bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of RNA-binding proteins in mammalian cellsby DNA-damaging agents.
Authors: Carrier F , Gatignol A , Hollander M C , Jeang K T , Fornace A J
Published: May 2006

A technique to detect RNA-binding proteins (RBP)
involving hybridization of RNA probe to proteins transferred to a membrane was
used to study RBP in different mammalian cells and in cells after genotoxic
stress. With this approach, up to 13 proteins of different sizes were detected
in crude nuclear extracts by using a viral RNA probe consisting of the
trans-activation-responsive (TAR) element of human immunodeficiency virus type 1
(HIV-1). The TAR RNA probe contains a stem-loop structure found in nascent HIV-1
transcripts. A G+C-rich probe with similar structure also bound to many of these
RBP. Only a 102-kDa protein strongly bound to other RNA probes lacking this
structure, while a probe with an A+U-rich stem-loop structure fail to bind most
RBP, thus indicating a RNA secondary structure preference. The expression of
these RBP varied substantially in nine different human and hamster cell lines,
with no detectable RBP in two human myeloid lines. Evidence for induction of
these RBP was found in six of seven lines after treatment with DNA-damaging
agents; UV radiation was the most effective agent. In Chinese hamster ovary
cells, which showed the strongest response, all five RBP present in untreated
cells rapidly increased in activity after UV irradiation, and eight additional
RBP were detected. The induction of these RBP by DNA-damaging agents indicates
one or more possible roles for these proteins in the cellular response to
genotoxic stress and in viral activation after such stress.
Cytosine deaminations catalyzed by DNA cytosinemethyltransferases are unlikely to be the major cause of mutational hot spots atsites of cytosine methylation in Escherichia coli.
Authors: Wyszynski M , Gabbara S , Bhagwat A S
Published: May 2006

Sites of cytosine methylation are hot spots for C
to T mutations in Escherichia coli DNA. We have developed a genetic reversion
assay that allows direct selection of C to T mutations at a site of methylation.
Because the mutant gene is on a plasmid, this system can be used to study
mutational effects of biochemical agents in vitro as well as in vivo. Using this
system we show that in vitro an E. coli methyltransferase can cause C to U
deaminations at a site of methylation. Reaction conditions that are known to
inhibit a side reaction of the methyltransferase also suppress reversion
frequency, suggesting that this side reaction is required for deamination.
Furthermore, a mutation in the enzyme that eliminates its catalytic activity but
not its ability to bind DNA eliminates the ability of the enzyme to cause C to U
deaminations. Despite this, in vivo experiments strongly suggest that
enzyme-catalyzed deaminations of cytosine do not play a major role in making
methylation sites in E. coli hot spots for mutations. For example, although
uracil-DNA glycosylase (Ung) suppresses the occurrence of mutations due to C to
U deaminations, the frequency of C to T mutations at a methylation site remains
high in ung+ cells. Furthermore, the reversion frequencies in ung+ and ung-
cells are quite similar.
Creation and functional screening of a multi-usepeptide library.
Authors: Jayawickreme C K , Graminski G F , Quillan J M , Lerner M R
Published: May 2006

Studies of functional interactions between
transmembrane proteins such as G-protein-coupled receptors and ligands would
benefit from the ability to utilize synthetic molecule libraries. This is
realized here by the construction and application of a multi-use combinatorial
peptide library (MUPL). Peptides are liberated from their supports in a dry
state so that the problem of signal interference due to mixing of peptide
molecules, particularly agonists and antagonists, is avoided. In addition, the
peptides are released from their supports in a controlled manner so that
fractions are available for multiple independent tests, thus eliminating the
need for iterative library analysis and resynthesis. The MUPL concept was
validated with a functional screen which detects agonists to G-protein-coupled
receptors and led to the discovery of new ligands. It is expected that combining
MUPLs with functional assays will enhance both basic scientific research and the
rates of drug discovery and development.
Effects of ocular injury and administration ofbrain-derived neurotrophic factor on survival and regrowth of axotomized retinalganglion cells.
Authors: Mansour-Robaey S , Clarke D B , Wang Y C , Bray G M , Aguayo A J
Published: May 2006

Optic nerve transection in adult rats results in
the death of approximately 50% of the axotomized retinal ganglion cells (RGCs)
by 1 week and nearly 90% by 2 weeks after injury. The capacity of brain-derived
neurotrophic factor (BDNF) to prevent this early, severe loss of RGCs was
investigated in vivo by intravitreal injections of BDNF [5 micrograms in 5
microliters of bovine serum albumin/phosphate-buffered saline (BSA/PBS)] or
vehicle (5 microliters of BSA/PBS). Using quantitative anatomical techniques, we
show that (i) all RGCs survived 1 week after a single injection of BDNF at the
time of axotomy. (ii) RGC densities decreased in the BDNF-treated retinas by 2
weeks but remained significantly greater than in the untreated controls. (iii)
An enhanced RGC survival was obtained with single injections of BDNF from 6 days
before to 5 days after axotomy. (iv) Repeated injections resulted in greater
numbers of surviving RGCs, an effect that declined to undetectable levels by 6
weeks. (v) There were indications for an endogenous local source of trophic
support whose expression was triggered by ocular injury, particularly to the
anterior part of the eye. (vi) With multiple BDNF injections, there was profuse
axonal sprouting around the optic disc. This remarkable intraretinal growth was
not, however, reflected in increased RGC innervation of the peripheral nerve
grafts, which are known to facilitate regeneration when used as optic nerve
substitutes.
A visible marker for antisense mRNA expression inplants: inhibition of chlorophyll synthesis with a glutamate-1-semialdehydeaminotransferase antisense gene.
Authors: Höfgen R , Axelsen K B , Kannangara C G , Schüttke I , Pohlenz H D , Willmitzer L , Grimm B , von Wettstein D
Published: May 2006

