A cluster of 24 genes was identified that includes twelve genes likely to be involved in anaerobic benzoate degradation and additional genes that convert the related compounds 4-hydroxybenzoate and cyclohexanecarboxylate to benzoyl-CoA. Genes encoding benzoyl-CoA reductase, a novel enzyme able to overcome the resonance stability of the aromatic ring, were identified by directed mutagenesis. The gene encoding the ring-cleavage enzyme, 2-ketocyclohexanecarboxyl-CoA hydrolase, was identified by assaying the enzymatic activity of the protein expressed in
Escherichia coli
. Physiological data and DNA sequence analyses indicate that the benzoate pathway consists of unusual enzymes for ring reduction and cleavage interposed among enzymes homologous to those catalyzing fatty acid degradation. The cloned genes should be useful as probes to identify benzoate degradation genes from other metabolically distinct groups of anaerobic bacteria, such as denitrifying bacteria and sulfate-reducing bacteria.
GPIIIa-(49–66) is a major pathophysiologically relevant antigenic determinant for anti-platelet GPIIIa of HIV-1-related immunologic thrombocytopenia
Authors: Nardi Michael A. , Liu Lin-Xing , Karpatkin Simon
Published: July 2002

High-affinity (
K
d
= 1 × 10
−9
M) anti-platelet GPIIIa has been isolated from serum immune complexes of immunologic thrombocytopenic HIV-1-infected patients (HIV-1-ITP). Affinity-purified anti-platelet antibody reacted with a recombinant GPIIIa-(1–200) and -(1–66) fusion peptide and with an 18-mer GPIIIa-(49–66) peptide but not with seven other GPIIIa peptides spanning the length of GPIIIa. Most of the anti-platelet antibody (≈85%) could be adsorbed to and eluted from a GPIIIa-(49–66) affinity column. Binding of antibody to platelets could be inhibited by GPIIIa-(49–66) or an equimolar peptide-albumin conjugate (IC
50
= 2 μM). Sera from 7 control subjects and 10 classic autoimmune thrombocytopenic patients gave background reactivity with GPIIIa-(49–66). HIV-1-ITP sera from 16 patients reacted with a mean OD 6-fold greater than background (range, 4- to 9-fold). Serum anti-GPIIIa-(49–66) concentration correlated inversely with platelet count,
R
2
= 0.51,
n
= 31,
P
< 0.0001. Because mouse platelet GPIIIa-(49–66) has 83% homology with human GPIIIa and mouse monocytes contain Fc receptors for the human IgG1-κ/λ antibody, we determined the
in vivo
effect of human anti-GPIIIa on mouse platelets. Affinity-purified antibody, 25–50 μg given i.p., resulted in a precipitous drop in platelet count to 30% of baseline, with nadir at 4 hr and return to normal in 36 hr. No effect was noted with control IgG. Acute thrombocytopenia could be prevented or reversed by the injection of the GPIIIa-(49–66) albumin conjugate at zero time or 2 hr after antibody, respectively, but not with a scrambled peptide-albumin conjugate. Thus HIV-1-ITP patients have high-affinity anti-platelet GPIIIa against a major antigenic determinant, GPIIIa-(49–66), which correlates inversely with platelet count and induces thrombocytopenia in mice.
A single serine residue controls the cation dependence of substrate transport by the rat serotonin transporter
Authors: Sur Cyrille , Betz Heinrich , Schloss Patrick
Published: July 2002

The serotonin transporter (SERT) is a member of the
Na
+
/Cl
−
-dependent neurotransmitter
transporter family and constitutes the target of several clinically
important antidepressants. Here, replacement of serine-545 in the
recombinant rat SERT by alanine was found to alter the cation
dependence of serotonin uptake. Substrate transport was now driven as
efficiently by LiCl as by NaCl without significant changes in serotonin
affinity. Binding of the antidepressant [
3
H]imipramine
occurred with 1/5th the affinity, whereas
[
3
H]citalopram binding was unchanged. These results
indicate that serine-545 is a crucial determinant of both the cation
dependence of serotonin transport by SERT and the imipramine binding
properties of SERT.
Abnormal skeletal patterning in embryos lacking a single
Cbp
allele: A partial similarity with Rubinstein–Taybi syndrome
Authors: Tanaka Yasunori , Naruse Ichiro , Maekawa Toshio , Masuya Hiroshi , Shiroishi Toshihiko , Ishii Shunsuke
Published: July 2002

