bluuebunny/crossref_metadata_2025 huggingface dataset:
Liver fatty acid-binding protein: specific mediator of the mitogenesis induced by two classes of carcinogenic peroxisome proliferators.
Authors: Khan S H , Sorof S
Published: May 2006

Peroxisome proliferators (PP) are a diverse group of chemicals that induce dramatic increases in peroxisomes in rodent hepatocytes, followed by hypertrophy, hepatomegaly, alterations in lipid metabolism, mitogenesis, and finally hepatocarcinomas. Termed nongenotoxic carcinogens, they do not interact with DNA, are not mutagenic in bacterial assays, and fail to elicit many of the phenotypes associated with classic genotoxic carcinogens. We report here that the mitogenesis induced by the major PP class, the amphipathic carboxylates, and by the tetrazole-substituted acetophenones specifically requires liver fatty acid-binding protein (L-FABP) in cultured rat hepatoma cells transfected with the sense cDNA of L-FABP, in contrast to L-FABP-nonexpressing cells transfected with its antisense cDNA. The mitogenic actions of L-FABP were protein-specific, inasmuch as no other protein in the nonexpressing cells could act like L-FABP. L-FABP was previously shown not only (i) to interact covalently with metabolites of the two genotoxic carcinogens 2-acetylaminofluorene and aminoazo dyes during liver carcinogenesis, but also (ii) to bind noncovalently the two classes of PP in vitro with avidities that correlate with their abilities to elicit peroxisomal enzymatic responses, and (iii) together with unsaturated fatty acids, especially linoleic acid, to promote multiplication of the transfected hepatoma cells in culture. The convergence of the two types of genotoxic carcinogens with the two classes of PP nongenotoxic carcinogens, and also with unsaturated fatty acids, at L-FABP actions in inducing mitogenesis allows the following hypothesis. During tumor promotion of carcinogenesis in vivo, these groups of genotoxic and nongenotoxic carcinogens act on the normal process by which L-FABP, functioning as a specific receptor of unsaturated fatty acids or their metabolites, promotes hepatocyte proliferation.
Molecular cloning and expression of the Saccharomyces cerevisiae RFC3 gene, an essential component of replication factor C.
Authors: Li X , Burgers P M
Published: May 2006

Yeast replication factor C (RF-C) is a multi-polypeptide complex required for processive DNA replication by DNA polymerases delta and epsilon. The gene encoding the 40-kDa subunit of the Saccharomyces cerevisiae RF-C (RFC3) has been cloned. The RFC3 gene is required for yeast cell growth and has been mapped to the left arm of chromosome XIV. The deduced amino acid sequence of the RFC3 gene shows a high homology to the 36-, 37-, and 40-kDa subunits of human RF-C (also called activator 1), with the highest homology to the 36-kDa subunit. Among the conserved regions are the A motif of ATP binding proteins; the "DEAD box," common to DNA helicases and other ATPases; and the "RFC box," an approximately 15-amino acid domain virtually identical in the yeast and human RF-C subunits. Limited homology to the functional homologs of the Escherichia coli replication apparatus was also observed. The steady-state mRNA levels of RFC3 do not change significantly during the mitotic cell cycle of yeast. The intact form of the RFC3 gene product (Rfc3p) has been overproduced in E. coli and purified to homogeneity. Purified Rfc3p has an ATPase activity that is markedly stimulated by single-stranded DNA but not by double-stranded DNA or RNA.
Cytokine therapeutics: lessons from interferonalpha.
Authors: Gutterman J U
Published: May 2006

Cytokines are soluble proteins that allow for
communication between cells and the external environment. Interferon (IFN)
alpha, the first cytokine to be produced by recombinant DNA technology, has
emerged as an important regulator of growth and differentiation, affecting
cellular communication and signal transduction pathways as well as immunological
control. This review focuses on the biological and clinical activities of the
cytokine. Originally discovered as an antiviral substance, the efficacy of
IFN-alpha in malignant, viral, immunological, angiogenic, inflammatory, and
fibrotic diseases suggests a spectrum of interrelated pathophysiologies. The
principles learned from in vivo studies will be discussed, particularly hairy
cell leukemia, chronic myelogenous leukemia, certain angiogenic diseases, and
hepatitis. After the surprising discovery of activity in a rare B-cell neoplasm,
IFN-alpha emerged as a prototypic tumor suppressor protein that represses the
clinical tumorigenic phenotype in some malignancies capable of differentiation.
Regulatory agencies throughout the world have approved IFN-alpha for treatment
of 13 malignant and viral disorders. The principles established with this
cytokine serve as a paradigm for future development of natural proteins for
human disease.
Autoregulation of the NF-kappa B transactivator RelA(p65) by multiple cytoplasmic inhibitors containing ankyrinmotifs.
Authors: Sun S C , Ganchi P A , Béraud C , Ballard D W , Greene W C
Published: May 2006

