bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromatin structure and gene expression.
Authors: Felsenfeld G , Boyes J , Chung J , Clark D , Studitsky V
Published: July 2002

It is now well understood that chromatin structure is perturbed in the neighborhood of expressed genes. This is most obvious in the neighborhood of promoters and enhancers, where hypersensitivity to nucleases marks sites that no longer carry canonical nucleosomes, and to which transcription factors bind. To study the relationship between transcription factor binding and the generation of these hypersensitive regions, we mutated individual cis-acting regulatory elements within the enhancer that lies between the chicken beta- and epsilon-globin genes. Constructions carrying the mutant enhancer were introduced by stable transformation into an avian erythroid cell line. We observed that weakening the enhancer resulted in creation of two classes of site: those still completely accessible to nuclease attack and those that were completely blocked. This all-or-none behavior suggests a mechanism by which chromatin structure can act to sharpen the response of developmental systems to changing concentrations of regulatory factors. Another problem raised by chromatin structure concerns the establishment of boundaries between active and inactive chromatin domains. We have identified a DNA element at the 5' end of the chicken beta-globin locus, near such a boundary, that has the properties of an insulator; in test constructions, it blocks the action of an enhancer on a promoter when it is placed between them. We describe the properties and partial dissection of this sequence. A third problem is posed by the continued presence of nucleosomes on transcribed genes, which might prevent the passage of RNA polymerase. We show, however, that a prokaryotic polymerase can transcribe through a histone octamer on a simple chromatin template. The analysis of this process reveals that an octamer is capable of transferring from a position in front of the polymerase to one behind, without ever losing its attachment to the DNA.
Involvement of integrins alpha v beta 3 and alpha v beta 5 in ocular neovascular diseases.
Authors: Friedlander M , Theesfeld C L , Sugita M , Fruttiger M , Thomas M A , Chang S , Cheresh D A
Published: July 2002

Angiogenesis underlies the majority of eye diseases that result in catastrophic loss of vision. Recent evidence has implicated the integrins alpha v beta 3 and alpha v beta 5 in the angiogenic process. We examined the expression of alpha v beta 3 and alpha v beta 5 in neovascular ocular tissue from patients with subretinal neovascularization from age-related macular degeneration or the presumed ocular histoplasmosis syndrome or retinal neovascularization from proliferative diabetic retinopathy (PDR). Only alpha v beta 3 was observed on blood vessels in ocular tissues with active neovascularization from patients with age-related macular degeneration or presumed ocular histoplasmosis, whereas both alpha v beta 3 and alpha v beta 5 were present on vascular cells in tissues from patients with PDR. Since we observed both integrins on vascular cells from tissues of patients with retinal neovascularization from PDR, we examined the effects of a systemically administered cyclic peptide antagonist of alpha v beta 3 and alpha v beta 5 on retinal angiogenesis in a murine model. This antagonist specifically blocked new blood vessel formation with no effect on established vessels. These results not only reinforce the concept that retinal and subretinal neovascular diseases are distinct pathological processes, but that antagonists of alpha v beta 3 and/or alpha v beta 5 may be effective in treating individuals with blinding eye disease associated with angiogenesis.
Promiscuous translocations into immunoglobulin heavy chain switch regions in multiple myeloma
Authors: Bergsagel P. Leif , Chesi Marta , Nardini Elena , Brents Leslie A. , Kirby Suzanne L. , Kuehl W. Michael
Published: July 2002

In multiple myeloma, karyotypic 14q32 translocations have been identified at a variable frequency (10–60% in different studies). In the majority of cases, the partner chromosome has not been identified (14q+), and in the remaining cases, a diverse array of chromosomal partners has been implicated, with 11q13 being the most common. We developed a comprehensive Southern blot assay to identify and distinguish different kinds of immunoglobulin heavy chain (IgH) switch recombination events. Illegitimate switch recombination fragments (defined as containing sequences from only one switch region) are potential markers of translocation events into IgH switch regions and were identified in 15 of 21 myeloma cell lines, including seven of eight karyotyped lines that have no detectable 14q32 translocation. From all nine lines or tumor samples analyzed further, cloned illegitimate switch recombination fragments were confirmed to be IgH switch translocation breakpoints. In three of these cases, the translocation breakpoint was shown to be present in the primary tumor. These translocation breakpoints involve six chromosomal loci: 4p16.3 (two lines and the one tumor); 6; 8q24.13; 11q13.3 (in three lines); 16q23.1; and 21q22.1. We suggest that translocations into the IgH locus (i) are frequent (karyotypic 14q32 translocations and/or illegitimate switch recombination fragments are present in primary tumor samples and in 19 of 21 lines that we have analyzed); (ii) occur mainly in switch regions; and (iii) involve a diverse but nonrandom array (i.e., frequently 11q13 or 4p16) of chromosomal partners. This appears to be the most frequent genetic abnormality in multiple myeloma.
Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase.
Authors: Hu J , Seeger C
Published: July 2002

The heat shock protein Hsp90 is known as an essential component of several signal transduction pathways and has now been identified as an essential host factor for hepatitis B virus replication. Hsp90 interacts with the viral reverse transcriptase to facilitate the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA ligand. This RNP complex is required early in replication for viral assembly and initiation of DNA synthesis through a protein-priming mechanism. These results thus invoke a role for the Hsp90 pathway in the formation of an RNP.
The treatment of chronic progressive multiple sclerosis with cladribine.
Authors: Beutler E , Sipe J C , Romine J S , Koziol J A , McMillan R , Zyroff J
Published: July 2002