CBP is a transcriptional coactivator required by many transcription factors for transactivation. Rubinstein–Taybi syndrome, which is an autosomal dominant syndrome characterized by abnormal pattern formation, has been shown to be associated with mutations in the
Cbp
gene. Furthermore,
Drosophila
CBP is required in
hedgehog
signaling for the expression of
decapentapleigic
, the
Drosophila
homologue of bone morphogenetic protein. However, no direct evidence exists to indicate that loss of one copy of the mammalian
Cbp
gene affects pattern formation. Here, we show that various abnormalities occur at high frequency in the skeletal system of heterozygous
Cbp
-deficient mice resulting from a C57BL/6-CBA × BALB/c cross. In support of a conserved signaling pathway for pattern formation in insects and mammals, the expression of
Bmp7
was found to be reduced in the heterozygous mutants. The frequency of the different abnormalities was significantly lower in a C57BL/6-CBA background, suggesting that the genetic background is an important determinant of the variability and severity of the anomalies seen in Rubinstein–Taybi syndrome patients.
Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures
Authors: Bengzon Johan , Kokaia Zaal , Elmér Eskil , Nanobashvili Avtandil , Kokaia Merab , Lindvall Olle
Published: July 2002

Neuronal apoptosis was observed in the rat dentate gyrus in two experimental models of human limbic epilepsy. Five hours after one hippocampal kindling stimulation, a marked increase ofin situterminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) of fragmented DNA was observed in nuclei located within and on the hilar border of the granule cell layer and in the polymorphic region. Forty kindling stimulations with 5-min interval produced higher numbers of labeled nuclei compared with one stimulation. The increase of TUNEL-positive nuclei was prevented by the protein synthesis inhibitor cycloheximide but not affected by theN-methyl-d-aspartate receptor antagonist MK-801. Kainic acid-induced seizures lead to a pattern of labeling in the hippocampal formation identical to that evoked by kindling. A large proportion of cells displaying TUNEL-positive nuclei was double-labeled by the neuron-specific antigen NeuN, demonstrating the neuronal identity of apoptotic cells. Either 1 or 40 kindling stimulations also gave rise to a marked increase of the number of cells double-labeled with the mitotic marker bromodeoxyuridine and NeuN in the subgranular zone and on the hilar border of the dentate granule cell layer. The present data show that single and intermittent, brief seizures induce both apoptotic death and proliferation of dentate gyrus neurons. We hypothesize that these processes, occurring early during epileptogenesis, are primary events in the development of hippocampal pathology in animals and possibly also in patients suffering from temporal lobe epilepsy.
Efficient exchange of the primary quinone acceptor Q
A
in isolated reaction centers of
Rhodopseudomonas viridis
Authors: Breton Jacques
Published: July 2002