RelA (p65) functions as the critical
transactivating component of the heterodimeric p50-p65 NF-kappa B complex and
contains a high-affinity binding site for its cytoplasmic inhibitor, I kappa B
alpha. After cellular activation, I kappa B alpha is rapidly degraded in concert
with the induced nuclear translocation of NF-kappa B. The present study
demonstrates that tumor necrosis factor alpha-induced degradation of I kappa B
alpha in human T cells is preceded by its rapid phosphorylation in vivo.
However, these effects on I kappa B alpha result in nuclear mobilization of only
a fraction of the entire cytoplasmic pool of RelA. Subsequent studies have
revealed that (i) cytoplasmic RelA is stably associated not only with I kappa B
alpha but also with other ankyrin motif-rich proteins including the products of
the NF-kappa B2 (p100) and NF-kappa B1 (p105) genes; (ii) in contrast to RelA-I
kappa B alpha, RelA-p100 cytoplasmic complexes are not dissociated following
tumor necrosis factor alpha activation; (iii) p100 functions as a potent
inhibitor of RelA-mediated transcription in vivo; (iv) the interaction of RelA
and p100 involves the conserved Rel homology domain of both proteins but not the
nuclear localization signal of RelA, which is required for I kappa B alpha
binding; (v) p100 inhibition of RelA function requires the C-terminal ankyrin
motif domain, which mediates cytoplasmic retention of RelA; and (vi) as observed
with I kappa B alpha, nuclear RelA stimulates p100 mRNA and protein expression.
These findings thus reveal the presence of a second inducible autoregulated
inhibitory pathway that helps ensure the rapid but transient action of nuclear
NF-kappa B.
Tityustoxin K alpha blocks voltage-gatednoninactivating K+ channels and unblocks inactivating K+ channels blocked byalpha-dendrotoxin in synaptosomes.
Authors: Rogowski R S , Krueger B K , Collins J H , Blaustein M P
Published: May 2006

Two nonhomologous polypeptide toxins, tityustoxin
K alpha (TsTX-K alpha) and tityustoxin K beta (TsTX-K beta), purified from the
venom of the Brazilian scorpion Tityus serrulatus, selectively block
voltage-gated noninactivating K+ channels in synaptosomes (IC50 values of 8 nM
and 30 nM, respectively). In contrast, alpha-dendrotoxin (alpha-DTX) and
charybdotoxin (ChTX) block voltage-gated inactivating K+ channels in
synaptosomes (IC50 values of 90 nM and 40 nM, respectively). We studied
interactions among these toxins in 125I-alpha-DTX binding and 86Rb efflux
experiments. Both TsTX-K alpha and ChTX completely displaced specifically bound
125I-alpha-DTX from synaptic membranes, but TsTX-K beta had no effect on bound
alpha-DTX. TsTX-K alpha and TsTX-K beta blocked the same noninactivating
component of 100 mM K(+)-stimulated 86Rb efflux in synaptosomes. Both alpha-DTX
and ChTX blocked the same inactivating component of the K(+)-stimulated 86Rb
efflux in synaptosomes. Both the inactivating and the noninactivating components
of the 100 mM K(+)-stimulated 86Rb efflux were completely blocked when 200 nM
TsTX-K beta and either 600 nM alpha-DTX or 200 nM ChTX were present. The effects
of TsTX-K alpha and ChTX on 86Rb efflux were also additive. When TsTX-K alpha
was added in the presence of alpha-DTX, however, only the noninactivating
component of the K(+)-stimulated efflux was blocked. The inactivating component
could then be blocked by ChTX, which is structurally homologous to TsTX-K alpha.
We conclude that TsTX-K alpha unblocks the voltage-gated inactivating K+
channels in synaptosomes when they are blocked by alpha-DTX, but not when they
are blocked by ChTX. TsTX-K alpha binds to a site on the inactivating K+ channel
that does not occlude the pore; its binding apparently prevents alpha-DTX (7054
Da), but not ChTX (4300 Da), from blocking the pore. The effects of TsTX-K alpha
on 125I-alpha-DTX binding and 86Rb efflux are mimicked by noxiustoxin, which is
homologous to TsTX-K alpha and ChTX.
Heterodimerization of epidermal growth factor receptorand wild-type or kinase-deficient Neu: a mechanism of interreceptor kinaseactivation and transphosphorylation.
Authors: Qian X , LeVea C M , Freeman J K , Dougall W C , Greene M I
Published: May 2006

We have shown that members of the erbB family
undergo homodimer and heterodimer formation. The rat p185c-neu and the epidermal
growth factor receptor (EGFR) can associate into an active heterodimeric
tyrosine kinase. Overexpression of these two receptors also results in a
transformed phenotype. We now show that mutant Neu proteins resulting from a
point mutation at the ATP-binding site (N757) or cytoplasmic domain deletions
(N691stop) are still able to undergo EGF-induced heterodimerization with EGFR.
Analysis of heterodimer formation between EGFR and truncated Neu proteins
revealed that heterodimerization is preferred over homodimerization of EGFR.
N757 can be transphosphorylated by associated EGFR upon EGF stimulation.
However, the heterodimer composed of EGFR and N691stop is kinase inactive. These
results provided evidence that the Neu ectodomain is sufficient to associate
with EGFR physically, and the cytoplasmic domain interaction is required for
heterodimeric kinase activation, indicating that Neu/c-erbB2 is not just a
simple substrate for EGFR but a transactivator as well.
Distinct effects of thioredoxin and antioxidants onthe activation of transcription factors NF-kappa B and AP-1.
Authors: Schenk H , Klein M , Erdbrügger W , Dröge W , Schulze-Osthoff K
Published: May 2006

The transcription factors NF-kappa B and AP-1 have
been implicated in the inducible expression of a variety of genes involved in
responses to oxidative stress and cellular defense mechanisms. Here, we report
that thioredoxin, an important cellular protein oxidoreductase with antioxidant
activity, exerts different effects on the activation of NF-kappa B and AP-1.
Transient expression or exogenous application of thioredoxin resulted in a
dose-dependent inhibition of NF-kappa B activity, as demonstrated in gel shift
and transactivation experiments. AP-1-dependent transactivation, in contrast was
strongly enhanced by thioredoxin. A similar increase of AP-1 activity was also
observed with other, structurally unrelated antioxidants such as pyrrolidine
dithiocarbamate and butylated hydroxyanisole, indicating that the
thioredoxin-induced increase of AP-1 activation was indeed based on an
antioxidant effect. Moreover, the stimulatory effect on AP-1 activity was found
to involve de novo transcription of the c-jun and c-fos components but to be
independent of protein kinase C activation. These results suggest that
thioredoxin plays an important role in the regulation of transcriptional
processes and oppositely affects NF-kappa B and AP-1 activation.
Ligand and p185c-neu density govern receptorinteractions and tyrosine kinase activation.
Authors: Samanta A , LeVea C M , Dougall W C , Qian X , Greene M I
Published: May 2006