A 2-year, placebo-controlled, double-blind, crossover study was started in 1992 to evaluate cladribine, an immunosuppressive drug, in the treatment of chronic progressive multiple sclerosis. In the first year patients were given cladribine 0.10 mg/kg per day for 7 days as four monthly courses for a total of 2.8 mg/kg or placebo. During the second year patients treated with placebo during the first year were given i.v. infusions of 0.10 mg, 0.05 mg, and 0.05 mg of cladribine per kg of body weight per day for 7 consecutive days in three successive monthly courses, for a total dose of 1.4 mg/kg. Patients who had been treated previously with cladribine were crossed over to placebo. Analysis of the results revealed a favorable influence on the neurological performance scores, both in the Kurtze extended disability status and the Scripps neurological rating scale, and on MRI findings in patients treated with cladribine. In the first year the most striking finding was that while clinical deterioration continued in the placebo-treated patients, the condition of patients who received cladribine stabilized or even improved slightly. Toxicity and therapeutic response were dose-related.
Environmental services of biodiversity.
Authors: Myers N
Published: July 2002

Humans derive many utilitarian benefits from the environmental services of biotas and ecosystems. This is often advanced as a prime argument to support conservation of biodiversity. There is much to be said for this viewpoint, as is documented in this paper through a summary assessment of several categories of environmental services, including regulation of climate and biogeochemical cycles, hydrological functions, soil protection, crop pollination, pest control, recreation and ecotourism, and a number of miscellaneous services. It is shown that the services are indeed significant, whether in ecological or economic senses. Particularly important is the factor of ecosystem resilience, which appears to underpin many of the services. It should not be supposed, however, that environmental services stem necessarily and exclusively from biodiversity. While biodiversity often plays a key role, the services can also derive from biomass and other attributes of biotas. The paper concludes with a brief overview assessment of economic values at issue and an appraisal of the implications for conservation planning.
Discovery of a second 15
S
-lipoxygenase in humans
Authors: Brash Alan R. , Boeglin William E. , Chang Min S.
Published: July 2002

The lipoxygenase metabolism of arachidonic acid occurs in specific blood cell types and epithelial tissues and is activated in inflammation and tissue injury. In the course of studying lipoxygenase expression in human skin, we detected and characterized a previously unrecognized enzyme that at least partly accounts for the 15
S
-lipoxygenase metabolism of arachidonic acid in certain epithelial tissues. The cDNA was cloned from human hair roots, and expression of the mRNA was detected also in prostate, lung, and cornea; an additional 16 human tissues, including peripheral blood leukocytes, were negative for the mRNA. The cDNA encodes a protein of 676 amino acids with a calculated molecular mass of 76 kDa. The amino acid sequence has approximately 40% identity to the known human 5
S
-, 12
S
-, and 15
S
-lipoxygenases. When expressed in HEK 293 cells, the newly discovered enzyme converts arachidonic acid exclusively to 15
S
-hydroperoxyeicosatetraenoic acid, while linoleic acid is less well metabolized. These features contrast with the previously reported 15
S
-lipoxygenase, which oxygenates arachidonic acid mainly at C-15, but also partly at C-12, and for which linoleic acid is an excellent substrate. The different catalytic activities and tissue distribution suggest a distinct function for the new enzyme compared with the previously reported human 15
S
-lipoxygenase.
AtMRP1
gene of
Arabidopsis
encodes a glutathione
S
-conjugate pump: Isolation and functional definition of a plant ATP-binding cassette transporter gene
Authors: Lu Yu-Ping , Li Ze-Sheng , Rea Philip A.
Published: July 2002

Because plants produce cytotoxic compounds to which they, themselves, are susceptible and are exposed to exogenous toxins (microbial products, allelochemicals, and agrochemicals), cell survival is contingent on mechanisms for detoxifying these agents. One detoxification mechanism is the glutathione
S
-transferase-catalyzed glutathionation of the toxin, or an activated derivative, and transport of the conjugate out of the cytosol. We show here that a transporter responsible for the removal of glutathione
S
-conjugates from the cytosol, a specific Mg
2+
-ATPase, is encoded by the
AtMRP1
gene of
Arabidopsis thaliana
. The sequence of
AtMRP1
and the transport capabilities of membranes prepared from yeast cells transformed with plasmid-borne
AtMRP1
demonstrate that this gene encodes an ATP-binding cassette transporter competent in the transport of glutathione
S
-conjugates of xenobiotics and endogenous substances, including herbicides and anthocyanins.
Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes
Authors: Wang Ming , Roberts David L. , Paschke Rosemary , Shea Thomas M. , Masters Bettie Sue Siler , Kim Jung-Ja P.
Published: July 2002

Microsomal NADPH–cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in
Escherichia coli
and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin–NADP
+
reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 Å. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome
c
and the physiological redox partners, including cytochromes P450 and
b
5 and heme oxygenase.
A dynamin GTPase mutation causes a rapid and reversible temperature-inducible locomotion defect in
C. elegans
Authors: Clark Scott G. , Shurland Dixie-Lee , Meyerowitz Elliot M. , Bargmann Cornelia I. , van der Bliek Alexander M.
Published: July 2002