A key step in the conversion of solar energy into chemical energy by photosynthetic reaction centers (RCs) occurs at the level of the two quinones, Q
A
and Q
B
, where electron transfer couples to proton transfer. A great deal of our understanding of the mechanisms of these coupled reactions relies on the seminal work of Okamura
et al.
[Okamura, M. Y., Isaacson, R. A., & Feher, G. (1975)
Proc. Natl. Acad. Sci. USA
88, 3491–3495], who were able to extract with detergents the firmly bound ubiquinone Q
A
from the RC of
Rhodobacter sphaeroides
and reconstitute the site with extraneous quinones. Up to now a comparable protocol was lacking for the RC of
Rhodopseudomonas viridis
despite the fact that its Q
A
site, which contains 2-methyl-3-nonaprenyl-1,4-naphthoquinone (menaquinone-9), has provided the best x-ray structure available. Fourier transform infrared difference spectroscopy, together with the use of isotopically labeled quinones, can probe the interaction of Q
A
with the RC protein. We establish that a simple incubation procedure of isolated RCs of
Rp. viridis
with an excess of extraneous quinone allows the menaquinone-9 in the Q
A
site to be almost quantitatively replaced either by vitamin K
1
, a close analogue of menaquinone-9, or by ubiquinone. To our knowledge, this is the first report of quinone exchange in bacterial photosynthesis. The Fourier transform infrared data on the quinone and semiquinone vibrations show a close similarity in the bonding interactions of vitamin K
1
with the protein at the Q
A
site of
Rp. viridis
and
Rb. sphaeroides
, whereas for ubiquinone these interactions are significantly different. The results are interpreted in terms of slightly inequivalent quinone–protein interactions by comparison with the crystallographic data available for the Q
A
site of the two RCs.
A hierarchical neuronal network for planning behavior
Authors: Dehaene Stanislas , Changeux Jean-Pierre
Published: July 2002

Planning a goal-directed sequence of behavior is a higher function of the human brain that relies on the integrity of prefrontal cortical areas. In the Tower of London test, a puzzle in which beads sliding on pegs must be moved to match a designated goal configuration, patients with lesioned prefrontal cortex show deficits in planning a goal-directed sequence of moves. We propose a neuronal network model of sequence planning that passes this test and, when lesioned, fails in a way that mimics prefrontal patients’ behavior. Our model comprises a descending planning system with hierarchically organized plan, operation, and gesture levels, and an ascending evaluative system that analyzes the problem and computes internal reward signals that index the correct/erroneous status of the plan. Multiple parallel pathways connecting the evaluative and planning systems amend the plan and adapt it to the current problem. The model illustrates how specialized hierarchically organized neuronal assemblies may collectively emulate central executive or supervisory functions of the human brain.
Angiogenesis promoted by vascular endothelial growth factor: Regulation through α1β1and α2β1 integrins
Authors: Senger Donald R. , Claffey Kevin P. , Benes Julie E. , Perruzzi Carole A. , Sergiou Ageliki P. , Detmar Michael
Published: July 2002

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors—the α1β1and α2β1integrins—through induction of mRNAs encoding the α1and α2subunits. In contrast, VEGF did not induce increased expression of the α3β1integrin, which also has been implicated in collagen binding. Integrin α1-blocking and α2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and α1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of α1β1and α2β1expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of α1-blocking and α2-blocking Abs.In vivo, a combination of α1-blocking and α2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of α1β1and α2β1expression by EC is an important mechanism by which VEGF promotes angiogenesis and that α1β1and α2β1antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.
Role of B cells in antigen presentation of the hepatitis B core
Authors: Milich David R. , Chen Margaret , Schödel Florian , Peterson Darrell L. , Jones Joyce E. , Hughes Janice L.
Published: July 2002

The hepatitis B virus (HBV) nucleocapsid or core antigen (HBcAg) is extremely immunogenic during infection and after immunization. For example, during many chronic infections, HBcAg is the only antigen capable of eliciting an immune response, and nanogram amounts of HBcAg elicit antibody production in mice. Recent structural analysis has revealed a number of characteristics that may help explain this potent immunogenicity. Our analysis of how the HBcAg is presented to the immune system revealed that the HBcAg binds to specific membrane Ig (mIg) antigen receptors on a high frequency of resting, murine B cells sufficiently to induce B7.1 and B7.2 costimulatory molecules. This enables HBcAg-specific B cells from unprimed mice to take up, process, and present HBcAg to naive Th cellsin vivoand to T cell hybridomasin vitroapproximately 105times more efficiently than classical macrophage or dendritic antigen-presenting cells (APC). These results reveal a structure–function relation for the HBcAg, confirm that B cells can function as primary APC, explain the enhanced immunogenicity of HBcAg, and may have relevance for the induction and/or maintenance of chronic HBV infection.
Scaling regions for food web properties
Authors: Bersier Louis-Félix , Sugihara George
Published: July 2002

