bluuebunny/crossref_metadata_2025 huggingface dataset:
Exogenous Mtv-7 superantigen transgene expression in major histocompatibility complex class II I-E- mice reconstituted with embryonic stem cell-derived hematopoietic stem cells.
Authors: Chambers C A , Kang J , Pawling J , Huber B , Hozumi N , Nagy A
Published: May 2006

Direct genetic manipulation of hematopoietic cells is limited by the lack of an established hematopoietic stem cell line. It has been demonstrated that embryonic stem (ES) cell<-->tetraploid embryos are completely ES cell-derived and that fetal liver (FL) cells from these embryos support hematopoiesis in lethally irradiated recipients. In this report, we demonstrate that FL cells from ES cell<-->tetraploid embryos support normal lymphopoiesis and T-cell repertoire development. Moreover, the introduction of the Mtv-7 superantigen transgene coding for minor lymphocyte stimulatory antigen 1 into murine hematopoietic cells via reconstitution with ES cell<-->tetraploid FL cells demonstrates that this method can effectively confer stable genetic changes into the hematopoietic tissues without going through the germ line. Long-term and secondary reconstitution with ES cell<-->tetraploid FL cells expressing the Mtv-7 superantigen transgene clonally deleted minor lymphocyte stimulatory antigen 1-reactive T-cell receptor V beta 6+, -8.1+, and -9+ T cells, but not V beta 7+ T cells, in H-2b (I-E-) mice. This model system will be extremely important for analyzing structure-function relationships of molecules involved in proliferation, differentiation, and selection of hematopoietic cells in vivo and for examining hematopoiesis-specific effects of mutations that are lethal during embryogenesis.
Oxidative susceptibility of low density lipoprotein subfractions is related to their ubiquinol-10 and alpha-tocopherol content.
Authors: Tribble D L , van den Berg J J , Motchnik P A , Ames B N , Lewis D M , Chait A , Krauss R M
Published: May 2006

The conjugated polyene fatty acid parinaric acid (PnA) undergoes a stoichiometric loss in fluorescence upon oxidation and can be used to directly monitor peroxidative stress within lipid environments. We evaluated the course of potentially atherogenic oxidative changes in low density lipoproteins (LDL) by monitoring the oxidation of PnA following its incorporation into buoyant (p = 1.026-1.032 g/ml) and dense (p = 1.040-1.054 g/ml) LDL subfractions. Copper-induced oxidation of LDL-associated PnA exhibited an initial lag phase followed by an increased rate of loss until depletion. Increased PnA oxidation occurred immediately after the antioxidants ubiquinol-10 and alpha-tocopherol were consumed but before there were marked elevations in conjugated dienes. Despite differences in sensitivity to early oxidation events, PnA oxidation and conjugated diene lag times were correlated (r = 0.582; P = 0.03), and both indicated a greater susceptibility of dense than buoyant LDL in accordance with previous reports. The greater susceptibility of PnA in dense LDL was attributed to reduced levels of ubiquinol-10 and alpha-tocopherol, which were approximately 50% lower than in buoyant LDL (mol of antioxidant/mol of LDL) and together accounted for 80% of the variation in PnA oxidation lag times. These results suggest that PnA is a useful probe of LDL oxidative susceptibility and may be superior to conjugated dienes for monitoring the initial stages of LDL lipid peroxidation. Differences in oxidative susceptibility among LDL density subfractions are detected by the PnA assay and are due in large part to differences in their antioxidant content.
Supramolecular organization of the mammalian translation system.
Authors: Negrutskii B S , Stapulionis R , Deutscher M P
Published: May 2006

Although evidence suggests that the protein synthetic machinery is organized within cells, this point has been difficult to prove because any organization that might exist is lost upon preparation of the cell-free systems usually used to study translation in vitro. To examine this process under conditions more representative of the intact cell, we have developed an active protein-synthesizing system using Chinese hamster ovary (CHO) cells permeabilized with the plant glycoside saponin. This procedure renders cells permeable to trypan blue and exogenous tRNA, but there is little release of endogenous macromolecules. Protein synthesis in this system proceeds at the same rate as that in intact cells and is about 40-fold faster than that in a cell-free system prepared from the same cells. Active protein synthesis in this system requires the addition of only Mg2+, K+, and creatine phosphate, with a small further stimulation by ATP and an amino acid mixture; no exogenous macromolecules are necessary. The proteins synthesized in this system are indistinguishable from those made by the intact cell, and the channeling of aminoacyl-tRNA observed in vivo is maintained. Our data suggest that the permeabilized cell system retains the protein-synthesizing capabilities of the intact cell and presumably its internal structure as well. Studies with this system demonstrate that the protein-synthesizing apparatus is highly organized and that its macromolecular components are not freely diffusible in mammalian cells.
Multiple forms of poly(A) polymerases in human cells.
Authors: Thuresson A C , Aström J , Aström A , Grönvik K O , Virtanen A
Published: May 2006