Drosophila shibire
and its mammalian homologue dynamin regulate an early step in endocytosis. We identified a
Caenorhabditis elegans
dynamin gene,
dyn-1
, based upon hybridization to the
Drosophila
gene. The
dyn-1
RNA transcripts are
trans
-spliced to the spliced leader 1 and undergo alternative splicing to code for either an 830- or 838-amino acid protein. These
dyn-1
proteins are highly similar in amino acid sequence, structure, and size to the
Drosophila
and mammalian dynamins: they contain an N-terminal GTPase, a pleckstrin homology domain, and a C-terminal proline-rich domain. We isolated a recessive temperature-sensitive
dyn-1
mutant containing an alteration within the GTPase domain that becomes uncoordinated when shifted to high temperature and that recovers when returned to lower temperatures, similar to
D. shibire
mutants. When maintained at higher temperatures,
dyn-1
mutants become constipated, egg-laying defective, and produce progeny that die during embryogenesis. Using a
dyn-1
::
lacZ
gene fusion, a high level of dynamin expression was observed in motor neurons, intestine, and pharyngeal muscle. Our results suggest that
dyn-1
function is required during development and for normal locomotion.
Immunocompetence, ornamentation, and viability of male barn swallows (Hirundo rustica)
Authors: Saino Nicola , Bolzern Anna Maria , Møller Anders Pape
Published: July 2002

Immunocompetence (i.e., the ability to produce an immune response to pathogens) can be predicted to influence the chances that organisms have to survive and reproduce. In this study we simulated a challenge to the immune systems of male barn swallows (Hirundo rustica) by injecting them intraperitoneally with a multigenic antigen, sheep red blood cells, and we analyzed long-term survival in relation to their immunocompetence. Males were assigned to four groups that differed for the treatment of the length of the outermost tail feathers, a sexually dimorphic ornamental character that is currently under directional sexual selection. Immunocompetence was measured as change of concentration of gamma globulins relative to plasma proteins. The intensity of the immune response was independent of age. Males that showed the highest short-term response to sheep red blood cells were more likely to survive until the breeding season following that in which they had been inoculated, a pattern consistently observed within each experimental group. Males with comparatively long tails were more likely to survive than those with short tails. To our knowledge, the results of this study are the first to demonstrate that immunocompetence can predict long-term survival in a free-ranging vertebrate. Moreover, they are compatible with current models of parasite-mediated sexual selection because long-tailed males are more immunocompetent than short-tailed ones, and females, by preferring to mate with the most ornamented males, may acquire the “good genes” for high immunocompetence and, hence, for high viability of their offspring.
Telomere lengthening and telomerase activation during human B cell differentiation
Authors: Weng Nan-ping , Granger Lawrence , Hodes Richard J.
Published: July 2002

The function of the immune system is highly dependent on cellular differentiation and clonal expansion of antigen-specific lymphocytes. However, little is known about mechanisms that may have evolved to protect replicative potential in actively dividing lymphocytes during immune differentiation and response. Here we report an analysis of telomere length and telomerase expression, factors implicated in the regulation of cellular replicative lifespan, in human B cell subsets. In contrast to previous observations, in which telomere shortening and concomitant loss of replicative potential occur in the process of somatic cell differentiation and cell division, it was found that germinal center (GC) B cells, a compartment characterized by extensive clonal expansion and selection, had significantly longer telomeric restriction fragments than those of precursor naive B cells. Furthermore, it was found that telomerase, a telomere-synthesizing enzyme, is expressed at high levels in GC B cells (at least 128-fold higher than those of naive and memory B cells), correlating with the long telomeres in this subset of B cells. Finally, both naive and memory B cells were capable of up-regulating telomerase activityin vitroin response to activation signals through the B cell antigen receptor in the presence of CD40 engagement and/or interleukin 4. These observations suggest that a novel process of telomere lengthening, possibly mediated by telomerase, functions in actively dividing GC B lymphocytes and may play a critical role in humoral immune response by maintaining the replicative potential of GC and descendant memory B cells.
Cockayne syndrome group B protein enhances elongation by RNA polymerase II
Authors: Selby Christopher P. , Sancar Aziz
Published: July 2002

Cockayne syndrome (CS) is characterized by impaired physical and mental development. Two complementation groups, CSA and CSB, have been identified. Here we report that the
CSB
gene product enhances elongation by RNA polymerase II. CSB stimulated the rate of elongation on an undamaged template by a factor of about 3. A thymine-thymine cyclobutane dimer located in the template strand is known to be a strong block to transcription. Addition of CSB to the blocked polymerase resulted in addition of one nucleotide to the nascent transcript. Finally, addition of transcription factor IIS is known to cause polymerase blocked at a thymine-thymine cyclobutane dimer to digest its nascent transcript, and CSB counteracted this transcript shortening action of transcription factor IIS. Thus a deficiency in transcription elongation may contribute to the CS phenotype.
A structural census of the current population of protein sequences
Authors: Gerstein Mark , Levitt Michael
Published: July 2002