The robustness of eight common food web properties is examined with respect to web size. We show that the current controversy concerning the scale dependence or scale invariance of these properties can be resolved by accounting for scaling constraints introduced by webs of very small size. We demonstrate statistically that the most robust way to view these properties is not to lump webs of all sizes, but to divide them into two distinct categories. For the present data set, small webs containing 12 or fewer species exhibit scale dependence, and larger webs containing more than 12 species exhibit scale invariance.
Antisense oligodeoxynucleotide-mediated disruption of hippocampal cAMP response element binding protein levels impairs consolidation of memory for water maze training
Authors: Guzowski John F. , McGaugh James L.
Published: July 2002

Extensive evidence suggests that long term memory (LTM) formation is dependent on the activation of neuronal second messenger systems and requires protein synthesis. The cAMP response element binding protein (CREB) is a constitutively expressed regulatory transcription factor that couples changes in second messenger levels to changes in cellular transcription. Several recent studies suggest that CREB and related transcription factors regulate gene expression necessary for neuronal plasticity and LTM. However, the role of CREB, within defined mammalian brain structures, in mediating the cellular events underlying LTM formation has not been investigated. We examined whether CREB-mediated transcription within the dorsal hippocampus is critical to LTM consolidation of water maze spatial training, which is known to depend on dorsal hippocampal function. Pretraining infusions of antisense oligodeoxynucleotides (ODN) directed against CREB mRNA were used to disrupt hippocampal CREB protein levels in adult rats. Control groups received pretraining infusions of ODN of the same base composition but in a randomized order (scrambled ODN) or buffer. Task acquisition and memory up to 4 h (i.e., short term memory) were similar in CREB antisense ODN and control groups. In contrast, CREB antisense ODN-infused rats exhibited significantly impaired memory 48 h later (i.e., LTM). Moreover, administration of antisense ODN 1 day after training did not affect subsequent retention performance. These findings provide the first evidence that CREB-mediated transcription is integral to hippocampal-dependent memory consolidation processes.
Cell-free tumor antigen peptide-based cancer vaccines
Authors: Schmidt Walter , Buschle Michael , Zauner Wolfgang , Kirlappos Helen , Mechtler Karl , Trska Barbara , Birnstiel Max L.
Published: July 2002

The central role that tumor antigen-derived peptides play in induction of antitumor immunity makes them ideal candidates for peptide-based cancer vaccines. We have demonstrated that “transloading” is an efficient strategy for importing short peptide ligands into antigen-presenting cellsin vitro. Postulating that the transloading procedure might effect peptide uptake by antigen-presenting cellsin vivoas well, we tested this approach for the generation of peptide-based cancer vaccines. In the P815 mastocytoma system, we vaccinated mice by s.c. injection of a single, known natural peptide derived from JAK-1 kinase. Whereas vaccination with peptide alone or mixed with incomplete Freund’s adjuvant was ineffective, application of the peptide in conjunction with the polycation poly-l-lysine protected a significant number of animals against tumor challenge. Dependent upon the type of poly-l-lysine applied, protection against tumor take was comparable to that achieved with irradiated whole-cell vaccines, genetically modified to secrete granulocyte–macrophage colony-stimulating factor. In the murine melanoma M-3, a combination of four putative tumor antigen-derived peptides was tested as a cancer vaccine. Administered in combination with polycations, these peptides evoked potent antitumor immunity that could not be obtained with the peptides alone or peptides emulsified in incomplete Freund’s adjuvant. However, peptide–polycation vaccines applied to the M-3 model were not as efficient as cellular control vaccines, consisting of irradiated interleukin 2 or granulocyte–macrophage colony-stimulating factor-secreting tumor cells.
Oxidative stress mediates impairment of muscle function in transgenic mice with elevated level of wild-type Cu/Zn superoxide dismutase
Authors: Peled-Kamar M. , Lotem J. , Wirguin I. , Weiner L. , Hermalin A. , Groner Y.
Published: July 2002