The neu protooncogene (also known as c-erbB2, NGL,
and HER2) encodes a 185-kDa transmembrane glycoprotein with intrinsic tyrosine
kinase activity that resembles the receptor for epidermal growth factor. The
p185 gene and protein were originally identified in the brain and are thought to
play a critical role in neurogenesis. Aberrant c-erbB2 protein overexpression
also occurs in several human adenocarcinomas. A ligand for p185, neu-activating
factor (NAF), specifically binds to neu receptor and increases the p185c-neu
tyrosine phosphorylation in vitro and in vivo in a dose-dependent manner. We now
show that NAF specifically binds to purified p185 expressed in baculovirus.
Direct binding analysis showed that NAF binds with high affinity (Kd = 1.3 nM).
We have investigated changes in the structure and association state of
baculovirus-produced neu holoreceptor that are induced by ligand binding. In
this study, we used sucrose gradients to show that purified p185c-neu exists
mainly in the monomeric form at low concentrations, whereas at higher
concentrations p185c-neu exists as dimers or multimers. At low concentrations,
but in the presence of ligand, p185c-neu sediments as a dimeric or multimeric
form. Monomer-oligomer interconversion is absolutely ligand dependent at low
receptor concentrations. The high molecular weight form of the receptor is
enzymatically more active, as a consequence of ligand-driven activation of the
receptor kinase. Oncogenic p185neu receptors sediment predominantly as high
molecular weight forms and have constitutively active kinases.
Antitumor bisdioxopiperazines inhibit yeast DNAtopoisomerase II by trapping the enzyme in the form of a closed proteinclamp.
Authors: Roca J , Ishida R , Berger J M , Andoh T , Wang J C
Published: May 2006

The mechanism of inhibition of eukaryotic DNA
topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] by a member
of the bisdioxopiperazine family of anticancer drugs, ICRF-193, was investigated
by using purified yeast DNA topoisomerase II. In the absence of ATP, ICRF-193
has little effect on the binding of the enzyme to various forms of DNA. In the
presence of ATP, the drug converts the enzyme to a form incapable of binding
circular DNA. Incubation of a preformed circular DNA-enzyme complex with
ICRF-193 and ATP converts the complex to a form stable in molar concentrations
of salt. These results can be interpreted in terms of the ATP-modulated
protein-clamp model of type II DNA topoisomerases [Roca, J. & Wang, J. C.
(1992) Cell 71, 833-840]; ICRF-193 can bind to the closed-clamp form of the
enzyme and prevents its conversion to the open-clamp form. This interpretation
is further supported by the finding that whereas both ATP and the drug are
needed to form the salt-stable circular DNA-enzyme complex, ATP is not needed
for maintaining this complex; furthermore, a signature of the closed-clamp form
of the enzyme, Staphylococcus aureus strain V8 endoproteinase cleavage site at
Glu-680, is observed if the enzyme is incubated with both ATP and ICRF-193.
Inhibition of interconversion between the open- and closed-clamp forms of type
II DNA topoisomerases offers a new mechanism in the selection and design of
therapeutics targeting this class of enzymes.
Details of the evolutionary history from invertebratesto vertebrates, as deduced from the sequences of 18S rDNA.
Authors: Wada H , Satoh N
Published: May 2006

Almost the entire sequences of 18S rDNA were
determined for two chaetognaths, five echinoderms, a hemichordate, and two
urochordates (a larvacean and a salp). Phylogenetic comparisons of the
sequences, together with those of other deuterostomes (an ascidian, a
cephalochordate, and vertebrates) and protostomes (an arthropod and a mollusc),
suggest the monophyly of the deuterostomes, with the exception of the
chaetognaths. Chaetognaths may not be a group of deuterostomes. The deuterostome
group closest to vertebrates was the group of cephalochordates. Ascidians,
larvaceans, and salps seem to form a discrete group (urochordates), in which the
early divergence of larvaceans is evident. These results support the hypothesis
that chordates evolved from free-living ancestors.
Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization.
Authors: Kallioniemi A , Kallioniemi O P , Piper J , Tanner M , Stokke T , Chen L , Smith H S , Pinkel D , Gray J W , Waldman F M
Published: May 2006

Comparative genomic hybridization was applied to 5 breast cancer cell lines and 33 primary tumors to discover and map regions of the genome with increased DNA-sequence copy-number. Two-thirds of primary tumors and almost all cell lines showed increased DNA-sequence copy-number affecting a total of 26 chromosomal subregions. Most of these loci were distinct from those of currently known amplified genes in breast cancer, with sequences originating from 17q22-q24 and 20q13 showing the highest frequency of amplification. The results indicate that these chromosomal regions may contain previously unknown genes whose increased expression contributes to breast cancer progression. Chromosomal regions with increased copy-number often spanned tens of Mb, suggesting involvement of more than one gene in each region.
Molecular cloning, characterization, and overexpression of ERG7, the Saccharomyces cerevisiae gene encoding lanosterol synthase.
Authors: Corey E J , Matsuda S P , Bartel B
Published: May 2006