We examine the occurrence of the ≈300 known protein folds in different groups of organisms. To do this, we characterize a large fraction of the currently known protein sequences (≈140,000) in structural terms, by matching them to known structures via sequence comparison (or by secondary-structure class prediction for those without structural homologues). Overall, we find that an appreciable fraction of the known folds are present in each of the major groups of organisms (e.g., bacteria and eukaryotes share 156 of 275 folds), and most of the common folds are associated with many families of nonhomologous sequences (i.e., >10 sequence families for each common fold). However, different groups of organisms have characteristically distinct distributions of folds. So, for instance, some of the most common folds in vertebrates, such as globins or zinc fingers, are rare or absent in bacteria. Many of these differences in fold usage are biologically reasonable, such as the folds of metabolic enzymes being common in bacteria and those associated with extracellular transport and communication being common in animals. They also have important implications for database-based methods for fold recognition, suggesting that an unknown sequence from a plant is more likely to have a certain fold (e.g., a TIM barrel) than an unknown sequence from an animal.
Increased fluid secretion after adenoviral-mediated transfer of the aquaporin-1 cDNA to irradiated rat salivary glands
Authors: Delporte Christine , O’Connell Brian C. , He Xinjun , Lancaster Henry E. , O’Connell Anne C. , Agre Peter , Baum Bruce J.
Published: July 2002

A replication-deficient, recombinant adenovirus encoding human aquaporin-1 (hAQP1), the archetypal water channel, was constructed. This virus, AdhAQP1, directed hAQP1 expression in several epithelial cell lines
in vitro
. In polarized MDCK cell monolayers, hAQP1 was localized in the apical and basolateral plasma membranes. Fluid movement across monolayers infected by AdhAQP1 in response to an osmotic gradient was ≈4-fold that seen with uninfected monolayers or monolayers infected by a control virus. When AdhAQP1 was administered to rat submandibular glands by retrograde ductal instillation, significant hAQP1 expression was observed by Western blot analysis in crude plasma membranes and by immunohistochemical staining in both acinar and ductal cells. Three or four months after exposure to a single radiation dose (17.5 or 21 Gy, respectively), AdhAQP1 administration to rat submandibular glands led to a two- to threefold increase in salivary secretion compared with secretion from glands administered a control virus. These results suggest that hAQP1 gene transfer may have potential as an unique approach for the treatment of postradiation salivary hypofunction.
Expression of RBM in the nuclei of human germ cells is dependent on a critical region of the Y chromosome long arm
Authors: Elliott D. J. , Millar M. R. , Oghene K. , Ross A. , Kiesewetter F. , Pryor J. , McIntyre M. , Hargreave T. B. , Saunders P. T. K. , Vogt P. H. , Chandley A. C. , Cooke H.
Published: July 2002

The association of abnormal spermatogenesis in men with Y chromosome deletions suggests that genes important for spermatogenesis have been removed from these individuals. Recently, genes encoding two putative RNA-binding proteins (RBM and DAZ/SPGY) have been mapped to two different regions of the human Y chromosome. Both of these genes encode proteins that contain a single RNA recognition motif and a (different) internally repeating sequence. Y-linked RBM homologues are found in all mammalian species. We have raised an antiserum to RBM and used it to show that RBM is a nuclear protein expressed in fetal, prepubertal, and adult male germ cells. The distribution of RBM protein in the adult correlates with the pattern of transcriptional activity in spermatogenesis, suggesting that RBM is involved in the nuclear metabolism of newly synthesized RNA. RBM sequences are found on both arms of the Y chromosome making genotype–phenotype correlations difficult for this gene family. To address the location of the functional genes and the consequences of their deletion, we examined a panel of men with Y chromosome deletions and known testicular pathologies using this antiserum. This approach enabled us to map a region of the Y chromosome essential for RBM expression. In the absence of detectable RBM expression we see stages of germ cell development up to early meiosis, but not past this point into the haploid phase of spermatogenesis.
A statistical mechanical model for β-hairpin kinetics
Authors: Muñoz Victor , Henry Eric R. , Hofrichter James , Eaton William A.
Published: July 2002

Understanding the mechanism of protein secondary structure
formation is an essential part of the protein-folding puzzle. Here, we
describe a simple statistical mechanical model for the formation of a
β-hairpin, the minimal structural element of the antiparallel
β-pleated sheet. The model accurately describes the thermodynamic and
kinetic behavior of a 16-residue, β-hairpin-forming peptide,
successfully explaining its two-state behavior and apparent negative
activation energy for folding. The model classifies structures
according to their backbone conformation, defined by 15 pairs of
dihedral angles, and is further simplified by considering only the 120
structures with contiguous stretches of native pairs of backbone
dihedral angles. This single sequence approximation is tested by
comparison with a more complete model that includes the 2
15
possible conformations and 15 × 2
15
possible kinetic
transitions. Finally, we use the model to predict the equilibrium
unfolding curves and kinetics for several variants of the β-hairpin
peptide.
Default taxonomy: Ernst Mayr’s view of the microbial world
Authors: Woese Carl R.
Published: July 2002