Cases of familial amyotrophic lateral sclerosis (fALS; a neurodegenerative disorder) have been reported in which the gene for Cu/Zn superoxide dismutase (CuZnSOD) was mutated. Several studies with the fALS mutant CuZnSOD in transgenic mice and cells showed that the fALS mutations act through an as yet undefined dominant gain-of-function mechanism. Wild-type CuZnSOD catalyzes the dismutation of superoxide (O
2⨪
) but also produces hydroxyl radicals (
•
OH) with H
2
O
2
as substrate. Two laboratories have recently demonstrated that the
•
OH production ability was preferentially enhanced by the fALS mutant CuZnSOD, suggesting that this might be the function gained in fALS. In this study, we used transgenic CuZnSOD (Tg-CuZnSOD) mice with elevated levels of CuZnSOD to determine whether overexpression of wild-type CuZnSOD was also associated with increased
•
OH production and impaired muscle function. Enhanced formation of
•
OH was detected, by spin trapping, in brain and muscle extracts of the Tg-CuZnSOD mice. Three independently derived Tg-CuZnSOD lines showed muscle abnormalities, reflected by altered electromyography (EMG) and diminished performance in the rope grip test. After treatment with paraquat (PQ), a widely used herbicide and O
2⨪
-generating compound, muscle disability significantly deteriorated in Tg-CuZnSOD mice but not in control mice. The results indicate that elevated levels of CuZnSOD cause indigenous long-term oxidative stress leading to impairment of muscle function. These findings may provide valuable clues about the concurred role of indigenous oxidative stress and exogenous agents in the etiology of sporadic ALS and several other neurodegenerative diseases in which a specific subset of neurons is affected.
Disaggregation of Alzheimer β-amyloid by site-directed mAb
Authors: Solomon Beka , Koppel Rela , Frankel Dan , Hanan-Aharon Eilat
Published: July 2002

In Alzheimer disease, β-amyloid peptide accumulates in the brain as insoluble amyloid plaques. Amyloid filaments, similar to those found in amyloid plaques, can be assembledin vitrofrom chemically synthesized β-peptides. In this study, we report that antibodies raised against the N-terminal region (1–28) of the β-amyloid peptide bind to thein vitro-formed β-amyloid assemblies, leading to disaggregation of the fibrils and partial restoration of the peptide’s solubility. The concomitant addition of fibrillar β-amyloid with these antibodies to PC 12 cells leads to the inhibition of the neurotoxic effects of β-amyloid. Some of the mAbs raised against soluble β-peptide (1–28) have been found to preventin vitrofibrillar aggregation of β-amyloid peptide. These experimental data suggest that site-directed mAbs interfere with the aggregation of β-amyloid and trigger reversal to its nontoxic, normal components. The above findings give hints on how to convertin vivosenile plaques into nontoxic, diffuse components and may have therapeutic interest for those studying Alzheimer disease and other human diseases related to amyloidogenic properties of physiological peptides and proteins.
Defective “pacemaker” current (
I
h
) in a zebrafish mutant with a slow heart rate
Authors: Baker Keith , Warren Kerri S. , Yellen Gary , Fishman Mark C.
Published: July 2002

At a cellular level, cardiac pacemaking, which sets the rate and rhythm of the heartbeat, is produced by the slow membrane depolarization that occurs between action potentials. Several ionic currents could account for this pacemaker potential, but their relative prominence is controversial, and it is not known which ones actually play a pacemaking role
in vivo
. To correlate currents in individual heart cells with the rhythmic properties of the intact heart, we have examined
slow mo (smo)
, a recessive mutation we discovered in the zebrafish
Danio rerio
. This mutation causes a reduced heart rate in the embryo, a property we can quantitate because the embryo is transparent. We developed methods for culture of cardiocytes from zebrafish embryos and found that, even in culture, cells from
smo
continue to beat relatively slowly. By patch-clamp analysis, we discovered that a large repertoire of cardiac currents noted in other species are present in these cultured cells, including sodium, T-type, and L-type calcium and several potassium currents, all of which appear normal in the mutant. The only abnormality appears to be in a hyperpolarization-activated inward current with the properties of
I
h
, a current described previously in the nervous system, pacemaker, and other cardiac tissue.
smo
cardiomyocytes have a reduction in
I
h
that appears to result from severe diminution of one kinetic component of the
I
h
current. This provides strong evidence that
I
h
is an important contributor to the pacemaking behavior of the intact heart.
Ultrarapid, highly efficient viral gene transfer to the heart
Authors: Donahue J. Kevin , Kikkawa Kohei , Johns David C. , Marban Eduardo , Lawrence John H.
Published: July 2002