We report the cloning, characterization, and overexpression of Saccharomyces cerevisiae ERG7, which encodes lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7], the enzyme responsible for the complex cyclization/rearrangement step in sterol biosynthesis. Oligonucleotide primers were designed corresponding to protein sequences conserved between Candida albicans ERG7 and the related Arabidopsis thaliana cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A PCR product was amplified from yeast genomic DNA using these primers and was used to probe yeast libraries by hybridization. Partial-length clones homologous to the two known epoxysqualene mutases were isolated, but a full-length sequence was found neither in cDNA nor genomic libraries, whether in phage or plasmids. Two overlapping clones were assembled to make a functional reconstruction of the gene, which contains a 2196-bp open reading frame capable of encoding an 83-kDa protein. The reconstruction complemented the erg7 mutation when driven from either its native promoter or the strong ADH1 promoter.
In vivo hepatic gene therapy: complete albeit transient correction of factor IX deficiency in hemophilia B dogs.
Authors: Kay M A , Landen C N , Rothenberg S R , Taylor L A , Leland F , Wiehle S , Fang B , Bellinger D , Finegold M , Thompson A R
Published: May 2006

Hemophilia B is a bleeding disorder caused by mutations in the factor IX gene. The disorder is X-linked recessive with a prevalence of about 1 in 30,000 Caucasian males. Factor IX is naturally synthesized in the liver and secreted into blood. Here we report the construction of recombinant adenoviral vectors containing the canine factor IX cDNA that are capable of transducing hepatocytes in mice at high efficiencies in vivo without partial hepatectomy. The recombinant viral vector was used to treat hemophilia B dogs by direct vector infusion into the portal vasculature of deficient animals. Plasma factor IX concentrations in the treated hemophilia B dogs increased from 0 to 300% of the level present in normal dogs, resulting in complete amelioration of the disease as demonstrated by normal blood coagulation and hemostatic measurements. Although plasma factor IX concentration started to decline after a few days, therapeutic levels of factor IX persisted for 1-2 months in the treated animals. The results validate the principle of in vivo hepatic gene delivery to reconstitute the genetic deficiency in a large animal model and suggest that gene therapy is achievable when long-acting vectors are developed.
Dengue: the risk to developed and developing countries.
Authors: Monath T P
Published: May 2006

Dengue viruses are members of the Flaviviridae, transmitted principally in a cycle involving humans and mosquito vectors. In the last 20 years the incidence of dengue fever epidemics has increased and hyperendemic transmission has been established over a geographically expanding area. A severe form, dengue hemorrhagic fever (DHF), is an immunopathologic disease occurring in persons who experience sequential dengue infections. The risk of sequential infections, and consequently the incidence of DHF, has risen dramatically, first in Asia and now in the Americas. At the root of the emergence of dengue as a major health problem are changes in human demography and behavior, leading to unchecked populations of and increased exposure to the principal domestic mosquito vector, Aedes aegypti. Virus-specified factors also influence the epidemiology of dengue. Speculations on future events in the epidemiology, evolution, and biological expression of dengue are presented.
Impact of malaria on genetic polymorphism and genetic diseases in Africans and African Americans.
Authors: Miller L H
Published: May 2006

The high mortality from malaria in sub-Sahara Africa selected multiple genes that give the population a selective advantage. Identification of the genetic basis for resistance may suggest unusual approaches to development of malarial vaccines and antimalarial drugs. Some of these genes may be deleterious, although of selective advantage within the African setting, and need to be identified for counseling for disease prevention.
ocs element promoter sequences are activated by auxin and salicylic acid in Arabidopsis.
Authors: Zhang B , Singh K B
Published: May 2006

ocs elements are a group of promoter elements that have been exploited by two distinct groups of plant pathogens, Agrobacterium and certain viruses, to express genes in plants. We examined the activity of single and multiple ocs elements linked to a minimal plant promoter and the uidA reporter gene in transgenic Arabidopsis. beta-Glucuronidase activity was detected only in root tips and in callus tissue after auxin treatment. A more sensitive assay revealed that auxin treatment also increased ocs element activity in aerial parts of the plant, although the absolute levels of ocs element activity were greater in roots. The response of ocs elements to exogenous auxin began within 1 h. Salicylic acid, a disease-resistance signal in plants, also increased ocs element activity in both roots and aerial parts of the plant. The question of whether the induction in ocs element activity is mediated through auxin and/or salicylic acid signal transduction pathways or is part of a more general stress response is discussed.
Schwann cells transplanted in the lateral ventricles prevent the functional and anatomical effects of monocular deprivation in the rat.
Authors: Pizzorusso T , Fagiolini M , Fabris M , Ferrari G , Maffei L
Published: May 2006

We investigated whether the transplant of Schwann cells prevents the physiological and morphological effects of monocular deprivation in the rat. On the day of eye opening in rats (postnatal day 14), we transplanted Schwann cells in the lateral ventricles and sutured the eyelids of one eye. After 20-30 days, at the end of the critical period for the visual system development, we analyzed the functional properties of visual cortical neurons. Spontaneous discharge, orientation selectivity, and receptive field size of visual cortical neurons in transplanted animals were in the normal range. Transplantation of Schwann cells prevented the detrimental effects of monocular deprivation on ocular dominance and binocularity of cortical neurons. Visual acuity of the deprived eye estimated by visually evoked potentials was also normal. Schwann cells derived from adult animals were as effective as those derived from neonates. The effects of Schwann cells on monocular deprivation were dependent upon the number of cells present in the transplant so that 10(6) Schwann cells were sufficient to prevent the effect of monocular deprivation, whereas 10(5) and 3.3 x 10(5) Schwann cells were ineffective, and 6.3 x 10(5) cells gave variable results. Shrinkage of the deprived lateral geniculate neurons was prevented by a transplant of 10(6) cells. In rats transplanted with hybridoma cells producing an antibody that functionally blocks nerve growth factor (NGF), we found that the effect of cotransplanted Schwann cells on monocular deprivation was partly counteracted. We conclude that transplantation of Schwann cells prevents both functional and anatomical effects of monocular deprivation, presumably acting through the production of NGF. We propose that transplants of Schwann cells could be a promising technique for clinical applications.
Lysine synthesis and catabolism are coordinately regulated during tobacco seed development.
Authors: Karchi H , Shaul O , Galili G
Published: May 2006