This perspective is a response to a taxonomic proposal by E. Mayr
[“Two empires or three?” (1998)
Proc. Natl. Acad.
Sci.
USA
95, 9720–9723]. Mayr has suggested
that the now accepted classification of life into three primary
domains, Archaea, Bacteria, and Eucarya—originally proposed by myself
and others—be abandoned in favor of the earlier Prokaryote–Eukaryote
classification. Although the matter appears a taxonomic quibble, it is
not that simple. At issue here are differing views as to the nature of
biological classification, which are underlain by differing views as to
what biology is and will be—matters of concern to all biologists.
TcpP protein is a positive regulator of virulence gene expression in
Vibrio cholerae
Authors: Häse Claudia C. , Mekalanos John J.
Published: July 2002

The production of several virulence factors in
Vibrio cholerae
O1, including cholera toxin and the pilus colonization factor TCP (toxin-coregulated pilus), is strongly influenced by environmental conditions. To specifically identify membrane proteins involved in these signal transduction events, we examined a transposon library of
V. cholerae
generated by Tn
bla
mutagenesis for cells that produce TCP when grown under various nonpermissive conditions. To select for TCP-producing cells we used the recently described bacteriophage CTXΦ-Kan, which uses TCP as its receptor and carries a gene encoding resistance to kanamycin. Among the isolated mutants was a transposon insertion in a gene homologous to
nqrB
from
Vibrio alginolyticus
, which encodes a subunit of a Na
+
-translocating NADH:ubiquinone oxidoreductase, and
tcpI
, encoding a chemoreceptor previously implicated in the negative regulation of TCP production. A third transposon mutant had an insertion in
tcpP
, which is in an operon with
tcpH
, a known positive regulator of TCP production. However, TcpP was shown to be essential for TCP production in
V. cholerae
, as a
tcpP
-deletion strain was deficient in pili production. The amino-terminal region of TcpP shows sequence homology to the DNA-binding domains of several regulatory proteins, including ToxR from
V. cholerae
and PsaE from
Yersinia pestis
. Like ToxR, TcpP activates transcription of the
toxT
gene, an essential activator of
tcp
operon transcription. Furthermore, TcpH, with its large periplasmic domain and inner membrane anchor, has a structure similar to that of ToxS and was shown to enhance the activity of TcpP. We propose that TcpP/TcpH constitute a pair of regulatory proteins functionally similar to ToxR/ToxS and PsaE/PsaF that are required for
toxT
transcription in
V. cholerae
.
Target cell-specific modulation of transmitter release at terminals from a single axon
Authors: Scanziani Massimo , Gähwiler Beat H. , Charpak Serge
Published: July 2002

In the hippocampus, a CA3 pyramidal cell forms excitatory synapses with thousands of other pyramidal cells and inhibitory interneurons. By using sequential paired recordings from three connected cells, we show that the presynaptic properties of CA3 pyramidal cell terminals, belonging to the same axon, differ according to the type of target cell. Activation of presynaptic group III metabotropic glutamate receptors decreases transmitter release only at terminals contacting CA1 interneurons but not CA1 pyramidal cells. Furthermore, terminals contacting distinct target cells show different frequency facilitation. On the basis of these results, we conclude that the pharmacological and physiological properties of presynaptic terminals are determined, at least in part, by the target cells.
A
Vibrio cholerae
pathogenicity island associated with epidemic and pandemic strains
Authors: Karaolis David K. R. , Johnson Judith A. , Bailey Camella C. , Boedeker Edgar C. , Kaper James B. , Reeves Peter R.
Published: July 2002

The bacterial species
Vibrio cholerae
includes harmless aquatic strains as well as strains capable of causing epidemics and global pandemics of cholera. While investigating the relationship between pathogenic and nonpathogenic strains, we identified a chromosomal pathogenicity island (PAI) that is present in epidemic and pandemic strains but absent from nonpathogenic strains. Initially, two ToxR-regulated genes (
aldA
and
tagA
) were studied and were found to be associated with epidemic and pandemic strains but absent in nontoxigenic strains. The region containing
aldA
and
tagA
comprises 13 kb of previously unidentified DNA and is part of a PAI that contains a regulator of virulence genes (ToxT) and a gene cluster encoding an essential colonization factor and the cholera toxin phage receptor (toxin-coregulated pilus; TCP). The PAI is 39.5 kb in size, has low %G+C (35%), contains putative integrase and transposase genes, is flanked by
att
sites, and inserts near a 10Sa RNA gene (
ssrA
), suggesting it may be of bacteriophage origin. We found this PAI in two clinical non-O1/non-O139 cholera toxin-positive strains, suggesting that it can be transferred within
V. cholerae
. The sequence within this PAI includes an ORF with homology to a gene associated with the type IV pilus gene cluster of enteropathogenic
Escherichia coli
, a transposase from
Vibrio anguillarum
, and several ORFs with no known homology. As the PAI contains the CTXΦ receptor, it may represent the initial genetic factor required for the emergence of epidemic and pandemic cholera. We propose to call this island VPI (
V. cholerae
pathogenicity island).
Derepression of the C/EBPα gene during adipogenesis: Identification of AP-2α as a repressor
Authors: Jiang Man-Shiow , Tang Qi-Qun , McLenithan John , Geiman Deb , Shillinglaw Wendy , Henzel William J. , Lane M. Daniel
Published: July 2002