Gene therapy for common myocardial diseases will require effective and homogeneous gene delivery throughout the intact heart. We created two experimental models to identify and optimize parameters important for adenovirus-mediated cardiac gene transfer. In cultured rabbit ventricular myocytes, the percentage of infected cells increased with higher absolute numbers of virus particles, longer durations of virus exposure, physiological temperatures, and specific culture media compositions. Simulating the
in vitro
conditions, we delivered adenovirus to intact rabbit hearts by intracoronary perfusion. The percentage of infected cells increased with higher coronary flow rates, longer virus exposure times, and higher virus concentrations. Under optimal conditions, nearly 100% of myocytes expressed the reporter gene β-galactosidase after
ex vivo
infection. This novel delivery method, the first to demonstrate virtually complete transduction of any intact organ, could be adapted to achieve widespread gene transfer
in vivo
.
Stimulation of HIV-1-neutralizing antibodies in simian HIV-IIIB-infected macaques
Authors: Müller Sybille , Margolin David H. , Nara Peter L. , Alvord W. Gregory , Köhler Heinz
Published: July 2002

Previously we have discovered a public idiotope, designated 1F7, that is expressed on antibodies against HIV type 1 (HIV-1) in human and nonhuman primates. To test the potential of mouse monoclonal antibody (mAb) 1F7 as a therapeutic anti-clonotypic antibody in HIV-1-infected patients, we used the simian HIV-IIIB macaque infection model, which mimics several immunological and pathological characteristics of HIV-1 infection in humans. Four healthy simian HIV-infected rhesus monkeys (Macaca mulatta) expressing the 1F7 marker on anti-gp120 antibodies were selected for this study. Three monkeys of this group were immunized several times with the murine mAb 1F7 i.v., and one monkey received as control an isotype-matched antibody, TEPC183. No serious side effect or allergic reaction was encountered. Blood collected before and during the immunization and over several months afterward were analyzed for neutralizing antibodies. Significant increases in breadth and potency of HIV-1-neutralizing antibody titers to one or more virus strains were detected in all three of the 1F7-immunized monkeys, but not in the control monkey immunized with TEPC183. These results show that an antibody, recognizing a public idiotope associated with anti-HIV-1 antibodies can function in chronically infected primates as an anti-clonotypic immunogen to boost antibodies that neutralize homologous and heterologous virus strains. This study represents a first step toward the preclinical evaluation of 1F7 as a therapeutic AIDS vaccine.
Genomic evidence for two functionally distinct gene classes
Authors: Rivera Maria C. , Jain Ravi , Moore Jonathan E. , Lake James A.
Published: July 2002

Analyses of complete genomes indicate that a massive prokaryotic gene transfer (or transfers) preceded the formation of the eukaryotic cell. In comparisons of the entire set of
Methanococcus jannaschii
genes with their orthologs from
Escherichia coli
,
Synechocystis 6803
, and the yeast
Saccharomyces cerevisiae
, it is shown that prokaryotic genomes consist of two different groups of genes. The deeper, diverging informational lineage codes for genes which function in translation, transcription, and replication, and also includes GTPases, vacuolar ATPase homologs, and most tRNA synthetases. The more recently diverging operational lineage codes for amino acid synthesis, the biosynthesis of cofactors, the cell envelope, energy metabolism, intermediary metabolism, fatty acid and phospholipid biosynthesis, nucleotide biosynthesis, and regulatory functions. In eukaryotes, the informational genes are most closely related to those of
Methanococcus
, whereas the majority of operational genes are most closely related to those of
Escherichia
, but some are closest to
Methanococcus
or to
Synechocystis
.
The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis
Authors: Smyth Mark J. , Krasovskis Erika , Sutton Vivien R. , Johnstone Ricky W.
Published: July 2002