The regulation of synthesis and accumulation of the essential amino acid lysine was studied in seeds of transgenic tobacco plants expressing, in a seed-specific manner, two feedback-insensitive bacterial enzymes: dihydrodipicolinate synthase (EC 4.2.1.52) and aspartate kinase (EC 2.7.2.4). High-level expression of the two bacterial enzymes resulted in only a slight increase in free lysine accumulation at intermediate stages of seed development, while free lysine declined to the low level of control plants toward maturity. To test whether enhanced catabolism may have contributed to the failure of free lysine to accumulate in seeds of transgenic plants, we analyzed the activity of lysine-ketoglutarate reductase (EC 1.5.1.7), an enzyme that catabolizes lysine into saccharopine. In both the control and the transgenic plants, the timing of appearance of lysine-ketoglutarate reductase activity correlated very closely with that of dihydrodipicolinate synthase activity, suggesting that lysine synthesis and catabolism were coordinately regulated during seed development. Notably, the activity of lysine-ketoglutarate reductase was significantly higher in seeds of the transgenic plants than in the controls. Coexpression of both bacterial enzymes in the same plant resulted in a significant increase in the proportions of lysine and threonine in seed albumins. Apparently, the normal low steady-state levels of free lysine and threonine in tobacco seeds may be rate limiting for the synthesis of seed proteins, which are relatively rich in these amino acids.
Molecular and physiological alterations in murine ventricular dysfunction.
Authors: Rockman H A , Ono S , Ross R S , Jones L R , Karimi M , Bhargava V , Ross J , Chien K R
Published: May 2006

The present study reports the development and characterization of a murine model of right ventricular dysfunction following graded constriction in the pulmonary artery via microsurgical approaches. To analyze in vivo ventricular function, a technique of x-ray contrast microangiography was developed to allow the quantitative analysis of ventricular volumes and of ejection fraction in normal and pressure-overloaded right ventricle. Severe, chronic pulmonary arterial banding for 14 days resulted in right ventricular dilatation and dysfunction, associated with right atrial enlargement, and angiographic evidence of tricuspid regurgitation. These effects were dependent on the extent of hemodynamic overload, since more moderate pulmonary arterial constriction resulted in hypertrophy with maintenance of right ventricular function. With severe pulmonary artery constriction, the murine right ventricle displays a failing heart phenotype including chamber dilation with reduced function that resembles right ventricular dysfunction in man during chronic pulmonary arterial hypertension. Northern and immunoblot analyses demonstrate a marked down-regulation of phospholamban mRNA and its corresponding protein with both levels of constriction, while a less pronounced but significant depression of sarcoplasmic reticulum Ca(2+)-ATPase protein was observed with severe overload, suggesting that this pattern is an early genetic marker of ventricular dysfunction. By coupling mouse genetics with this murine model and the ability to assess cardiac function in vivo, one should be able to test the role of the down-regulation of phospholamban and other defined alterations in the cardiac muscle gene program in the onset of the failing heart phenotype.
Receptor regulation of G-protein palmitoylation.
Authors: Mumby S M , Kleuss C , Gilman A G
Published: May 2006

Many alpha subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) are palmitoylated. Exposure of cells to the beta-adrenergic agonist isoproterenol increased incorporation of [3H]palmitate specifically into alpha s, the alpha subunit that mediates stimulation of adenylyl cyclase. Pulse-chase experiments suggested that isoproterenol increased turnover of alpha s-bound palmitate. Mutagenesis of Cys-3 in alpha s or alpha o (a homologous alpha subunit) prevented palmitoylation of these proteins. Differing results were obtained when mutations of Cys-3 in alpha s or alpha o were expressed in cells and assayed for their distribution between soluble and membrane fractions. Some alpha subunits, including alpha o, are myristoylated at the amino-terminal glycine residue. Mutation of this glycine prevented both myristoylation and palmitoylation of alpha o, indicating that myristoylation precedes palmitoylation of dually acylated alpha subunits. The amino-terminal sequences and fatty acylation properties of dually acylated alpha subunits are strikingly similar to those of some members of the Src family of protein-tyrosine kinases. The amino-terminal sequence Met-Gly-Cys-Xaa-Xaa-Ser/Cys shared by these proteins may represent a motif for cotranslational and posttranslational processing that includes myristoylation of the glycine residue and reversible palmitoylation of the cysteine residue.
Dynamics and species richness of tropical rain forests.
Authors: Phillips O L , Hall P , Gentry A H , Sawyer S A , Vásquez R
Published: May 2006