During adipogenesis, CCAAT/enhancer binding protein α (C/EBPα) serves as a pleiotropic transcriptional activator of adipocyte genes. Previously, we identified dual repressive elements in the C/EBPα gene and a putative transacting factor (
C
/EBPα
u
ndifferentiated
p
rotein, or CUP) expressed by preadipocytes, but not adipocytes, that bind to these elements. In the present investigation, CUP was purified 17,000-fold from nuclear extracts of 3T3-L1 preadipocytes. Amino acid sequence and mass spectral analysis of tryptic peptides derived from purifed CUP (molecular mass ≈50 kDa) revealed that the repressor is (or contains) an isoform of the transcription factor, AP-2α. Electrophoretic mobility shift and Western blot analysis on purified CUP and preadipocyte nuclear extracts confirmed the identity of CUP as AP-2α. Both AP-2α protein and CUP binding activity are expressed by preadipocytes and then decrease concomitantly during differentiation of 3T3-L1 preadipocytes into adipocytes. Consistent with a repressive role of AP-2α/CUP, an AP-2α1 expression vector, cotransfected with a C/EBPα promoter-reporter construct into 3T3-L1 adipocytes, inhibited reporter gene transcription. Taken together with previous results, these findings suggest that in preadipocytes the C/EBPα gene is repressed by AP-2α/CUP, which, upon induction of differentiation, is down-regulated, allowing expression of the gene.
Sexually transmitted chemical defense in a moth
(Utetheisa ornatrix)
Authors: González Andrés , Rossini Carmen , Eisner Maria , Eisner Thomas
Published: July 2002

The arctiid moth
Utetheisa ornatrix
is protected against predation by pyrrolizidine alkaloids (PA) that it sequesters as a larva from its food plant. Earlier work had shown that males transmit PA to the female with the sperm package and that the female bestows part of this gift on the eggs, protecting these against predation as a result. We now show that the female herself derives protection from the gift. Females deficient in PA are vulnerable to predation from spiders (
Lycosa ceratiola
and
Nephila clavipes
). If mated with a PA-laden male, the females become unacceptable as prey. The effect takes hold promptly and endures; females are unacceptable to spiders virtually from the moment they uncouple from the male and remain unacceptable as they age. Chemical data showed that the female allocates the received PA quickly to all body parts. We predict that other instances will be found of female insects being rendered invulnerable by receipt of sexually transmitted chemicals.
The tumor-suppressor gene
FHIT
is involved in the regulation of apoptosis and in cell cycle control
Authors: Sard Laura , Accornero Paola , Tornielli Silvana , Delia Domenico , Bunone Giuseppe , Campiglio Manuela , Colombo Mario P. , Gramegna Marcella , Croce Carlo M. , Pierotti Marco A. , Sozzi Gabriella
Published: July 2002

Alteration of the
FHIT
(fragile histidine triad) gene occurs as an early and frequent event in lung carcinogenesis.
FHIT
gene transfer into lung cancer cell line H460 lacking Fhit protein expression resulted in reversion of tumorigenicity. To gain insight into the biological function of
FHIT
, we compared the H460 cell line with its Fhit transfectants (H460/
FHIT
). A significant inhibition of cell growth was observed in H460/
FHIT
cells. The analysis of apoptosis by
in situ
terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling revealed a high rate of apoptosis-induced DNA strand breaks in stable clones.
In situ
results were confirmed by FACScan analysis that showed an apoptotic rate of 44–47% compared with a 15% level in the control H460 cells. Analysis of cell cycle-phase distribution indicated a significant G
0
/G
1
arrest and the presence of a sub-G
1
peak in the stable clones. No significant changes in Bcl2, BclX, and Bax protein expression level were observed in the transfected clones as compared with the control H460 cells whereas a 2-fold increase in Bak protein levels was noticed. An increased level of p21
waf
protein paralleled by an up-regulation of p21
waf
transcripts also was found in Fhit-expressing clones compared with the H460 cell line. No differences in p53 levels were observed in the same cells, suggesting a p53-independent effect. These data suggest that the observed growth-inhibitory effect in
FHIT
-reexpressing cells could be related to apoptosis and cell cycle arrest and link the tumor-suppressor activity of
FHIT
to its proapoptotic function.
Persistence of an alternate chromatin structure at silenced loci
in vitro
Authors: Ansari Athar , Gartenberg Marc R.
Published: July 2002

In
Saccharomyces cerevisiae
, transcriptional repression at the
HM
mating-type loci and telomeres results from the formation of a heterochromatin-like structure. Silencing requires at least three Sir proteins (Sir2p–4p), which are recruited to chromatin by silencers at the
HM
loci and TG
1–3
tracts at telomeres. Sir proteins and telomeres colocalize at the nuclear periphery, suggesting that this subnuclear position may also contribute to transcriptional repression. To evaluate the contribution of nuclear context to silencing, we developed methodology to isolate silent chromatin for analysis
in vitro
. Site-specific recombination was used
in vivo
to produce DNA rings from the silent
HMR
locus, and differential centrifugation was used to isolate the rings from whole-cell lysate. The partially purified rings retained many of the intracellular hallmarks of transcriptionally repressed domains. Specifically, rings from repressed strains were resistant to restriction endonuclease digestion, bore an altered DNA topology, and were associated with Sir3p. The recombination approach also was used to form rings from
HMR
that lacked silencers. Despite the uncoupling of these cis-acting regulatory elements, similar but nonidentical results were obtained. We conclude that an alternate chromatin structure at silent loci can persist
in vitro
in the absence of silencers and nuclear compartmentalization.
Atmospheric oxygen over Phanerozoic time
Authors: Berner Robert A.
Published: July 2002