Glutamate 1-semialdehyde aminotransferase
[(S)-4-amino-5-oxopentanoate 4,5-aminomutase, EC 5.4.3.8] catalyzes the last
step in the conversion of glutamate to delta-aminolevulinate of which eight
molecules are needed to synthesize a chlorophyll molecule. Two full-length cDNA
clones that probably represent the homeologous Gsa genes of the two tobacco
(Nicotiana tabacum) genomes have been isolated. The deduced amino acid sequences
of the 468-residue-long precursor polypeptides differ by 10 amino acids. The
cDNA sequence of isoenzyme 2 was inserted in reverse orientation under the
control of a cauliflower mosaic virus 35S promoter derivative in an expression
vector and was introduced by Agrobacterium-mediated transformation into tobacco
plants. Antisense gene expression decreased the steady-state mRNA level of
glutamate 1-semialdehyde aminotransferase, the translation of the enzyme, and
chlorophyll synthesis. Remarkably, partial or complete suppression of the
aminotransferase mimics in tobacco a wide variety of chlorophyll variegation
patterns caused by nuclear or organelle gene mutations in different higher
plants. The antisense gene is inherited as a dominant marker.
Expression and localization of amiloride-sensitivesodium channel indicate a role for non-taste cells in tasteperception.
Authors: Li X J , Blackshaw S , Snyder S H
Published: May 2006

Salty taste is blocked by the diuretic amiloride,
which inhibits specific sodium channels. We have isolated an amiloride-sensitive
sodium channel (ASSC) from taste tissues by polymerase chain reaction and
screening of a cDNA library prepared from rat circumvallate papillae. Northern
analysis reveals ASSC in taste and non-taste tissues with the highest level of
expression of ASSC in the lung. In situ hybridization establishes ASSC
localizations in the epithelia of lung and colon as well as tongue epithelial
layers containing and lacking taste buds. These results support a model in which
ASSC in non-taste cells regulates responses of taste cells to salt as well as
other tastants.
Midline signaling in the primordium of the zebrafish anterior central nervous system.
Authors: Hatta K , Püschel A W , Kimmel C B
Published: May 2006

In all vertebrates the brain develops from the enlarged anterior part of the neural plate. However, in the zebrafish mutant cyclops, the girth of the central nervous system (CNS) is nearly uniform along its length. Changes in expression patterns of homeobox genes and neuronal markers reveal a massive deletion of the ventral forebrain, particularly the diencephalon, as well as its precursor region in the neural plate. The deletion is due to a nonautonomous action of the mutation: very few wild-type cells transplanted to the midline of a mutant embryo can rescue the forebrain phenotype, including cyclopia. Establishment of forebrain ventral positional coordinates may thus require inductive signaling by forebrain midline cells whose specification depends upon the cyclops gene product.
A gene encoding a DnaK/hsp70 homolog in Escherichia coli.
Authors: Seaton B L , Vickery L E
Published: May 2006

Eukaryotic organisms have been shown to have multiple forms of hsp70-class stress-related proteins, but only a single family member, DnaK, has been found in prokaryotes. We report here the identification of a heat shock cognate gene, designated hsc, in Escherichia coli. The amino acid sequence deduced from hsc predicts a 65,647-Da polypeptide having 41% sequence identity with DnaK from E. coli, and overexpression produces a protein (Hsc66) with properties similar to DnaK. In contrast to dnaK, however, the hsc gene lacks a consensus heat shock promoter sequence, and expression is not induced by elevated temperature. The hsc gene is located near 54 min on the physical map, immediately upstream of the fdx gene, which encodes a [2Fe-2S] ferredoxin; evidence is presented that the hsc and fdx genes make up a bicistronic operon in which expression of the ferredoxin is coupled to that of Hsc66. The function of Hsc66 is not known, but the coregulation of its expression with that of ferredoxin suggests the possibility of a specific role in association with the ferredoxin protein.
Use of Drosophila mutants to distinguish among volatile general anesthetics.
Authors: Campbell D B , Nash H A
Published: May 2006

The way general anesthetics cause their clinically useful effects on the nervous system is not known. Since the principal determinant of the potency of these agents is their solubility in oil, the role of chemical structure in affecting anesthetic targets has been obscured. In this work, we use an intense beam of light (Tinklenberg, J. A., Segal, I. S., Tianzhi, G. & Maze, M. (1991) Ann. N.Y. Acad. Sci. 625, 532-539) to assess the effect of general anesthetics on the capacity of fruit flies to sense a noxious stimulus and respond to it. By examining the effect of halothane-resistant (har) mutations on the potency of various volatile anesthetics in this assay, we establish similarities and differences between these agents that highlight the was chemical structure influences anesthetic action. In general, the potencies of anesthetics with different chemical structure are affected to different extents by one or more har mutations. However, three anesthetics of related structure (enflurane, isoflurane, and desflurane) show quantitatively indistinguishable alterations in potency in each of four genetic tests. These results not only identify important structural features of anesthetics but also place limits on the classical view that all anesthetics act at a common target.
Nuclear concentration and mitotic dispersion of the essential cell cycle protein, p13suc1, examined in living cells.
Authors: Hepler P K , Sek F J , John P C
Published: May 2006