We present a worldwide analysis of humid tropical forest dynamics and tree species richness. New tree mortality, recruitment, and species richness data include the most dynamic and diverse mature tropical forests known. Twenty-five sites show a strong tendency for the most species-rich forests to be dynamic and aseasonal. Mean annual tree mortality and recruitment-turnover-is the most predictive factor of species richness, implying that small-scale disturbance helps regulate tropical forest diversity. Turnover rates are also closely related to the amount of basal area turnover in mature tropical forests. Therefore the contribution of small-scale disturbance to maintaining tropical forest diversity may ultimately be driven by ecosystem productivity.
Primacy of liver glucosensors in the sympathetic response to progressive hypoglycemia.
Authors: Donovan C M , Hamilton-Wessler M , Halter J B , Bergman R N
Published: May 2006

The impact of hepatic glucose concentration on the sympathetic response to progressive hypoglycemia was examined in chronically cannulated conscious male dogs (n = 6). Graded hypoglycemia was induced via peripheral insulin infusion (30 pmol.kg-1.min-1) with either peripheral (PER) or portal (POR) glucose infusion. Over the 260-min experimental period, arterial glycemia was adjusted from 5.2 +/- 0.1 to 2.5 +/- 0.1 mM in decrements of approximately 0.5 mM every 40 min. Arterial glycemias were not significantly different between PER and POR at any measured level. However, hepatic glycemia was significantly elevated at all times during POR (8.4 +/- 0.8 to 3.4 +/- 0.2 mM) when compared to PER (5.2 +/- 0.2 to 2.5 +/- 0.1 mM). Plasma epinephrine values were significantly greater during PER vs. POR at all arterial glycemias below 4.0 mM. At the lowest level of arterial glycemia studied (2.5 +/- 0.2 mM) the epinephrine response above basal was 3-fold greater for PER (8.7 +/- 1.7 nM) when compared to POR (2.6 +/- 0.6 nM) (P < 0.01). Plasma norepinephrine results were similar for the two protocols, with PER demonstrating a 3-fold greater response above basal when compared to POR at 2.5 mM arterial glycemia (P < 0.05). While the sympathetic response was markedly different between protocols when expressed as a function of arterial glycemia, when expressed as a function of hepatic glycemia this discrepancy was largely eliminated. This latter observation supports the liver as the primary locus for glycemic detection relevant to the sympathoadrenal response when hypoglycemia develops slowly--i.e., over a period of 2-3 h. A comparison of the current findings with our previous observations suggests that the hepatic glucosensors may play a greater role in hypoglycemic counterregulation as the rate of fall in glycemia is less.
T lymphocytes synthesize and export heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor, mitogens for vascular cells and fibroblasts: differential production and release by CD4+ and CD8+ T cells.
Authors: Blotnick S , Peoples G E , Freeman M R , Eberlein T J , Klagsbrun M
Published: May 2006

T lymphocytes infiltrate wounds, tumors, and atherosclerotic plaques, pathophysiological processes characterized by the migration and proliferation of vascular cells and fibroblasts. Although T lymphocytes are known to produce cytokines for inflammatory cells, it has not been demonstrated that they synthesize growth factors that are mitogenic for vascular cells and fibroblasts. We demonstrate that cultured T lymphocytes isolated from normal human peripheral blood synthesize and export two well-characterized growth factors, heparin-binding epidermal growth factor-like growth factor (HB-EGF) and basic fibroblast growth factor (bFGF). This conclusion is based on mRNA expression analysis, heparin-affinity chromatography profiles, target-cell specificity, and functional inhibition by specific neutralizing antibodies. Atypically, a substantial amount of T-cell-derived bFGF-like activity appears to be constitutively released into conditioned medium, almost as much as is associated with T-cell lysates. bFGF is synthesized and exported by purified CD4+ and CD8+ T cells, whereas HB-EGF is synthesized and exported primarily by CD4+ T cells. The T-cell-derived HB-EGF and bFGF activities are potent mitogens for fibroblasts and smooth muscle cells, and the bFGF-like activity is also mitogenic for endothelial cells. These results suggest that T lymphocytes may play key roles in mediating smooth muscle hyperplasia associated with atherosclerosis and in angiogenesis associated with wound healing and tumor growth by acting locally to deliver vascular-cell growth factors to tissues.
Five SWI/SNF gene products are components of a large multisubunit complex required for transcriptional enhancement.
Authors: Peterson C L , Dingwall A , Scott M P
Published: May 2006

The Saccharomyces cerevisiae SWI1, SWI2 (SNF2), SWI3, SNF5, and SNF6 gene products play a crucial role in the regulation of transcription. We provide here direct biochemical evidence that all five SWI/SNF polypeptides are components of a large multisubunit complex. These five polypeptides coelute from a gel-filtration column with an apparent molecular mass of approximately 2 MDa. The five SWI/SNF polypeptides do not copurify when extracts are prepared from swi- or snf- mutants. We show that SWI/SNF polypeptides also remain associated during an affinity-chromatography step followed by gel filtration. Assembly of the SWI/SNF complex is not disrupted by a mutation in the putative APT-binding site of SWI2, although this mutation eliminates SWI2 function.
Colocalization of nitric oxide synthase and somatostatin immunoreactivity in rat dentate hilar neurons.
Authors: Dun N J , Dun S L , Wong R K , Förstermann U
Published: May 2006