Multidrug resistance mediated by the drug efflux protein, P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape death induced by chemotherapeutic agents. However, the mechanism by which P-gp confers resistance to a large variety of structurally diverse molecules has remained elusive. In this study, classical multidrug resistant human CEM and K562 tumor cell lines expressing high levels of P-gp were less sensitive to multiple forms of caspase-dependent cell death, including that mediated by cytotoxic drugs and ligation of Fas. The DNA fragmentation and membrane damage inflicted by these stimuli were defined as caspase dependent by various soluble peptide fluoromethylketone caspase inhibitors. Inhibition of P-gp function by the anti-P-gp mAb MRK-16 or verapamil could reverse resistance to these forms of cell death. Inhibition of P-gp function also enhanced drug or Fas-mediated activation of caspase-3 in drug-resistant CEM cells. By contrast, caspase-independent cell death events in the same cells, including those mediated by pore-forming proteins or intact NK cells, were not affected by P-gp expression. These observations suggest that, in addition to effluxing drugs, P-gp may play a specific role in regulating some caspase-dependent apoptotic pathways.
Propagation and recovery of intact, infectious Epstein–Barr virus from prokaryotic to human cells
Authors: Delecluse Henri-Jacques , Hilsendegen Tanja , Pich Dagmar , Zeidler Reinhard , Hammerschmidt Wolfgang
Published: July 2002

With current techniques, genetic alterations of herpesviruses are difficult to perform, mostly because of the large size of their genomes. To solve this problem, we have designed a system that allows the cloning of any γ-herpesvirus in
Escherichia coli
onto an F factor-derived plasmid. Immortalized B cell lines were readily established with recombinant Epstein–Barr virus (EBV), demonstrating that the F factor-cloned EBV genome has all the characteristics of wild-type EBV. Because any genetic modification is possible in
E. coli
, this experimental approach opens the way to the genetic analysis of all EBV functions. Moreover, it is now feasible to generate attenuated EBV strains
in vitro
such that vaccine strains can be designed. Because we incorporated the genes for hygromycin resistance and green fluorescent protein onto the
E. coli
cloned EBV genome, the still open question of the EBV target cells other than B lymphocytes will be addressed.
Mental chronometry using latency-resolved functional MRI
Authors: Menon Ravi S. , Luknowsky David C. , Gati Joseph S.
Published: July 2002

Vascular responses to neural activity are exploited as the basis of a number of brain imaging techniques. The vascular response is thought to be too slow to resolve the temporal sequence of events involved in cognitive tasks, and hence, imaging studies of mental chronometry have relied on techniques such as the evoked potential. Using rapid functional MRI (fMRI) of single trials of two simple behavioral tasks, we demonstrate that while the microvascular response to the onset of neural activity is delayed consistently by several seconds, the relative timing between the onset of the fMRI responses in different brain areas appears preserved. We examined a number of parameters that characterize the fMRI response and determined that its onset time is best defined by the inflection point from the resting baseline. We have found that fMRI onset latencies determined in this manner correlate well with independently measurable parameters of the tasks such as reaction time or stimulus presentation time and can be used to determine the origin of processing delays during cognitive or perceptual tasks with a temporal accuracy of tens of milliseconds and spatial resolution of millimeters.
Cyclooxygenase-2 inhibition prevents delayed death of CA1 hippocampal neurons following global ischemia
Authors: Nakayama Masaki , Uchimura Koichi , Zhu Raymond Li , Nagayama Tetsuya , Rose Marie E. , Stetler R. Anne , Isakson Peter C. , Chen Jun , Graham Steven H.
Published: July 2002