Stamen hair cells of Tradescantia virginiana have been microinjected with p13suc1 labeled with carboxyfluorescein (CF) and studied throughout the division cycle in living cells by using the confocal laser scanning microscope. The protein, p13suc1, is essential for the rapid inactivation of the key mitotic catalyst, p34cdc2 kinase, at anaphase and for completion of nuclear division. During interphase or prophase, CF-p13suc1 concentrates quickly (< 2 min) in nuclei, reaching levels that are approximately 2-fold greater than those in the cytoplasm. At nuclear envelope breakdown, CF-p13suc1 permeates throughout the entire spindle and nonspindle cytoplasm. The protein is excluded from the tightly condensed chromosomes but otherwise no regions accumulate or exclude the protein. It remains evenly distributed throughout metaphase, anaphase, and well into cytokinesis; however, during telophase CF-p13suc1 reconcentrates in the daughter nuclei.
Role of an Escherichia coli stress-response operon in stationary-phase survival.
Authors: Weiner L , Model P
Published: May 2006

The phage shock protein operon (pspABCE) of Escherichia coli is strongly expressed in response to stressful environmental conditions, such as heat shock, ethanol treatment, osmotic shock, and filamentous phage infection. We show that bacteria lacking the pspABC genes exhibit a substantial decrease in the ability to survive prolonged incubation in stationary phase under alkaline conditions (pH 9). The psp mutant bacteria grow approximately as well as wild-type strains in the alkaline medium, and stationary-phase survival of the psp mutants improves substantially at pH values closer to the optimal growth range (pH 6-8). In late stationary-phase (1- to 2-day-old) cells, the operon can be strongly induced under certain conditions, and PspA can become one of the most highly expressed bacterial proteins. The combination of stationary-phase starvation and alkaline pH is likely to place a severe strain on the maintenance of endogenous energy sources, and, consistent with these effects, we find that psp expression is also induced by uncouplers of oxidative phosphorylation and other agents that interfere with energy production. The death rate of psp mutants in stationary phase is accelerated by the presence of wild-type bacteria in the same culture, suggesting that the psp operon may play a significant role in enabling E. coli to compete for survival under nutrient- or energy-limited conditions.
Changes in human ecology and behavior in relation to the emergence of diarrheal diseases, including cholera.
Authors: Levine M M , Levine O S
Published: May 2006

Human populations throughout the world can be found in diverse conditions. A proportion of the population of developing countries lives in deprived conditions characterized by ramshackle housing, lack of piped water and sanitation, and widespread fecal contamination of the environment. Enteric infections, particularly due to bacterial pathogenes, are readily transmitted under these circumstances. In contrast, the majority of inhabitants of industrialized countries live in a sanitary environment that generally discourages the transmission of enteric pathogenes, particularly bacteria. In both these ecologic niches, changes in human ecology and behavior are leading to the emergence of certain enteric infections. Relevant factors in developing areas include urbanization (leading to periurban slums), diminished breastfeeding, and political upheaval that results in population migrations. In industrialized areas, large-scale food production (e.g., enormous poultry farms), distribution, and retailing (e.g., fast-food chains) create opportunities where widespread and extensive outbreaks of food-borne enteric infection can ensue if a breakdown in food hygiene occurs.
Herpes simplex virus infections of women and their offspring: implications for a developed society.
Authors: Whitley R J
Published: May 2006

Herpes simplex virus infections of humans have been known since ancient times. Contemporary society has witnessed a series of devastating manifestations of herpes simplex virus infections--namely, genital herpes simplex virus infection and neonatal herpes simplex virus infection. With the evolution of society, particularly advances in birth control and increasing promiscuity, the seroprevalence of herpes simplex virus type 2 infections has increased worldwide, however, more so in developed societies. As a consequence, individuals of child-bearing age are at risk for either reactivation of herpes simplex virus at termination of gestation or acquisition of a new primary infection at that time. The consequences of vertical transmission of herpes simplex virus from mother to child, resulting in neonatal herpes simplex virus infection, can be devastating. Current efforts, which are directed toward the treatment of neonatal herpes, have established the value of drugs such as vidarabine and acyclovir. However, the real emphasis for future programs is the prevention of herpes simplex virus infections to avoid person-to-person transmission either horizontally or vertically. The development of vaccines directed against herpes simplex virus may be of value toward this end.
Overexpression of the beta 1 thyroid receptor induces differentiation in neuro-2a cells.
Authors: Lebel J M , Dussault J H , Puymirat J
Published: May 2006