Distribution of nitric oxide synthase (NOS), somatostatin (SSN), and parvalbumin (PV) was studied in the rat hippocampus by immunohistochemical methods. The aim was to explore the interrelationship between SSN-immunoreactive (SSN-IR) neurons in the dentate hilus, which have been shown to be vulnerable to a number of pathophysiological insults, and the presence or absence of NOS and/or PV in the same subset of dentate hilar neurons. Small NOS-IR neurons were scattered in the pyramidal, oriens, and radiatum layers of the CA1-CA3 areas and in the subiculum, where larger NOS-IR neurons were occasionally noted. In the area dentata, NOS-IR neurons, which were composed of small and large polymorphic cells, appeared as a single file at the hilar border with the granule cell layer and clustered in the hilus in fairly high density. Double-labeling techniques showed that most NOS-IR neurons in the hilus were SSN-IR, whereas coexistence of NOS and PV immunoreactivity or SSN and PV immunoreactivity was low in dentate hilar neurons. In other areas of the hippocampus, colocalization of NOS and SSN in the same neurons was much less frequent. Thus, SSN-IR neurons in the dentate hilus constitute a population of neurons that contain the enzyme NOS as well. The presence of NOS coupled to the lack or low level of PV in this group of neurons may provide a neurochemical basis for their high susceptibility to certain pathophysiological insults.
Tyrosine kinase JAK1 is associated with the granulocyte-colony-stimulating factor receptor and both become tyrosine-phosphorylated after receptor activation.
Authors: Nicholson S E , Oates A C , Harpur A G , Ziemiecki A , Wilks A F , Layton J E
Published: May 2006

Granulocyte-colony-stimulating factor (G-CSF) stimulates the proliferation and differentiation of cells of the neutrophil lineage by interaction with a specific receptor. Early signal transduction events following G-CSF receptor activation were studied. We detected tyrosine phosphorylation of both the G-CSF receptor and the protein tyrosine kinase JAK1 following G-CSF binding to the human G-CSF receptor. In vitro, the kinase activity of JAK1 was increased by G-CSF stimulation. Coimmunoprecipitation of JAK1 with the G-CSF receptor suggested a physical association which existed prior to G-CSF stimulation.
Identification of a brefeldin A-insensitive guanine nucleotide-exchange protein for ADP-ribosylation factor in bovine brain.
Authors: Tsai S C , Adamik R , Moss J , Vaughan M
Published: May 2006

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that participate in vesicular transport in the Golgi and other intracellular compartments and stimulate cholera toxin ADP-ribosyltransferase activity. ARFs are active in the GTP-bound form; hydrolysis of bound GTP to GDP, possibly with the assistance of a GTP hydrolysis (GTPase)-activating protein results in inactivation. Exchange of GDP for GTP and reactivation were shown by other workers to be enhanced by Golgi membranes in a brefeldin A-sensitive reaction, leading to the proposal that the guanine nucleotide-exchange protein (GEP) was a target of brefeldin A. In the studies reported here, a soluble GEP was partially purified from bovine brain. Exchange of nucleotide on ARFs 1 and 3, based on increased ARF activity in a toxin assay and stimulation of binding of guanosine 5'-[gamma-[35S]thio]triphosphate, was dependent on phospholipids, with phosphatidylserine being more effective than cardiolipin. GEP appeared to increase the rate of nucleotide exchange but did not affect the affinity of ARF for GTP. Whereas the crude GEP had a size of approximately 700 kDa, the partially purified GEP behaved on Ultrogel AcA 54 as a protein of 60 kDa. With purification, the GEP activity became insensitive to brefeldin A, consistent with the conclusion that, in contrast to earlier inferences, the exchange protein is not itself the target of brefeldin A.
Isolation of the human peroxisomal acyl-CoA oxidase gene: organization, promoter analysis, and chromosomal localization.
Authors: Varanasi U , Chu R , Chu S , Espinosa R , LeBeau M M , Reddy J K
Published: May 2006

Peroxisomal acyl-CoA oxidase (ACOX; EC 1.3.3.6) is the first enzyme of the fatty acid beta-oxidation pathway, which catalyzes the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs, and it donates electrons directly to molecular oxygen, thereby producing H2O2. The discovery of carcinogenic peroxisome proliferators, which markedly increase the levels of this H2O2-producing ACOX in rat and mouse liver, generated interest in peroxisomal beta-oxidation system genes. The present study deals with the structural organization of human ACOX gene. This gene spans approximately 33 kb and consists of 14 exons and 13 introns. Primer-extension analysis revealed three principal cap sites, which were mapped at 50, 52, and 53 nt upstream of the initiator methionine codon. The 5' flanking region of the ACOX gene was sequenced up to 500 bp upstream of the cap sites. This promoter region is G + C-rich and contains three copies of the "GC box" hexanucleotides. Multiple GC boxes are a characteristic feature of the rat ACOX and bifunctional protein genes of the beta-oxidation system. A + T-rich TATA-boxlike sequences, TTTATTT and TTATT, have also been identified in this human ACOX gene, but typical CCAAT motifs are absent. This ACOX gene has been mapped to chromosome 17q25 by in situ hybridization, using a biotinlabeled probe.
Activation-dependent phosphorylation of the T-lymphocyte surface receptor CD28 and associated proteins.
Authors: Hutchcroft J E , Bierer B E
Published: May 2006

CD28 is a costimulatory receptor that can provide the second signal necessary for T-cell activation and function in response to stimulation through the T-cell antigen receptor/CD3 complex. We found that a distinct array of proteins was phosphorylated on tyrosine following stimulation with anti-CD28 monoclonal antibody, as detected by immune-complex kinase assays. Anti-CD28 stimulation of in vitro kinase activity was detergent-dependent, occurring in immune complexes prepared with Brij 96 but not Nonidet P-40. Pretreatment of cells with low concentrations of phorbol ester increased the activation-independent phosphorylation of proteins in CD28 immune complexes. Reimmunoprecipitation studies indicated that the cytoplasmic protein-tyrosine kinases Lck and Fyn were associated with CD28. CD28 itself was phosphorylated both in vitro and in vivo in an activation-dependent manner, as detected by nonreducing/reducing SDS/PAGE analyses. The activation-stimulated phosphorylation of CD28 may play a key role in signaling through this receptor.
Drosophila syndecan: conservation of a cell-surface heparan sulfate proteoglycan.
Authors: Spring J , Paine-Saunders S E , Hynes R O , Bernfield M
Published: May 2006