It is quite possible that the level of
atmospheric oxygen has varied (roughly between 15 and 30%
O
2
) over the past 550 million years. This variation is
suggested by modeling of the carbon and sulfur cycles, by the excessive
sediment burial of organic matter that accompanied the advent of large
vascular land plants, and by recent physiological studies that relate
to biological evolution.
The structure of the human βII-tryptase tetramer: Fo(u)r better or worse
Authors: Sommerhoff Christian P. , Bode Wolfram , Pereira Pedro J. B. , Stubbs Milton T. , Stürzebecher Jörg , Piechottka Gerd P. , Matschiner Gabriele , Bergner Andreas
Published: July 2002

Tryptases, the predominant serine proteinases of human mast cells, have recently been implicated as mediators in the pathogenesis of allergic and inflammatory conditions, most notably asthma. Their distinguishing features, their activity as a heparin-stabilized tetramer and resistance to most proteinaceous inhibitors, are perfectly explained by the 3-Å crystal structure of human βII-tryptase in complex with 4-amidinophenylpyruvic acid. The tetramer consists of four quasiequivalent monomers arranged in a flat frame-like structure. The active centers are directed toward a central pore whose narrow openings of approximately 40 Å × 15 Å govern the interaction with macromolecular substrates and inhibitors. The tryptase monomer exhibits the overall fold of trypsin-like serine proteinases but differs considerably in the conformation of six surface loops arranged around the active site. These loops border and shape the active site cleft to a large extent and form all contacts with neighboring monomers via two distinct interfaces. The smaller of these interfaces, which is exclusively hydrophobic, can be stabilized by the binding of heparin chains to elongated patches of positively charged residues on adjacent monomers or, alternatively, by high salt concentrationsin vitro. On tetramer dissociation, the monomers are likely to undergo transformation into a zymogen-like conformation that is favored and stabilized by intramonomer interactions. The structure thus provides an improved understanding of the unique properties of the biologically active tryptase tetramer in solution and will be an incentive for the rational design of mono- and multifunctional tryptase inhibitors.
Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes
Authors: Foretz Marc , Guichard Colette , Ferré Pascal , Foufelle Fabienne
Published: July 2002

Hepatic glucokinase plays a key role in glucose metabolism as underlined by the anomalies associated with glucokinase mutations and the consequences of tissue-specific knock-out. In the liver, glucokinase transcription is absolutely dependent on the presence of insulin. The cis-elements and trans-acting factors that mediate the insulin effect are presently unknown; this is also the case for most insulin-responsive genes. We have shown previously that the hepatic expression of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) is activated by insulin. We show here in primary cultures of hepatocytes that the adenovirus-mediated transduction of a dominant negative form of SREBP-1c inhibits the insulin effect on endogenous glucokinase expression. Conversely, in the absence of insulin, the adenovirus-mediated transduction of a dominant positive form of SREBP-1c overcomes the insulin dependency of glucokinase expression. Hepatic fatty acid synthase and Spot-14 are insulin/glucose-dependent genes. For this latter class of genes, the dominant positive form of SREBP-1c obviates the necessity for the presence of insulin, whereas glucose potentiates the effect of SREBP-1c on their expression. In addition, the insulin dependency of lipid accumulation in cultured hepatocytes is overcome by the dominant positive form of SREBP-1c. We propose that SREBP-1c is a major mediator of insulin action on hepatic gene expression and a key regulator of hepatic glucose/lipid metabolism.
Mice mutant for glucokinase regulatory protein exhibit decreased liver glucokinase: A sequestration mechanism in metabolic regulation
Authors: Farrelly Dennis , Brown Karen S. , Tieman Aaron , Ren Jianming , Lira Sergio A. , Hagan Deborah , Gregg Richard , Mookhtiar Kasim A. , Hariharan Narayanan
Published: July 2002

The importance of glucokinase (GK; EC
2.7.1.12
) in glucose homeostasis has been demonstrated by the association of GK mutations with diabetes mellitus in humans and by alterations in glucose metabolism in transgenic and gene knockout mice. Liver GK activity in humans and rodents is allosterically inhibited by GK regulatory protein (GKRP). To further understand the role of GKRP in GK regulation, the mouse GKRP gene was inactivated. With the knockout of the GKRP gene, there was a parallel loss of GK protein and activity in mutant mouse liver. The loss was primarily because of posttranscriptional regulation of GK, indicating a positive regulatory role for GKRP in maintaining GK levels and activity. As in rat hepatocytes, both GK and GKRP were localized in the nuclei of mouse hepatocytes cultured in low-glucose-containing medium. In the presence of fructose or high concentrations of glucose, conditions known to relieve GK inhibition by GKRP
in vitro
, only GK was translocated into the cytoplasm. In the GKRP-mutant hepatocytes, GK was not found in the nucleus under any tested conditions. We propose that GKRP functions as an anchor to sequester and inhibit GK in the hepatocyte nucleus, where it is protected from degradation. This ensures that glucose phosphorylation is minimal when the liver is in the fasting, glucose-producing phase. This also enables the hepatocytes to rapidly mobilize GK into the cytoplasm to phosphorylate and store or metabolize glucose after the ingestion of dietary glucose. In GKRP-mutant mice, the disruption of this regulation and the subsequent decrease in GK activity leads to altered glucose metabolism and impaired glycemic control.
An ancestral MADS-box gene duplication occurred before the divergence of plants and animals
Authors: Alvarez-Buylla Elena R. , Pelaz Soraya , Liljegren Sarah J. , Gold Scott E. , Burgeff Caroline , Ditta Gary S. , Ribas de Pouplana Lluis , Martínez-Castilla León , Yanofsky Martin F.
Published: July 2002