The inducible isoform of the enzyme cyclooxygenase-2 (COX2) is an immediate early gene induced by synaptic activity in the brain. COX2 activity is an important mediator of inflammation, but it is not known whether COX2 activity is pathogenic in brain. To study the role of COX2 activity in ischemic injury in brain, expression of COX2 mRNA and protein and the effect of treatment with a COX2 inhibitor on neuronal survival in a rat model of global ischemia were determined. Expression of both COX2 mRNA and protein was increased after ischemia in CA1 hippocampal neurons before their death. There was increased survival of CA1 neurons in rats treated with the COX2-selective inhibitor SC58125 {1-[(4-methylsulfonyl) phenyl]-3-trifluoro-methyl-5-[(4-fluoro)phenyl] pyrazole} before or after global ischemia compared with vehicle controls. Furthermore, hippocampal prostaglandin E
2
concentrations 24 h after global ischemia were decreased in drug-treated animals compared with vehicle-treated controls. These results suggest that COX2 activity contributes to CA1 neuronal death after global ischemia.
Macrophage migration inhibitory factor is a critical mediator of the activation of immune cells by exotoxins of Gram-positive bacteria
Authors: Calandra Thierry , Spiegel Lori A. , Metz Christine N. , Bucala Richard
Published: July 2002

Discovered in the early 1960s as a T cell cytokine, the protein mediator known as macrophage migration inhibitory factor (MIF) has been found recently to be a pituitary peptide released during the physiological stress response, a proinflammatory macrophage cytokine secreted after LPS stimulation, and a T cell product expressed as part of the antigen-dependent activation response. We report herein that MIF also plays a critical role in the innate host response to staphylococcal and streptococcal exotoxins. In RAW 264.7 or elicited mouse peritoneal macrophages, peak MIF secretion was induced by concentrations of the staphylococcal toxic shock syndrome (TSS) toxin 1 (TSST-1) and the streptococcal pyrogenic exotoxin A as low as 10 pg/ml. Moreover, dose-response studies of splenocyte cytokine production showed that lower concentrations of TSST-1 (10 pg/ml) were needed to release MIF than to induce interleukin 2 or interferon-γ secretion (1 ng/ml). We also studied the effect of neutralizing anti-MIF antibodies on TSST-1-induced lymphocyte proliferation and lethal toxic shock. Pretreatment of C57BL/6 mice with anti-MIF antibody 2 hr before TSST-1 injection prevented spleen enlargement and reduced by 50% the proliferation of splenocytes measuredex vivo. In a lethal mouse model of TSST-1-induced shock, anti-MIF antibody increased survival from 8% to 54% (P< 0.0001). These studies indicate that Gram-positive exotoxins are extremely potent inducers of MIF secretion and establish a critical role for MIF and the macrophage in the pathogenesis of the TSSs and in the innate immune response.
The type 1 receptor (CD120a) is the high-affinity receptor for soluble tumor necrosis factor
Authors: Grell Matthias , Wajant Harald , Zimmermann Gudrun , Scheurich Peter
Published: July 2002

Tumor necrosis factor (TNF) can induce a variety of cellular responses at low picomolar concentrations. This is in apparent conflict with the published dissociation constants for TNF binding to TNF receptors in the order of 100–500 pM. To elucidate the mechanisms underlying the outstanding cellular sensitivity to TNF, we determined the binding characteristics of TNF to both human TNF receptors at 37°C. Calculation of the dissociation constant (Kd) from the association and dissociation rate constants determined at 37°C revealed a remarkable high affinity for TNF binding to the 60-kDa TNF type 1 receptor (TNF-R1;Kd= 1.9 × 10−11M) and a significantly lower affinity for the 80-kDa TNF type 2 receptor (TNF-R2;Kd= 4.2 × 10−10M). The high affinity determined for TNF-R1 is mainly caused by the marked stability of ligand–receptor complexes in contrast to the transient interaction of soluble TNF with TNF-R2. These data can readily explain the predominant role of TNF-R1 in induction of cellular responses by soluble TNF and suggest the stability of the TNF–TNF receptor complexes as a rationale for their differential signaling capability. In accordance with this reasoning, the lower signaling capability of homotrimeric lymphotoxin, compared with TNF, correlates with a lower stability of the lymphotoxin–TNF-R1 complex at 37°C.