To determine the functions of the alpha 1 and beta 1 thyroid hormone receptors (TRs) in neural differentiation, we have established stable transfected neuronal cell lines (Neuro-2a) that overexpress either TR alpha 1 or TR beta 1. 3,5,3'-Triiodothyronine (T3) treatment of cells that overexpress TR beta 1 blocks proliferation by an arrest of cells in G0/G1 and induces morphological and functional differentiation of Neuro-2a cells as indicated by the marked increase in the number of perisomatal filopodia-like neurites and in acetylcholinesterase (AChE) activity. The effect on AChE activity was dose-dependent, and the time-course analysis reveals that this effect occurs after 24 hr of T3 treatment, with a maximal increase occurring after 48 hr of treatment. The increase of AChE activity is paralleled by an increase of AChE mRNAs. Last, we present evidence that shows that the effects of T3 on differentiation are independent of its effect on proliferation. T3 had no effect on the differentiation of Neuro-2a cells that overexpressed TR alpha 1. Our results indicate that TR beta 1 may play a key role in the effects of T3 in neuroblastoma cell differentiation.
Rice tungro bacilliform virus encodes reverse transcriptase, DNA polymerase, and ribonuclease H activities.
Authors: Laco G S , Beachy R N
Published: May 2006

Rice tungro bacilliform virus (RTBV) is a newly described badnavirus and proposed member of the plant pararetrovirus group. RTBV open reading frame 3 is predicted to encode a capsid protein, protease (PR), and reverse transcriptase (RT) and has the capacity to encode other proteins of as yet unknown function. To study the possible enzymatic activities encoded by open reading frame 3, a DNA fragment containing the putative PR and RT domains was used to construct the recombinant baculovirus PR/RT-BBac. Trichoplusia ni insect cells infected with PR/RT-BBac were used in pulse-labeling experiments and demonstrated synthesis of an 87-kDa polyprotein that corresponds in molecular mass to that predicted from the PR/RT DNA coding sequence. The 87-kDa polyprotein was processed with concomitant accumulation of 62-kDa (p62) and 55-kDa (p55) proteins. Amino-terminal sequencing of p62 and p55 determined that they mapped to the PR/RT domain and shared common amino termini. p62 and p55 were purified and exhibited both RT and DNA polymerase activities using synthetic primer/template substrates. Only p55 had detectable ribonuclease H activity, an activity intrinsic to all reverse transcriptases studied to date. Characterization of the RTBV RT provides a biochemical basis for classifying RTBV as a pararetrovirus and will lead to further studies of these proteins and their role in virus replication.
Enterochromaffin cells of the digestive system: cellular source of guanylin, a guanylate cyclase-activating peptide.
Authors: Cetin Y , Kuhn M , Kulaksiz H , Adermann K , Bargsten G , Grube D , Forssmann W G
Published: May 2006

Guanylin, a bioactive peptide, has recently been isolated from the intestine; this peptide activates intestinal guanylate cyclase (i.e., guanylate cyclase C) and thus is potentially involved in the regulation of water/electrolyte transport in the gastrointestinal mucosa. As yet, the cells involved in synthesis, storage, or secretion of guanylin have not been identified by immunocytochemistry. We raised antisera against guanylin and investigated the entire gastrointestinal tract of guinea pigs by light and electron microscopical immunocytochemistry. Extracts of various intestinal segments and plasma analyzed on a Western blot revealed a peptide band corresponding to the molecular mass of guanylin. Localization studies in the entire digestive tract showed that guanylin is exclusively confined to enterochromaffin (EC) cells. Remarkably, most EC cells contacted the gut lumen by cell processes that were highly immunoreactive for guanylin. In addition to the well known secretion in an endocrine fashion, EC cells by circumstantial evidence may release guanylin into the gut lumen to activate guanylate cyclase C that is immediately located on the brush border of adjacent enterocytes. The unique localization of guanylin in EC cells may indicate that these cells are involved in the regulation of fluid secretion in the gastrointestinal mucous membrane.
Requirement for a conserved, tertiary interaction in the core of 23S ribosomal RNA.
Authors: Aagaard C , Douthwaite S
Published: May 2006

A putative base-pairing interaction that determines the folding of the central region of 23S rRNA has been investigated by mutagenesis. Each of the possible base substitutions has been made at the phylogenetically covariant positions adenine-1262 (A1262) and U2017 in Escherichia coli 23S rRNA. Every substitution that disrupts the potential for Watson-Crick base pairing between these positions reduces or abolishes the participation of 23S rRNA in protein synthesis. All mutant 23S rRNAs are assembled into 50S subunits, but the mutant subunits are less able to stably interact with 30S subunits to form translationally active ribosomes. The function of 23S rRNA is largely reestablished by introduction of an alternative G1262.C2017 or U1262.A2017 pair, although neither of these supports polysome formation quite as effectively as the wild-type pair. A 23S rRNA with a C1262.G2017 pair is nonfunctional. In contrast to the considerable effect the mutations have on function, they impart only slight structural changes on the naked rRNA, and these are limited to the immediate vicinity of the mutations. The data show that positions 1262 and 2017 pair in a Watson-Crick manner, but the data also indicate that these nucleotides engage in additional interactions within the ribosome and that these interactions determine what base pairs are acceptable there.
Synaptic disinhibition during maintenance of long-term potentiation in the CA1 hippocampal subfield.
Authors: Stelzer A , Simon G , Kovacs G , Rai R
Published: May 2006