We have cloned human poly(A) polymerase (PAP) mRNA as cDNA in Escherichia coli. The primary structure of the mRNA was determined and compared to the bovine PAP mRNA sequence. The two sequences were 97% identical at the nucleotide level, which translated into 99% similarity at the amino acid level. Polypeptides representing recombinant PAP were expressed in E. coli, purified, and used as antigens to generate monoclonal antibodies. Western blot analysis using these monoclonal antibodies as probes revealed three PAPs, having estimated molecular masses of 90, 100, and 106 kDa in HeLa cell extracts. Fractionation of HeLa cells showed that the 90-kDa polypeptide was nuclear while the 100- and 106-kDa species were present in both nuclear and cytoplasmic fractions. The 106-kDa PAP was most likely a phosphorylated derivative of the 100-kDa species. PAP activity was recovered in vitro by using purified recombinant human PAP. Subsequent mutational analysis revealed that both the N- and C-terminal regions of PAP were important for activity and suggested that cleavage and polyadenylylation specificity factor (CPSF) interacted with the C-terminal region of PAP. Interestingly, tentative phosphorylation sites have been identified in this region, suggesting that phosphorylation/dephosphorylation may regulate the interaction between the two polyadenylylation factors PAP and CPSF.
Concerted evolution at the population level: pupfish HindIII satellite DNA sequences.
Authors: Elder J F , Turner B J
Published: May 2006

The canonical monomers (approximately 170 bp) of an abundant (1.9 x 10(6) copies per diploid genome) satellite DNA sequence family in the genome of Cyprinodon variegatus, a "pupfish" that ranges along the Atlantic coast from Cape Cod to central Mexico, are divergent in base sequence in 10 of 12 samples collected from natural populations. The divergence involves substitutions, deletions, and insertions, is marked in scope (mean pairwise sequence similarity = 61.6%; range = 35-95.9%), is largely confined to the 3' half of the monomer, and is not correlated with the distance among collecting sites. Repetitive cloning and direct genomic sequencing experiments failed to detect intrapopulation and intraindividual variation, suggesting high levels of sequence homogeneity within populations. The satellite sequence has therefore undergone "concerted evolution," at the level of the local population. Concerted evolution has previously almost always been discussed in terms of the divergence of species or higher taxa; its intraspecific occurrence apparently has not been reported previously. The generality of the observation is difficult to evaluate, for although satellite DNAs from a large number of organisms have been studied in detail, there appear to be little or no other data on their sequence variation in natural populations. The relationship (if any) between concerted, population level, satellite DNA divergence and the extent of gene flow/genetic isolation among conspecific natural populations remains to be established.
Retroviruses in invertebrates: the gypsyretrotransposon is apparently an infectious retrovirus of Drosophilamelanogaster.
Authors: Kim A , Terzian C , Santamaria P , Pélisson A , Purd'homme N , Bucheton A
Published: May 2006

Retroviruses are commonly considered to be
restricted to vertebrates. However, the genome of many eukaryotes contains
mobile sequences known as retrotransposons with long terminal repeats (LTR
retrotransposons) or viral retrotransposons, showing similarities with
integrated proviruses of retroviruses, such as Ty elements in Saccharomyces
cerevisiae, copia-like elements in Drosophila, and endogenous proviruses in
vertebrates. The gypsy element of Drosophila melanogaster has LTRs and contains
three open reading frames, one of which encodes potential products similar to
gag-specific protease, reverse transcriptase, and endonuclease. It is more
similar to typical retroviruses than to LTR retrotransposons. We report here
experiments showing that gypsy can be transmitted by microinjecting egg plasma
from embryos of a strain containing actively transposing gypsy elements into
embryos of a strain originally devoid of transposing elements. Horizontal
transfer is also observed when individuals of the "empty" stock are
raised on medium containing ground pupae of the stock possessing transposing
elements. These results suggest that gypsy is an infectious retrovirus and
provide evidence that retroviruses also occur in invertebrates.
Structure, function, and phylogeny of [Arg8]vasotocinreceptors from teleost fish and toad.
Authors: Mahlmann S , Meyerhof W , Hausmann H , Heierhorst J , Schönrock C , Zwiers H , Lederis K , Richter D
Published: May 2006

[Arg8]Vasotocin (AVT) is considered to be the most
primitive known vertebrate neurohypophyseal peptide of the vasopressin/oxytocin
hormone family and may thus be ancestral to all the other vertebrate peptide
hormones. The molecular evolution of the corresponding receptor family has now
been studied by cloning an AVT receptor, consisting of 435 amino acid residues,
from the teleost fish, the white sucker Catostomus commersoni. Frog oocytes
injected with the AVT receptor-encoding cRNA respond to the application of AVT,
but not to its structural and functional counterpart isotocin, by an induction
of membrane chloride currents indicating the coupling of the AVT receptor to the
inositol phosphate/calcium pathway. The pharmacological properties of the
expressed AVT receptor show that it represents, or is closely related to, an
ancestral nonapeptide receptor: oxytocin, aspargtocin, mesotocin, and
vasopressin activated the receptor, but other members of the
vasopressin/oxytocin family tested showed little or no potency; antagonists of
the mammalian vasopressin V1 and oxytocin receptors blocked the AVT response.
Comparison of AVT receptor sequences spanning transmembrane domains two to five,
deduced by cloning cDNAs from the Pacific salmon Oncorhynchus kisutch, the
cave-dwelling fish Astyanax fasciatus, and the anuran Xenopus laevis, with those
of their mammalian counterparts emphasizes amino acid residues that are involved
in hormone binding. The presence of a 5.0-kb transcript in various teleost
tissues (pituitary, liver, gills, swim bladder, and lateral line) points to a
physiological role for the fish AVT receptor in metabolic, osmoregulatory, and
sensory processes.
Chloride ions catalyze the formation of cis adducts inthe binding of anti-benzo[a]pyrene diol epoxide to nucleicacids.
Authors: Wolfe A R , Yamamoto J , Meehan T
Published: May 2006