Changes in genes encoding transcriptional regulators can alter development and are important components of the molecular mechanisms of morphological evolution. MADS-box genes encode transcriptional regulators of diverse and important biological functions. In plants, MADS-box genes regulate flower, fruit, leaf, and root development. Recent sequencing efforts in
Arabidopsis
have allowed a nearly complete sampling of the MADS-box gene family from a single plant, something that was lacking in previous phylogenetic studies. To test the long-suspected parallel between the evolution of the MADS-box gene family and the evolution of plant form, a polarized gene phylogeny is necessary. Here we suggest that a gene duplication ancestral to the divergence of plants and animals gave rise to two main lineages of MADS-box genes: TypeI and TypeII. We locate the root of the eukaryotic MADS-box gene family between these two lineages. A novel monophyletic group of plant MADS domains (AGL34 like) seems to be more closely related to previously identified animal SRF-like MADS domains to form TypeI lineage. Most other plant sequences form a clear monophyletic group with animal MEF2-like domains to form TypeII lineage. Only plant TypeII members have a K domain that is downstream of the MADS domain in most plant members previously identified. This suggests that the K domain evolved after the duplication that gave rise to the two lineages. Finally, a group of intermediate plant sequences could be the result of recombination events. These analyses may guide the search for MADS-box sequences in basal eukaryotes and the phylogenetic placement of new genes from other plant species.
Aβ1–40-related reduction in functional hyperemia in mouse neocortex during somatosensory activation
Authors: Niwa Kiyoshi , Younkin Linda , Ebeling Christine , Turner Sherry K. , Westaway David , Younkin Steven , Ashe Karen Hsiao , Carlson George A. , Iadecola Costantino
Published: July 2002

Peptides derived from proteolytic processing of the β-amyloid precursor protein (APP), including the amyloid-β peptide (Aβ), play a critical role in the pathogenesis of Alzheimer's dementia. We report that transgenic mice overexpressing APP and Aβ have a profound attenuation in the increase in neocortical blood flow elicited by somatosensory activation. The impairment is highly correlated with brain Aβ concentration and is reproduced in normal mice by topical neocortical application of exogenous Aβ1–40 but not Aβ1–42. Overexpression of M146L mutant presenilin-1 in APP mice enhances the production of Aβ1–42 severalfold, but it does not produce a commensurate attenuation of the hyperemic response. APP and Aβ overexpression do not diminish the intensity of neural activation, as reflected by the increase in somatosensory cortex glucose usage. Thus, Aβ-induced alterations in functional hyperemia produce a potentially deleterious mismatch between substrate delivery and energy demands imposed by neural activity.
Serological identification of embryonic neural proteins as highly immunogenic tumor antigens in small cell lung cancer
Authors: Güre Ali O. , Stockert Elisabeth , Scanlan Matthew J. , Keresztes Roger S. , Jäger Dirk , Altorki Nasser K. , Old Lloyd J. , Chen Yao-Tseng
Published: July 2002

Serological analysis of expression cDNA libraries (SEREX) derived from two small cell lung cancer (SCLC) cell lines using pooled sera of SCLC patients led to the isolation of 14 genes, including 4
SOX
group B genes (
SOX1
,
SOX2
,
SOX3
, and
SOX21
) and
ZIC2. SOX
group B genes and
ZIC2
encode DNA-binding proteins; SOX group B proteins regulate transcription of target genes in the presence of cofactors, whereas ZIC2 is also suspected to be a transcriptional regulator. These genes are expressed at early developmental stages in the embryonic nervous system, but are down-regulated in the adult. Although
SOX2
mRNA can be detected in some adult tissues,
ZIC2
is expressed only in brain and testis, and
SOX1
,
SOX3
, and
SOX21
transcripts are not detectable in normal adult tissues. Of SCLC cell lines tested, 80% expressed
ZIC2
mRNA, and SOX1, SOX2, and SOX3 expression was detected in 40%, 50%, and 10%, respectively. SOX group B and ZIC2 antigens elicited serological responses in 30–40% of SCLC patients in this series, at titers up to 1:10
6
. In sera from 23 normal adults, no antibody was detected against SOX group B or ZIC2 proteins except for one individual with low-titer anti-SOX2 antibody. Seroreactivity against SOX1 and 2 was consistently higher titered than SOX3 and 21 reactivity, suggesting SOX1 and/or SOX2 as the main antigens eliciting anti-SOX responses. Although paraneoplastic neurological syndromes have been associated with several SCLC antigens, neurological symptoms have not been observed in patients with anti-SOX or anti-ZIC2 antibodies.