Long-term potentiation (LTP) in the CA1 region of the hippocampus is widely believed to occur through a strengthening of efficacy of excitatory synapses between afferent fibers and pyramidal cells. An alternative mechanism of LTP, reduction of efficacy of synaptic inhibition, was examined in the present report. The present study demonstrates that the maintenance of LTP in the CA1 hippocampal subfield of guinea pigs is accompanied by impairment of type A gamma-aminobutyric acid (GABA) receptor function, particularly at apical dendritic sites of CA1 pyramidal cells. Enhanced excitability of GABAergic interneurons during LTP represents a strengthening of inhibitory efficacy. The net effect of opposite modifications of synaptic inhibition during LTP of CA1 pyramidal cells is an overall impairment of the strength of GABAergic inhibition, and disinhibition could contribute importantly to CA1 pyramidal cell LTP.
Enhancement of antigen-induced T-cell proliferation by soluble CD26/dipeptidyl peptidase IV.
Authors: Tanaka T , Duke-Cohan J S , Kameoka J , Yaron A , Lee I , Schlossman S F , Morimoto C
Published: May 2006

The addition of a soluble recombinant CD26 (sCD26) enhanced proliferation of peripheral blood lymphocytes induced by the recall antigen tetanus toxoid. sCD26 itself did not provide a mitogenic signal and did not augment the proliferative response of T cells to other mitogenic stimuli such as phytohemagglutinin and anti-CD3. Dipeptidyl peptidase IV-negative sCD26 did not have this enhancement effect, implying a requirement for enzyme activity. It was found that there exists a large variation in the levels of human plasma sCD26/dipeptidyl peptidase IV in vivo which may regulate T-cell activity. Peripheral blood lymphocytes from individuals whose plasma sCD26 was high and responded strongly to tetanus toxoid stimulation were insensitive to the enhancing effects of exogenously added sCD26. This suggests that plasma sCD26 had modulated the responsiveness of T cells of these individuals in vivo and that the endogenous plasma sCD26 regulates immune responses by allowing antigen-specific T cells to exert a maximal response to their specific antigen.
Biosynthesis of lipooligosaccharide nodulation factors: Rhizobium NodA protein is involved in N-acylation of the chitooligosaccharide backbone.
Authors: Röhrig H , Schmidt J , Wieneke U , Kondorosi E , Barlier I , Schell J , John M
Published: May 2006

Rhizobium meliloti interacts symbiotically with alfalfa by forming root nodules in which the bacteria fix nitrogen. The Rhizobium nodulation genes nodABC are involved in the synthesis of lipooligosaccharide symbiotic signal molecules, which are mono-N-acylated chitooligosaccharides. These bacterial signals elicit nodule organogenesis in roots of legumes. To elucidate the role of the NodA protein in lipooligosaccharide biosynthesis, we prepared a radiolabeled tetrasaccharide precursor carrying an amino group as a potential attachment site for N-acylation at the nonreducing glucosamine residue. Various criteria demonstrate that NodA is involved in the attachment of a fatty acyl chain to this tetrasaccharide precursor, yielding a biologically active nodulation factor.
Domain swapping: entangling alliances between proteins.
Authors: Bennett M J , Choe S , Eisenberg D
Published: May 2006

The comparison of monomeric and dimeric diphtheria toxin (DT) reveals a mode for protein association which we call domain swapping. The structure of dimeric DT has been extensively refined against data to 2.0-A resolution and a three-residue loop has been corrected as compared with our published 2.5-A-resolution structure. The monomeric DT structure has also been determined, at 2.3-A resolution. Monomeric DT is a Y-shaped molecule with three domains: catalytic (C), transmembrane (T), and receptor binding (R). Upon freezing in phosphate buffer, DT forms a long-lived, metastable dimer. The protein chain tracing discloses that upon dimerization an unprecedented conformational rearrangement occurs: the entire R domain from each molecule of the dimer is exchanged for the R domain from the other. This involves breaking the noncovalent interactions between the R domain and the C and T domains, rotating the R domain by 180 degrees with atomic movements up to 65 A, and re-forming the same noncovalent interactions between the R domain and the C and T domains of the other chain of the dimer. This conformational transition explains the long life and metastability of the DT dimer. Several other intertwined, dimeric protein structures satisfy our definition of domain swapping and suggest that domain swapping may be the molecular mechanism for evolution of these oligomers and possibly of oligomeric proteins in general.
Allelic polymorphism of emm loci provides evidence for horizontal gene spread in group A streptococci.
Authors: Bessen D E , Hollingshead S K
Published: May 2006

Group A streptococci have a virulence regulon containing a single emm locus or two or three distinct and adjacent loci of structurally related emm family genes. The products of the emm gene cluster consist of fibrillar surface proteins, at least some of which are known to contain determinants of type specificity located in their NH2-terminal regions, lying distal to the cell surface. The emm genes can be categorized into four major subfamilies (SFs), based on structural differences within their 3' regions encoding the peptidoglycan-spanning domain. In this study, we investigate the polymorphism within the 5' region of SF-4 and SF-3 emm genes (which occupy the first and last emm positions of the gene cluster, respectively) in 22 strains representing different serotypes. Our findings indicate that unlike the centrally positioned SF-1 or SF-2 genes, SF-3 and SF-4 genes each display only limited polymorphism in their 5' regions, suggesting that their gene products may not be major contributors to type specificity. Two forms of the SF-3 gene (SF3a, SF3b) and two forms of the SF-4 gene (SF4a, SF4b) are found to exist in all four possible combinations (SF3aSF4a, SF3aSF4b, SF3bSF4a, SF3bSF4b), strongly suggesting that horizontal gene spread has contributed to the evolution of emm genes and to the generation of emm gene diversity in group A streptococci.
A tumor suppressor locus within 3p14-p12 mediates rapid cell death of renal cell carcinoma in vivo.
Authors: Sanchez Y , el-Naggar A , Pathak S , Killary A M
Published: May 2006