The alkylation of DNA by racemic
7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE)
exhibits a strong preference for formation of trans adducts between the N2
deoxyguanosine alkylation site and the (+)-enantiomer of anti-BPDE. In the
presence of 10 mM buffer with no added salt, 98% of the adducts formed with
native calf thymus DNA result from trans opening of the epoxide ring. The strong
selectivity for trans adduct formation obtained with duplex DNA at low salt
concentration is found to a lesser degree with poly(G) but is nearly absent with
dAMP. When DNA adducts are formed in 10 mM MgCl2 or 1 M NaCl, the proportion of
cis adducts increases to approximately 7 and approximately 26%, respectively. At
low salt, 10 mM MgCl2, and 1 M NaCl, deoxyguanosine adducts are approximately
1%, 6%, and 24% cis, whereas deoxyadenosine adducts are approximately 11%, 14%,
and 37% cis, respectively. NaCl also increases the proportion of cis adducts
formed with poly(G) and dAMP. It is proposed that the increase in cis-adduct
formation due to salt results from SN1 attack of chloride ion on the BPDE
carbocation, forming a trans chlorohydrin, followed by SN2 attack of
DNA.
Increased body temperature accelerates aggregation ofthe Leu-68-->Gln mutant cystatin C, the amyloid-forming protein in hereditarycystatin C amyloid angiopathy.
Authors: Abrahamson M , Grubb A
Published: May 2006

Hereditary cystatin C amyloid angiopathy is a
dominantly inherited disorder, characterized by dementia, paralysis, and death
from cerebral hemorrhage in early adult life. A variant of the cysteine
proteinase inhibitor, cystatin C, is deposited as amyloid in the tissues of the
patients and their spinal-fluid level of cystatin C is abnormally low. The
disease-associated Leu-68-->Gln mutant (L68Q) cystatin C has been produced in
an Escherichia coli expression system and isolated by use of denaturing buffers,
immunosorption, and gel filtration. Parallel physicochemical and functional
investigations of L68Q-cystatin C and wild-type cystatin C revealed that both
proteins effectively inhibit the cysteine proteinase cathepsin B (equilibrium
constants for dissociation, 0.4 and 0.5 nM, respectively) but differ
considerably in their tendency to dimerize and form aggregates. While wild-type
cystatin C is monomeric and functionally active even after prolonged storage at
elevated temperatures, L68Q-cystatin C starts to dimerize and lose biological
activity immediately after it is transferred to a nondenaturing buffer. The
dimerization of L68Q-cystatin C is highly temperature-dependent, with a rise in
incubation temperature from 37 to 40 degrees C resulting in a 150% increase in
dimerization rate. The aggregation at physiological concentrations is likewise
increased at 40 compared to 37 degrees C, by approximately 60%. These properties
of L68Q-cystatin C have bearing upon our understanding of the pathophysiological
process of hereditary cystatin C amyloid angiopathy. They might also be of
clinical relevance, since medical intervention to abort febrile periods of
carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin
C aggregates.
Structure of S-lectin, a developmentally regulatedvertebrate beta-galactoside-binding protein.
Authors: Liao D I , Kapadia G , Ahmed H , Vasta G R , Herzberg O
Published: May 2006

The crystal structure of a 14-kDa bovine spleen
S-lectin complexed with the disaccharide N-acetyllactosamine at 1.9-A resolution
reveals a surprising structural relationship to legume lectins, despite the lack
of sequence homology. Two monomers associate to form an extended beta-sandwich,
each with the same jelly roll topology typical of legume lectins but with
dramatically trimmed loops and with different dimer association. Each monomer
binds one N-acetyllactosamine molecule in a topologically and spatially
different site than that of legume lectins. The carbohydrate-binding site
provides an unprecedented paradigm for carbohydrate binding, with a unique
network of salt bridges. The specificity for beta-galactose arises from
intricate interactions that constrain the position of the O4
atom.
Dynamic actin structures stabilized byprofilin.
Authors: Finkel T , Theriot J A , Dise K R , Tomaselli G F , Goldschmidt-Clermont P J
Published: May 2006

We describe the production and analysis of clonal
cell lines in which we have overexpressed human profilin, a small ubiquitous
actin monomer binding protein, to assess the role of profilin on actin function
in vivo. The concentration of filamentous actin is increased in cells with
higher profilin levels, and actin filament half-life measured in these cells is
directly proportional to the steady-state profilin concentration. The
distribution of actin filaments is altered by profilin overexpression. While
parallel actin bundles crossing the cells are virtually absent in cells
overexpressing profilin, the submembranous actin network of these cells is
denser than in control cells. These results suggest that in vivo profilin
regulates the stability, and thereby distribution, of specific dynamic actin
structures.
Design, creation, and characterization of a stable,monomeric triosephosphate isomerase.
Authors: Borchert T V , Abagyan R , Jaenicke R , Wierenga R K
Published: May 2006

Protein engineering on trypanosomal
triosephosphate isomerase (TIM) converted this oligomeric enzyme into a stable,
monomeric protein that is enzymatically active. Wild-type TIM consists of two
identical subunits that form a very tight dimer involving interactions of 32
residues of each subunit. By replacing 15 residues of the major interface loop
by another 8-residue fragment, a variant was constructed that is a stable and
monomeric protein with TIM activity. The length, sequence, and conformation of
the designed fragment were suggested by extensive modeling.
The second intron of the K-ras gene containsregulatory elements associated with mouse lung tumorsusceptibility.
Authors: Chen B , Johanson L , Wiest J S , Anderson M W , You M
Published: May 2006