In mammals, cell-surface heparan sulfate is required for the action of basic fibroblast growth factor, fibronectin, antithrombin III, as well as other effectors. The syndecans, a gene family of four transmembrane proteoglycans that participates in these interactions, are the major source of this heparan sulfate. Based on the conserved transmembrane and cytoplasmic domains of the mammalian syndecans, a single syndecan-like gene was detected and localized in the Drosophila genome. As in mammals, Drosophila syndecan is a heparan sulfate proteoglycan expressed at the cell surface that can be shed from cultured cells. The single Drosophila syndecan is expressed in embryonic tissues that correspond with those tissues in mammals that express distinct members of the syndecan family predominantly. Conservation of this class of molecules suggests that Drosophila, like mammals, uses cell-surface heparan sulfate as a receptor or coreceptor for extracellular effector molecules.
Cytotoxicity of immunoglobulins from amyotrophic lateral sclerosis patients on a hybrid motoneuron cell line.
Authors: Smith R G , Alexianu M E , Crawford G , Nyormoi O , Stefani E , Appel S H
Published: May 2006

Patients with amyotrophic lateral sclerosis possess antibodies (ALS IgGs) that bind to L-type skeletal muscle voltage-gated calcium channels (VGCCs) and inhibit L-type calcium current. To determine whether interaction of ALS IgGs with neuronal VGCCs might influence motoneuron survival, we used a motoneuron-neuroblastoma hybrid (VSC 4.1) cell line expressing binding sites for inhibitors of L-, N-, and P-type VGCCs. Using direct viable cell counts, quantitation of propidium iodide- and fluorescein diacetate-labeled cells, and lactate dehydrogenase release to assess cell survival, we document that ALS IgG kills 40-70% of cAMP-differentiated VSC 4.1 cells within 2 days. ALS IgG-mediated cytotoxicity is dependent on extracellular calcium and is prevented by peptide antagonists of N- or P-type VGCCs but not by dihydropyridine modulators of L-type VGCCs. Preincubating IgG with purified intact L-type VGCC or with isolated VGCC alpha 1 subunit also blocks ALS IgG-mediated cytotoxicity. These results suggest that ALS IgG may directly lead to motoneuron cell death by a mechanism requiring extracellular calcium and mediated by neuronal-type calcium channels.
Posttranscriptional modification of myosin heavy-chain gene expression in the hypertrophied rat myocardium.
Authors: Ojamaa K , Petrie J F , Balkman C , Hong C , Klein I
Published: May 2006

Hypertrophy of the myocardium in response to pressure or volume overload elicits a change in myofibrillar protein content as a result of changes in both transcriptional and translational regulation of gene expression. Hemodynamic overload caused by aortic constriction produced changes in the expression of the two isoforms of myosin heavy chain (MHC) with a 319% increase in beta-MHC mRNA and a 54% decrease in alpha-MHC mRNA (P < 0.01). Cardiac unloading as a result of heterotopic transplantation resulted in a decrease in cardiac mass and a similar shift in MHC isoform expression. In this study. We investigated cardiac gene transcription to understand how different hemodynamic stimuli produce similar cardiac phenotypes. We studied the in vivo activity of the alpha-MHC promoter (-2564 to +421 bp of the transcriptional start site) by directly injecting a recombinant expression plasmid (pAM3LUC) into the ventricular tissue of coarctated animals as well as into the unloaded heterotopic transplanted heart. When expressed as a function of the activity of a constitutively active viral promoter (pSVCAT), pAM3LUC activities were 18.4 +/- 2.9, 24.6 +/- 2.6, and 25.0 +/- 4.5 (x10(4)) luciferase/chloramphenicol acetyltransferase units in the hypertrophied ventricles of 2-, 3-, and 7-day coarctated animals, respectively. These values were not statistically different from pAM3LUC activity in control hearts of sham operated animals even though alpha-MHC mRNA content was decreased by 54% in the hypertrophied myocardium. This disparity between transcriptional activity and mRNA content suggests that alpha-MHC expression in the hypertrophic ventricle is in part regulated by a posttranscriptional mechanism. In contrast, alpha-MHC promoter activity in the unloaded transplanted hearts decreased significantly by 37% compared to control working hearts and suggests that a transcriptional mechanism of regulation of the alpha-MHC gene may account for the phenotypic expression observed in the unloaded myocardium.
Cell biological mechanisms of multidrug resistance in tumors.
Authors: Simon S M , Schindler M
Published: May 2006

Multidrug resistance (MDR) is a generic term for the variety of strategies tumor cells use to evade the cytotoxic effects of anticancer drugs. MDR is characterized by a decreased sensitivity of tumor cells not only to the drug employed for chemotherapy but also to a broad spectrum of drugs with neither obvious structural homology nor common targets. This pleiotropic resistance is one of the major obstacles to the successful treatment of tumors. MDR may result from structural or functional changes at the plasma membrane or within the cytoplasm, cellular compartments, or nucleus. Molecular mechanisms of MDR are discussed in terms of modifications in detoxification and DNA repair pathways, changes in cellular sites of drug sequestration, decreases in drug-target affinity, synthesis of specific drug inhibitors within cells, altered or inappropriate targeting of proteins, and accelerated removal or secretion of drugs.