High frequency loss of alleles and cytogenetic aberrations on the short arm of chromosome 3 have been documented in renal cell carcinoma (RCC). Potentially, three distinct regions on 3p could encode tumor suppressor genes involved in the genesis of this cancer. We report that the introduction of a centric fragment of 3p, encompassing 3p14-q11, into a highly malignant RCC cell line resulted in a dramatic suppression of tumor growth in athymic nude mice. Another defined deletion hybrid contained the region 3p12-q24 of the introduced human chromosome and failed to suppress tumorigenicity. These data functionally define a tumor suppressor locus, nonpapillary renal carcinoma-1 (NRC-1), within 3p14-p12, the most proximal region of high frequency allele loss in sporadic RCC as well as the region containing the translocation breakpoint in familial RCC. Furthermore, we provide functional evidence that NRC-1 controls the growth of RCC cells by inducing rapid cell death in vivo.
Borrelia burgdorferi swims with a planar waveform similar to that of eukaryotic flagella.
Authors: Goldstein S F , Charon N W , Kreiling J A
Published: May 2006

Borrelia burgdorferi is a motile spirochete with multiple internal periplasmic flagella (PFs) attached near each end of the cell cylinder; these PFs overlap in the cell center. We analyzed the shape and motion of wild type and PF-deficient mutants using both photomicrography and video microscopy. We found that swimming cells resembled the dynamic movements of eukaryotic flagella. In contrast to helically shaped spirochetes, which propagate spiral waves, translating B. burgdorferi swam with a planar waveform with occasional axial twists; waves had a peak-to-peak amplitude of 0.85 micron and a wavelength of 3.19 microns. Planar waves began full-sized at the anterior end and propagated toward the back end of the cell. Concomitantly, these waves gyrated counter-clockwise as viewed from the posterior end along the cell axis. In nontranslating cells, wave propagation ceased. Either the waveform of nontranslating cells resembled the translating form, or the cells became markedly contorted. Cells of the PF-deficient mutant isolated by Sadziene et al. [Sadziene, A., Thomas, D. D., Bundoc, V. G., Holt, S. C. & Barbour, A. G. (1991) J. Clin. Invest. 88, 82-92] were found to be relatively straight. The results suggest that the shape of B. burgdorferi is dictated by interactions between the cell body and the PFs. In addition, the PFs from opposite ends of the cell are believed to interact with one another so that during the markedly distorted nontranslational form, the PFs from opposite ends rotate in opposing directions around one another, causing the cell to bend.
Voltage-dependent Ca2+ influx into right-side-out plasma membrane vesicles isolated from wheat roots: characterization of a putative Ca2+ channel.
Authors: Huang J W , Grunes D L , Kochian L V
Published: May 2006

We report on the identification of a voltage-dependent Ca2+ transport system that mediates Ca2+ influx across the plasma membrane (PM) of wheat (Triticum aestivum) root cells. The experimental approach involved the imposition of transmembrane electrical potentials (via K+ diffusion potentials) in populations of purified, right-side-out PM vesicles isolated from wheat roots. Using 45Ca2+ to quantify Ca2+ influx into the PM vesicles, the voltage-dependent characteristics of Ca2+ transport were found to be similar to those exhibited by L-type voltage-gated Ca2+ channels in animal cells. The putative PM Ca2+ channel opened upon depolarization of the membrane potential, and Ca2+ flux increased to a maximum upon further depolarization and then decreased back to zero upon further successive depolarizations. This channel was found to be selective for Ca2+ over Mg2+, Sr2+, K+, and Na+; was blocked by very low concentrations of La3+; was unaffected by high concentrations of the K+ channel blocker tetraethylammonium; and exhibited Michaelis-Menten-type transport kinetics. Based on these transport properties, we argue that this transport system is a PM Ca2+ channel. We suggest that the use of radiotracer flux analysis of voltage-clamped PM vesicles derived from plant roots is a straightforward approach for the characterization of certain voltage-gated ion channels functioning in cellular membranes of higher plant cells.
Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor.
Authors: Kawakami Y , Eliyahu S , Delgado C H , Robbins P F , Rivoltini L , Topalian S L , Miki T , Rosenberg S A
Published: May 2006

By cDNA expression cloning we have isolated a gene encoding a shared human melanoma antigen recognized by HLA-A2 restricted autologous and allogenic tumor-infiltrating lymphocytes (TILs) from patients with metastatic melanoma. By using both transient and stable expression systems, transfection of this gene into non-antigen-expressing HLA-A2+ cell lines resulted in recognition by the antigen-specific TILs. The sequence of this cDNA revealed a previously undescribed putative transmembrane protein whose expression was restricted to melanoma and melanocyte cell lines and human retina but no other fresh or cultured normal tissues tested or other tumor histologies. Thus, we have identified a gene encoding a melanocyte lineage-specific protein (MART-1; melanoma antigen recognized by T cells 1) that is a widely shared melanoma antigen recognized by the T lymphocytes of patients with established malignancy. Identification of this gene opens possibilities for the development of immunotherapies for patients with melanoma.
"...the tyranny of the lattice...".
Authors: Dickerson R E , Goodsell D S , Neidle S
Published: May 2006