We have previously demonstrated the preferential
activation of the K-ras gene from the susceptible A/J parent in lung tumors from
F1 mouse hybrids. In the present study, the mechanism of this observation is
further investigated. Higher levels of expression of A/J K-ras allele were
detected in lung adenomas (30 of 30) from the C3A mouse. In addition, three
K-ras alleles, designated as susceptible (Ks), intermediate (Ki), or resistant
(Kr), were identified by sequence analysis of the second intron of the K-ras
gene from 32 strains of mice. These K-ras alleles are associated with
differences in mouse lung tumor susceptibility. All Kr alleles have a tandem
37-bp direct repeat (nt 282-355) in the second intron of the K-ras gene. Ks and
Ki alleles have only one copy of the 37-bp sequence (nt 282-318). Ks strains
have three base variations at nt 288, 296, and 494, and Ki strains have two base
variations at nt 288 and 494 in the second intron of the K-ras gene.
Differential protein-binding patterns were observed in gel-mobility-shift
experiments between the duplicated 37-bp sequence of the Kr allele and the
single 37-bp sequence of the Ks and Ki alleles. DNase I footprinting assay
revealed protein binding sites in the second intron of the K-ras gene that
correspond to the tandem repeat sequences. Our data suggest that higher
expression of the A/J allele relative to C3H allele may be responsible for the
allele-specific activation of the K-ras gene in lung tumors from F1 hybrid
mice.
Insulin-like growth factor I stimulates myofibrildevelopment and decreases smooth muscle alpha-actin of adultcardiomyocytes.
Authors: Donath M Y , Zapf J , Eppenberger-Eberhardt M , Froesch E R , Eppenberger H M
Published: May 2006

Adult rat cardiomyocytes in long-term culture
express type 1 insulin-like growth factor (IGF) receptors. In contrast to
insulin receptors, type 1 IGF receptors are up-regulated during culturing. IGF-I
added to the cells at plating increased granular density and pseudopodia number
per cell after 7 days. After 16 days, IGF-I-treated cells showed, as compared
with controls, a dramatic increase of the number of newly built sarcomeres and
were packed with myofibrils. At the same time, IGF-I suppressed the accumulation
of smooth muscle alpha-actin (sm-alpha-actin) in a dose-dependent manner. Under
the conditions of this in vitro system, growth hormone had no effect on cell
morphology or sm-alpha-actin. sm-alpha-Actin, a nonsarcomeric isoform of actin
expressed in early fetal cardiac development, reappears both during long-term
culture of adult rat cardiomyocytes and during heart hypertrophy. This study
shows that type 1 IGF receptors are up-regulated in adult rat cardiomyocytes in
long-term culture and that IGF-I enhances myofibril development and
concomitantly down-regulates sm-alpha-actin. This protein forms
stress-fiber-like structures and may temporarily serve as a scaffold for the
formation of new sarcomeres until myofibrils have developed throughout the cell
and the scaffold is no longer needed. Our findings thus allow us to propose
another hypothesis for the mechanism leading to overload heart
hypertrophy.
Induction and down-regulation of PLK, a humanserine/threonine kinase expressed in proliferating cells andtumors.
Authors: Holtrich U , Wolf G , Bräuninger A , Karn T , Böhme B , Rübsamen-Waigmann H , Strebhardt K
Published: May 2006

We have identified the nucleotide sequence of the
cDNA encoding the human counterpart of the mouse gene Plk (polo-like kinase).
The sequence of the human gene, PLK, predicts a serine/threonine kinase of 603
aa. Expression of PLK mRNA appeared to be strongly correlated with the mitotic
activity of cells. Resting peripheral lymphocytes did not express the gene at
all. When primary T cells were activated by phytohemagglutinin, a high level of
PLK transcripts resulted within 2-3 days. In some cases, addition of interleukin
2 to these cells increased the expression of PLK mRNA further. In contrast,
primary cultures of human peripheral macrophages, which were not dividing under
the culture conditions applied, showed very little or no PLK mRNA. Stimulation
of these cells by bacterial lipopolysaccharide, an inducer of several cytokines
in macrophages, totally abrogated the expression of PLK mRNA. In line with a
function of PLK mRNA expression in mitotically active cells is our finding that
six immortalized cell lines examined expressed the gene. In A-431 epidermoid
carcinoma cells this expression was down-regulated by serum starvation and
enhanced after serum was added again. Tumors of various origin (lung, colon,
stomach, smooth muscle, and esophagus as well as non-Hodgkin lymphomas)
expressed high levels of PLK transcripts in about 80% of the samples studied,
whereas PLK mRNA was absent in surrounding tissue, except for colon. The only
normal tissues where PLK mRNA expression was observed were colon and placenta,
both known to be mitotically active. No PLK transcripts were found in normal
adult lung, brain, heart, liver, kidney, skeletal muscle, and pancreas. In
Northern blot experiments with RNA from lymphocytes which were treated with
phytohemagglutinin and cycloheximide, PLK transcripts were not detectable,
suggesting that PLK is not an early growth-response gene.
Neuroanatomical correlates of retrieval in episodic memory: auditory sentence recognition.
Authors: Tulving E , Kapur S , Markowitsch H J , Craik F I , Habib R , Houle S
Published: May 2006

This study used positron emission tomography (PET) to investigate the neuroanatomical correlates of remembering previously experienced events. Twelve young healthy adults listened to "old" meaningful sentences which they had studied 24 hr previously. As a control task the subjects listened to comparable "new" sentences that they had never heard before. Regional cerebral blood flow associated with each task was measured by PET scans using 15O-labeled water. Comparison (old-sentence task minus new-sentence task) of the PET images revealed an extended strip of increased blood flow in the right dorsolateral prefrontal cortex (Brodmann's areas 10, 46, and 9) and the anterior portion of area 6. Other principal regions of increased blood flow were situated around the left anterior cingulate sulcus and bilaterally in the parietal lobes (areas 7 and 40). Major decreases in blood flow were situated bilaterally in the temporal lobes (areas 21, 22, 41, and 42). A high proportion of activity changes seemed to be located in the depths of cortical sulci. Increases in blood flow are seen as reflecting the operations of a widely distributed neuronal network involving prefrontal and parietal cortical regions that subserves the conscious recollection of previously experienced events. Decreases in blood flow in the temporal auditory areas are interpreted as reflecting auditory priming. The prevalence of sulcal blood-flow changes may reflect extensive cortical gyrification; it may also indicate that memory-related processes rely on the densely packed neuropil of sulcal regions.
An3 mRNA encodes an RNA helicase that colocalizes with nucleoli in Xenopus oocytes in a stage-specific manner.
Authors: Gururajan R , Mathews L , Longo F J , Weeks D L
Published: May 2006

An3 is a maternal mRNA localized to the animal hemisphere of oocytes and early embryos. We have analyzed the enzymatic activity and the subcellular localization of the protein encoded by An3 mRNA during Xenopus oogenesis. Antibodies raised using recombinant full-length and truncated An3 protein recognized a single protein in Xenopus and single proteins from HeLa cells, Drosophila, mouse testes, and Saccharomyces cerevisiae. An3 protein immunoprecipitated from stage IV and stage VI oocytes had ATP-dependent RNA helicase activity. The subcellular location of An3 protein changed during oocyte development. In previtellogenic oocytes, An3 was present throughout the nucleus; cytoplasmic localization was relatively sparse. Nuclear localization in mid-vitellogenic oocytes was primarily nucleolar; cytoplasmic staining increased relative to earlier stages. In stage VI oocytes, An3 protein was detected only in the cytoplasm. The temporal change in An3 protein localization is consistent with a role in the production of large maternal pools of rRNA during oogenesis.
Structure of the gene V protein of bacteriophage f1 determined by multiwavelength x-ray diffraction on the selenomethionyl protein.
Authors: Skinner M M , Zhang H , Leschnitzer D H , Guan Y , Bellamy H , Sweet R M , Gray C W , Konings R N , Wang A H , Terwilliger T C
Published: May 2006

The crystal structure of the dimeric gene V protein of bacteriophage f1 was determined using multiwavelength anomalous diffraction on the selenomethionine-containing wild-type and isoleucine-47-->methionine mutant proteins with x-ray diffraction data phased to 2.5 A resolution. The structure of the wild-type protein has been refined to an R factor of 19.2% using native data to 1.8 A resolution. The structure of the gene V protein was used to obtain a model for the protein portion of the gene V protein-single-stranded DNA complex.
Identification of Mycobacterium tuberculosis DNA in a pre-Columbian Peruvian mummy.
Authors: Salo W L , Aufderheide A C , Buikstra J , Holcomb T A
Published: May 2006

The existence of tuberculosis in the pre-Columbian Americas is controversial because the morphology of the lesion is not specific, the organism is culturally nonviable in ancient tissues, and nonpathogenic soil mycobacteria can contaminate buried bodies. We report the recovery of DNA unique to Mycobacterium tuberculosis from a lung lesion of a spontaneously mummified, 1000-year-old adult female body in southern Peru. This provides the most specific evidence possible for the pre-Columbian presence of human tuberculosis in the New World.
RNA polymerase binding using a strongly acidic hydrophobic-repeat region of sigma 54.
Authors: Tintut Y , Wong C , Jiang Y , Hsieh M , Gralla J D
Published: May 2006

sigma 54 is a rare bacterial protein that substitutes for sigma 70 in the case of Escherichia coli genes transcribed by certain activators with enhancer protein-like properties. It contains a strongly acidic region of previously unknown function. Gel mobility-shift assays using sigma 54 deletion mutants show that this region is essential for sigma 54 to bind the core RNA polymerase and recruit it to the promoter. Multiple-point mutational analysis shows that the acidic amino acids and overlapping periodic hydrophobic amino acids are necessary for this binding. Related sequences are not found within the core binding determinant of sigma 70, which binds the same core RNA polymerase. This comparison suggests that the core RNA polymerase interacts differently with the two sigma factors, likely contributing to the critical differences in transcription mechanism in the two cases.
Multiple origins of a spider radiation in Hawaii.
Authors: Gillespie R G , Croom H B , Palumbi S R
Published: May 2006