A systematic comparison of crystal structures of nine different B-DNA dodecamers, in three different space groups, with and without A-tracts, shows that crystal packing or lattice forces are of secondary importance for helix axis bending, minor-groove width, and propeller twist. While other local helix parameters may be influenced or even established by crystal packing, the properties just enumerated are determined primarily by base sequence. One and the same crystal packing scheme can accommodate a bend in one of two different directions, or no bend at all. A-tract regions of B-DNA are inherently straight and unbent, with base-pair inclination no different from that of general-sequence B-DNA. Where bends are observed at junctions between G.C and A.T regions, they always involve a roll about base-pair long axes in a direction that compresses the wide major groove and, hence, are 90 degrees away from that necessary for the correctness of the junction model of A-tract bending. The G.C/A.T junction appears to be a flexible hinge, capable of adopting either a straight or a bent conformation under the local influence of weak crystal packing forces. Such forces therefore are a source of information about DNA deformability and not a curse to be deplored. But as an indication of the weakness of crystal packing forces, introduction of a single bromine atom in the major groove is sufficient to eliminate a bend, although brominated and unbrominated crystals are isomorphous.
Pancreatic beta-cell replication and amelioration of surgical diabetes by Reg protein.
Authors: Watanabe T , Yonemura Y , Yonekura H , Suzuki Y , Miyashita H , Sugiyama K , Moriizumi S , Unno M , Tanaka O , Kondo H
Published: May 2006

We previously isolated from a rat regenerating islet cDNA library a gene named Reg, which is expressed in regenerating islets but is not expressed in normal islets. Here we examined the effect of rat Reg protein on pancreatic beta-cell replication using both 90% depancreatized rats and isolated islets. The depancreatized rats that received i.p. administration of recombinant rat Reg protein (1 mg/kg per day) for 2 months showed amelioration of the surgical diabetes, as evidenced by a significant decrease in blood glucose with an increased beta-cell mass in the residual pancreas. In isolated rat islets, Reg protein (18-180 nM: 0.3-3 micrograms/ml) significantly increased [3H]thymidine incorporation into the nuclei of beta cells. These results indicate that Reg protein is a growth factor for pancreatic beta cells and also suggest that the administration of Reg protein could be used as another therapeutic approach for diabetes mellitus.
Id-1 as a possible transcriptional mediator of muscle disuse atrophy.
Authors: Gundersen K , Merlie J P
Published: May 2006

Disuse of muscle leads to atrophy of the fibers. This atrophy is correlated with reduced transcription. We found that when muscle was denervated or paralyzed with a nerve impulse block, the mRNA for Id-1, a negative regulator of transcription, was increased 2- to 7-fold. To test the effect of high Id-1 levels in active muscles, we made transgenic mice in which Id-1 was overexpressed under control of regulatory elements which confer tissue- and fiber-type-specific expression in differentiated muscle cells. Fiber types with high transgene expression were atrophic compared to those in wild-type litter mates. In contrast, fiber types with low transgene expression displayed hypertrophy, presumably caused by an overload due to reduced strength in atrophic synergistic fibers. Apart from the selective effects on fiber caliber, the muscle tissue showed no signs of pathology, and apart from a characteristic slightly lower body weight, the transgenic animals looked and behaved normally. We suggest that in the mature muscle, Id-1 may be involved in regulating muscle fiber size at the transcriptional level during disuse.
A permutational approach toward protein-DNA recognition.
Authors: Huang L , Sera T , Schultz P G
Published: May 2006

The cI repressor of bacteriophage 434, known as 434 repressor, binds to 14-bp operator sequences by means of a helix-turn-helix motif. To probe the requirements for selective DNA recognition by this class of DNA binding proteins, as well as to generate new proteins with altered specificities, a library of approximately 3 x 10(6) mutants was generated that contains all permutations of five residues in the recognition helix (helix 3) of the repressor. These mutants were then selected in vivo for their ability to bind both wild-type (WT) and mutant operator sequences. The results of the selection demonstrate that four of these residues--Gln28, Gln29, Ser30, and Gln33--play a critical role in recognition of the WT operator. A number of repressors with mutations at Thr27 showed altered DNA binding affinities and specificities. The approach described here may also prove useful in studies of DNA recognition by other classes of DNA binding proteins.
Stereoelectronic effects of deoxyribose O4' on DNA conformation.
Authors: Egli M , Gessner R V
Published: May 2006

While B-DNA, the most common DNA conformation, displays rather regular twist angles and base stacking between successive base pairs, left-handed Z-DNA is characterized by the alternation of two different dinucleotide conformations with either a large twist and a small slide or a small twist and a large slide between adjacent base pairs. This results in poor stacking within the latter dinucleotide repeat that is in apparent contradiction to the rigidity and conformational stability of Z-DNA at high ionic strength. However, at d(CpG) steps the cytidine deoxyribose is situated such that its O4' sits directly over the six-membered ring of the guanine. We show here that the particular positionings of the two O4' lone-pair electrons provide stability through an intracytidine O4'...H6--C6 hydrogen bond and an n-->pi* interaction with the guanidinium system of the stacked base. Our model is based on the assumption of a strong polarization of the guanine bases in Z-DNA that is consistent with the Z-DNA-specific guanine O6 and N7 coordination to metal and organic cations and the proximity of its N2 and C8 positions to neighboring phosphate groups, as well as several other Z-DNA-specific conformational features.
Involvement of small GTP-binding proteins in defense signal-transduction pathways of higher plants.
Authors: Sano H , Ohashi Y
Published: May 2006