The Hawaiian Islands are renowned for some of the most spectacular species radiations in the world. Most of these radiations have been attributed to single colonization events, although the evidence supporting monophyletic origins is often poorly resolved and/or ambiguous. Without a concrete understanding of the origins of species radiations, it is impossible to understand the phylogenetic pattern of species proliferation or the spectrum of morphological, ecological, and behavioral modifications attributable to a single colonist. In this study we examined the species radiation of the spider genus Tetragnatha in Hawaii. Unlike their mainland congeners, the Hawaiian Tetragnatha are extremely diverse in morphology, ecology, and behavior. We tested whether this diversity arose from a single or multiple colonization events. We coupled morphological (37 characters) and molecular (sequence from the 12S ribosomal subunit of mitochondrial DNA) approaches to assess the phylogenetic position of the Hawaiian Tetragnatha relative to continental congeners and to examine evidence for monophyly. We provide evidence that the Hawaiian Tetragnatha emanate from multiple origins. At least two independent species radiations, the "spiny-leg" clade and the web-building species Tetragnatha stelarobusta and Tetragnatha acuta, have arisen from one or more founder events. Two additional natural colonizations have resulted in the establishment of non-speciose lineages, as represented by Tetragnatha hawaiensis and Doryonychus raptor.
Polymer-encapsulated cells genetically modified to secrete human nerve growth factor promote the survival of axotomized septal cholinergic neurons.
Authors: Winn S R , Hammang J P , Emerich D F , Lee A , Palmiter R D , Baetge E E
Published: May 2006

Effective treatments for neurodegenerative disorders are limited by our inability to alter the progression of the diseases. A number of proteins have specific neuroprotective activities in vitro; however, the delivery of these factors into the central nervous system over the long term at therapeutic levels has been difficult to achieve. BHK cells engineered to express and release human nerve growth factor were encapsulated in an immunoisolation polymeric device and transplanted into both fimbria-fornix-lesioned rat brains and naive controls. In the lesioned rat brain, chronic delivery of human nerve growth factor by the encapsulated BHK cells provided nearly complete protection of axotomized medial septal cholinergic neurons. Human nerve growth factor continued to be released by encapsulated cells upon removal from the aspirative site after 3 weeks or from normal rat striatum after 3 and 6 months in vivo. Long-term encapsulated cell survival was confirmed by histologic analysis. This encapsulated xenogeneic system may provide therapeutically effective amounts of a number of neurotrophic factors, alone or in combination, to virtually any site within the body.
Hepatitis viruses: changing patterns of human disease.
Authors: Purcell R H
Published: May 2006

Viral hepatitis is a disease of antiquity, but evidence for more than one etiologic agent has been recognized only since the 1940s, when two viruses (hepatitis A virus and hepatitis B virus) were thought to account for all disease. In the past 20 years, three additional hepatitis agents (hepatitis C virus, hepatitis D virus, and hepatitis E virus) have been discovered, and there is evidence for at least one additional virus. Each of the five recognized hepatitis viruses belongs to a different virus family, and each has a unique epidemiology. The medical impact of these viruses on society has been strongly influenced by changes in human ecology. This has resulted in some cases in diminished disease and in others in increases in the incidence of disease.
Phosphacan, a chondroitin sulfate proteoglycan of brain that interacts with neurons and neural cell-adhesion molecules, is an extracellular variant of a receptor-type protein tyrosine phosphatase.
Authors: Maurel P , Rauch U , Flad M , Margolis R K , Margolis R U
Published: May 2006

We have identified cDNA clones encoding a chondroitin sulfate proteoglycan of rat brain (previously designated 3F8 and now named phosphacan) that binds to neurons and neural cell-adhesion molecules. A sequence of 1616 amino acids deduced from a 4.8-kb open reading frame contains the N-terminal amino acid sequence of the 3F8 core glycoprotein as well as four internal CNBr, tryptic, and endoproteinase Lys-C peptide sequences from the proteoglycan. The deduced amino acid sequence, beginning with a 24-amino acid signal peptide, reveals an N-terminal domain of 255 amino acids homologous to carbonic anhydrases. The entire amino acid sequence deduced from our cDNA clones corresponds to the extracellular portion of a human receptor-type protein tyrosine phosphatase (RPTP zeta/beta) with which it has 76% identity, and the proteoglycan may represent an mRNA splicing variant of the larger transmembrane protein. RNA analysis demonstrated that a probe to the N-terminal carbonic anhydrase domain of the proteoglycan hybridizes with rat brain mRNA of 9.5, 8.4, and 6.4 kb, whereas probes to the phosphatase domains hybridize with only the 9.5-kb message and with the 6.4-kb message (which corresponds to a previously identified variant of the transmembrane protein in which half of the extracellular domain is deleted). The 30 N-terminal amino acids of the 3H1 chondroitin/keratan sulfate proteoglycan of brain are identical to those of the 3F8 proteoglycan, and six internal tryptic peptide sequences also matched those found in sequenced peptides of the 3F8 proteoglycan and/or amino acid sequences deduced from the cDNA clones. We therefore conclude that the 3H1 chondroitin/keratan sulfate proteoglycan and the 3F8 chondroitin sulfate proteoglycan represent glycosylation and possible extracellular splicing variants of a receptor-type protein tyrosine phosphatase. These proteoglycans may modulate cell interactions and other developmental processes in nervous tissue through heterophilic binding to cell-surface and extracellular matrix molecules, and by competition with ligands of the transmembrane phosphatase.
Isolation and characterization of a fourth Arabidopsis thaliana G-box-binding factor, which has similarities to Fos oncoprotein.
Authors: Menkens A E , Cashmore A R
Published: May 2006

A fourth member of the Arabidopsis G-box-binding factor (GBF) family of bZIP proteins, GBF4, has been isolated and characterized. In a manner reminiscent of the Fos-related oncoproteins of mammalian systems, GBF4 cannot bind to DNA as a homodimer, although it contains a basic region capable of specifically recognizing the G-box and G-box-like elements. However, GBF4 can interact with GBF2 and GBF3 to bind DNA as heterodimers. Mutagenesis of the leucine zipper of GBF4 indicates that the mutation of a single amino acid confers upon the protein the ability to recognize the G-box as a homodimer, apparently by altering the charge distribution within the leucine zipper.
A molecular model for the genetic and phenotypic characteristics of the mouse lethal yellow (Ay) mutation.
Authors: Michaud E J , Bultman S J , Klebig M L , van Vugt M J , Stubbs L J , Russell L B , Woychik R P
Published: May 2006

Lethal yellow (Ay) is a mutation at the mouse agouti locus in chromosome 2 that causes a number of dominant pleiotropic effects, including a completely yellow coat color, obesity, an insulin-resistant type II diabetic condition, and an increased propensity to develop a variety of spontaneous and induced tumors. Additionally, homozygosity for Ay results in preimplantation lethality, which terminates development by the blastocyst stage. The Ay mutation is the result of a 170-kb deletion that removes all but the promoter and noncoding first exon of another gene called Raly, which lies in the same transcriptional orientation as agouti and maps 280 kb proximal to the 3' end of the agouti gene. We present a model for the structure of the Ay allele that can explain the dominant pleiotropic effects associated with this mutation, as well as the recessive lethality, which is unrelated to the agouti gene.
Cloning and functional characterization of a third pituitary adenylate cyclase-activating polypeptide receptor subtype expressed in insulin-secreting cells.
Authors: Inagaki N , Yoshida H , Mizuta M , Mizuno N , Fujii Y , Gonoi T , Miyazaki J , Seino S
Published: May 2006

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide belonging to the vasoactive intestinal polypeptide/glucagon/secretin family. It is widely distributed in the body, and a variety of biological actions have been reported. PACAP exerts its biological effects by binding to specific receptors that are coupled to GTP-binding proteins. Recent studies have shown that there is a family of PACAP receptors (PACAPRs), and two members of this family have been identified. We report here the cloning, functional expression, and tissue distribution of a third PACAPR subtype, designated PACAPR-3. The cDNA encoding PACAPR-3 has been isolated from a mouse insulin-secreting beta-cell line MIN6 cDNA library. Mouse PACAPR-3 is a protein of 437 amino acids that has 50% and 51% identity with rat PACAP type I and type II receptors, respectively. Expression of recombinant mouse PACAPR-3 in mammalian cells shows that it binds to vasoactive intestinal polypeptide as well as PACAP-38 and -27, with a slightly higher affinity for PACAP-38, and is positively coupled to adenylate cyclase. The expression of PACAPR-3 in Xenopus oocytes indicates that calcium-activated chloride currents are evoked by PACAP and vasoactive intestinal polypeptide, suggesting that PACAPR-3 can also be coupled to phospholipase C. RNA blot analysis studies reveal that PACAPR-3 mRNA is expressed at high levels in MIN6, at moderate levels in pancreatic islets and other insulin-secreting cell lines, HIT-T15 and RINm5F, as well as in the lung, brain, stomach, and colon, and at low levels in the heart. Furthermore, insulin secretion from MIN6 cells is significantly stimulated by PACAP-38. These results suggest that the diverse biological effects of PACAP are mediated by a family of structurally related proteins and that PACAPR-3 participates in the regulation of insulin secretion.
A mouse B16 melanoma mutant deficient in glycolipids.
Authors: Ichikawa S , Nakajo N , Sakiyama H , Hirabayashi Y
Published: May 2006

Mouse B16 melanoma cell line, GM-95 (formerly designated as MEC-4), deficient in sialyllactosylceramide was examined for its primary defect. Glycolipids from the mutant cells were analyzed by high-performance TLC. No glycolipid was detected in GM-95 cells, even when total lipid from 10(7) cells was analyzed. In contrast, the content of ceramide, a precursor lipid molecule of glycolipids, was normal. Thus, the deficiency of glycolipids was attributed to the first glucosylation step of ceramide. The ceramide glucosyltransferase (EC 2.4.1.80) activity was not detected in GM-95 cells. There was no significant difference of sialyllactosylceramide synthase activity, however, between GM-95 and the parental cells. The deficiency of glycolipids in GM-95 cells was associated with changes of the cellular morphology and growth rate. The parental cells showed irregular shapes and tended to overlap each other. On the other hand, GM-95 cells exhibited an elongated fibroblastic morphology and parallel arrangement. The population-doubling times of GM-95 and the parental cells in serum-free medium were 28 hr and 19 hr, respectively.
Expression of recombinant human casein kinase II and recombinant heat shock protein 90 in Escherichia coli and characterization of their interactions.
Authors: Shi Y , Brown E D , Walsh C T
Published: May 2006

To assess the interaction of human casein kinase II (CKII) with the heat shock protein 90 (HSP90) class of chaperone proteins, human CKII alpha and beta subunits and beta S2A mutant were expressed and purified separately or from a tandem coexpression construct in Escherichia coli. Recombinant human HSP90 beta and recombinant yeast HSP90 as His6 constructs were also expressed in and purified from E. coli. The rhCKII S2A mutant removed the regulatory beta subunit autophosphorylation site but had no effect on catalytic efficiency with peptide or protein substrates. As a CKII substrate, recombinant hHSP90 beta displayed a Km of 9.8 microM and a kcat of 4.1 min-1 and was phosphorylated to 1.5 mol/mol, whereas ryHSP90, lacking the known serine CKII sites of hHSP90, was phosphorylated at a 19-fold lower kcat/Km ratio to levels of 0.8 mol/mol. The endoplasmic reticulum HSP90 family member Grp94 was phosphorylated to 1.4 mol/mol but, in contrast, HSC70 and FKBP25 chaperones were phosphorylated to < 0.01 mol/mol. Neither phospho nor dephospho forms of hHSP90 showed significant activation of CKII toward the peptide substrate RRREEETEEE in contrast to a previous report that activation was observed at high molar ratios of chaperone to kinase.
A geometric identity for Pappus' theorem.
Authors: Hawrylycz M
Published: May 2006