Small GTP-binding proteins play a critical role in the regulation of a range of cellular processes--including growth, differentiation, and intracellular transportation. Previously, we isolated a gene, rgp1, encoding a small GTP-binding protein, by differential screening of a rice cDNA library with probe DNAs from rice tissues treated with or without 5-azacytidine, a powerful inhibitor of DNA methylation. To determine the physiological role of rgp1, the coding region was introduced into tobacco plants. Transformants, with rgp1 in either sense or antisense orientations, showed distinct phenotypic changes with reduced apical dominance, dwarfism, and abnormal flower development. These abnormal phenotypes appeared to be associated with the higher levels of endogenous cytokinins that were 6-fold those of wild-type plants. In addition, the transgenic plants produced salicylic acid and salicylic acid-beta-glucoside in an unusual response to wounding, thus conferring increased resistance to tobacco mosaic virus infection. In normal plants, the wound- and pathogen-induced signal-transduction pathways are considered to function independently. However, the wound induction of salicylic acid in the transgenic plants suggests that expression of rgp1 somehow interfered with the normal signaling pathways and resulted in cross-signaling between these distinct transduction systems. The results imply that the defense signal-transduction system consists of a complicated and finely tuned network of several regulatory factors, including cytokinins, salicylic acid, and small GTP-binding proteins.
Transposon tagging of tobacco mosaic virus resistance gene N: its possible role in the TMV-N-mediated signal transduction pathway.
Authors: Dinesh-Kumar S P , Whitham S , Choi D , Hehl R , Corr C , Baker B
Published: May 2006

Plants can recognize and resist invading pathogens by signaling the induction of rapid defense responses. Often these responses are mediated by single dominant resistance genes (R genes). The products of R genes have been postulated to recognize the pathogen and trigger rapid host defense responses. Here we describe isolation of the classical resistance gene N of tobacco that mediates resistance to the well-characterized pathogen tobacco mosaic virus (TMV). The N gene was isolated by transposon tagging using the maize Activator (Ac) transposon. We confirmed isolation of the N gene by complementation of the TMV-sensitive phenotype with a genomic DNA fragment. Sequence analysis of the N gene shows that it encodes a protein with an amino-terminal domain similar to that of the cytoplasmic domains of the Drosophila Toll protein and the interleukin 1 receptor in mammals, a putative nucleotide-binding site and 14 imperfect leucine-rich repeats. The presence of these functional domains in the predicted N gene product is consistent with the hypothesis that the N resistance gene functions in a signal transduction pathway. Similarities of N to Toll and the interleukin 1 receptor suggest a similar signaling mechanism leading to rapid gene induction and TMV resistance.
Molecular mechanisms of defense by rhizobacteria against root disease.
Authors: Cook R J , Thomashow L S , Weller D M , Fujimoto D , Mazzola M , Bangera G , Kim D S
Published: May 2006

Genetic resistance in plants to root diseases is rare, and agriculture depends instead on practices such as crop rotation and soil fumigation to control these diseases. "Induced suppression" is a natural phenomenon whereby a soil due to microbiological changes converts from conducive to suppressive to a soilborne pathogen during prolonged monoculture of the susceptible host. Our studies have focused on the wheat root disease "take-all," caused by the fungus Gaeumannomyces graminis var. tritici, and the role of bacteria in the wheat rhizosphere (rhizobacteria) in a well-documented induced suppression (take-all decline) that occurs in response to the disease and continued monoculture of wheat. The results summarized herein show that antibiotic production plays a significant role in both plant defense by and ecological competence of rhizobacteria. Production of phenazine and phloroglucinol antibiotics, as examples, account for most of the natural defense provided by fluorescent Pseudomonas strains isolated from among the diversity of rhizobacteria associated with take-all decline. There appear to be at least three levels of regulation of genes for antibiotic biosynthesis: environmental sensing, global regulation that ties antibiotic production to cellular metabolism, and regulatory loci linked to genes for pathway enzymes. Plant defense by rhizobacteria producing antibiotics on roots and as cohabitants with pathogens in infected tissues is analogous to defense by the plant's production of phytoalexins, even to the extent that an enzyme of the same chalcone/stilbene synthase family used to produce phytoalexins is used to produce 2,4-diacetylphloroglucinol. The defense strategy favored by selection pressure imposed on plants by soilborne pathogens may well be the ability of plants to support and respond to rhizosphere microorganisms antagonistic to these pathogens.
Three-dimensional scroll waves of cAMP could direct cell movement and gene expression in Dictyostelium slugs.
Authors: Bretschneider T , Siegert F , Weijer C J
Published: May 2006

Complex three-dimensional waves of excitation can explain the observed cell movement pattern in Dictyostelium slugs. Here we show that these three-dimensional waves can be produced by a realistic model for the cAMP relay system [Martiel, J. L. & Goldbeter, A. (1987) Biophys J. 52, 807-828]. The conversion of scroll waves in the prestalk zone of the slug into planar wave fronts in the prespore zone can result from a smaller fraction of relaying cells in the prespore zone. Further, we show that the cAMP concentrations to which cells in a slug are exposed over time display a simple pattern, despite the complex spatial geometry of the waves. This cAMP distribution agrees well with observed patterns of cAMP-regulated cell type-specific gene expression. The core of the spiral, which is a region of low cAMP concentration, might direct expression of stalk-specific genes during culmination.