An expression in the exterior algebra of a Peano space yielding Pappus' theorem was originally given by Doubilet, Rota, and Stein [Doubilet, P., Rota, G.-C. & Stein, J. (1974) Stud. Appl. Math. 8, 185-216]. Motivated by an identity of Rota, I give an identity in a Grassmann-Cayley algebra of step 3, involving joins and meets alone, which expresses the theorem of Pappus.
Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells.
Authors: Yusibov V M , Steck T R , Gupta V , Gelvin S B
Published: May 2006

During the inception of crown gall tumorigenesis, the transferred DNA (T-DNA) is processed from the Ti (tumor inducing) plasmid of Agrobacterium tumefaciens and is transferred to plant cells. T-DNA processing and transfer require the induction of vir (virulence) genes by phenolic compounds secreted by wounded plant cells. After vir gene induction, both single-stranded (T-strands) and double-stranded forms of processed T-DNA accumulate in the bacteria. Although current models favor the transfer of T-strands to plants, there has yet been no experimental evidence to show this. In this paper, we show that T-strands disappear from acetosyringone-induced A. tumefaciens within 30 min of bacterial cocultivation with tobacco protoplasts. PCR analysis of T-DNA associated with protoplasts indicates that single-stranded, but not double-stranded, T-DNA can be detected in the plant cells within 30 min of bacterial cocultivation. Control experiments show that this T-DNA does not originate from lysed contaminating bacterial cells. T-DNA transfer depends on a functional bacterial virB operon. Protoplast infections using an A. tumefaciens virE mutant result in a low level of accumulation of T-strands in the plant cells.
Cellular requirements for tumor-specific immunity elicited by heat shock proteins: tumor rejection antigen gp96 primes CD8+ T cells in vivo.
Authors: Udono H , Levey D L , Srivastava P K
Published: May 2006

Purified preparations of 96-kDa heat shock proteins (gp96) have been previously shown to elicit tumor-specific immunity to the tumor from which gp96 is obtained but not to antigenically distinct chemically induced tumors. The cellular requirements of gp96-elicited immunity have been examined. It is observed that depletion of CD8+, but not CD4+, T cells in the priming phase abrogates the immunity elicited by gp96. The CD8+ T cells elicited by immunization with gp96 are active at least up to 5 weeks after immunization. Depletion of macrophages by treatment of mice with carrageenan during the priming phase also results in loss of gp96-elicited immunity. In the effector phase, all three compartments, CD4+ and CD8+ T cells and macrophages, are required. Immunity elicited by whole irradiated tumor cells shows a different profile of cellular requirements. In contrast to immunization with gp96, depletion of CD4+, but not CD8+, T cells during priming with whole tumor cells abrogates tumor immunity. Further, ablation of macrophage function during priming or effector phases has no effect on tumor immunity elicited by whole cells. Our results suggest the existence of a macrophage-dependent and a macrophage-independent pathway of tumor immunity. Our observations also show that in spite of exogenous administration, vaccination with gp96 preparations elicits a CD8+ T-cell response in vivo, and it is therefore a useful method of vaccination against cancer and infectious diseases.
Electrophysiological properties of newborn and adult rat spinal cord glycine receptors expressed in Xenopus oocytes.
Authors: Morales A , Nguyen Q T , Miledi R
Published: May 2006

The properties of glycine receptors (GlyRs) from newborn and adult rat spinal cord were studied in Xenopus oocytes injected with whole mRNA or the heavy (H) or light (L) mRNA fractions encoding their respective GlyRs. Mean open times and conductances of channels gated by H- or L-GlyRs were determined by noise analysis or voltage jumps. We found that adult H- and L-GlyRs opened channels that differed in their mean open time but had the same channel conductance. Both H- and L-GlyRs gated Cl- currents that displayed a similarly strong outward rectification. Nevertheless, single channels of adult H- and L-GlyRs did not rectify and their mean open times were only slightly altered by voltage. It follows that the outward rectification of adult GlyRs is due mainly to a reduction in the number of open channels. In contrast to H-GlyRs, whose characteristics seem to remain essentially unchanged with age, L-GlyRs from newborn and adult rats have different properties. Channels of newborn L-GlyRs have a higher conductance, longer open time, and greater voltage dependency than those from the adult. Interestingly, properties of newborn GlyRs expressed by whole mRNA were markedly different from those encoded by newborn or adult L or H mRNA. These results demonstrate that the functional heterogeneity of GlyRs is developmentally regulated.
Community-level competition: asymmetrical dominance.
Authors: Gilpin M
Published: May 2006

Ecological competition between entire communities of species occurs only when geographic barriers are suddenly removed. Recent empirical analysis suggests that, following the disappearance of a barrier, one community may swamp a second community, causing most or all of its species to go extinct. I provide theoretical insight into this result by showing that two "naive" competition communities mix randomly following the removal of a barrier. However, if the two communities have been "assembled," or self-organized, through a history of competitive exclusion, the communities are likely to battle as coordinated armies, with one or the other side ultimately claiming the entire landscape.