bluuebunny/crossref_metadata_2025 huggingface dataset:
Two additional potential retrotransposons isolated from a human L1 subfamily that contains an active retrotransposable element.
Authors: Dombroski B A , Scott A F , Kazazian H H
Published: May 2006

We have previously reported the isolation of a human retrotransposable L1 element. This element, allele L1.2B at the LRE-1 locus of chromosome 22, was shown by nucleotide sequence identity to be the direct precursor of a de novo retrotransposition event into the factor VIII gene on the X chromosome, resulting in hemophilia A in patient JH-27. We now report the isolation of the two remaining full-length members of the subfamily of L1 elements closely related to L1.2B present in the genome of the mother of JH-27. Since these elements, L1.3 and L1.4, are very similar in sequence to L1.2B and contain both open reading frames 1 and 2 intact, they are also likely to be active retrotransposable elements. This suggests that certain L1 subfamilies may contain multiple active elements.
Wavelengths effective in induction of malignant melanoma.
Authors: Setlow R B , Grist E , Thompson K , Woodhead A D
Published: May 2006

It is generally agreed that sunlight exposure is one of the etiologic agents in malignant melanoma of fair-skinned individuals. However, the wavelengths responsible for tumorigenesis are not known, although DNA is assumed to be the target because individuals defective in the repair of UV damage to DNA are several thousandfold more prone to the disease than the average population. Heavily pigmented backcross hybrids of the genus Xiphophorus (platyfish and swordtails) are very sensitive to melanoma induction by single exposures to UV. We irradiated groups of five 6-day-old fish with narrow wavelength bands at 302, 313, 365, 405, and 436 nm and scored the irradiated animals for melanomas 4 months later. We used several exposures at each wavelength to obtain estimates of the sensitivity for melanoma induction as a function of exposure and wavelength. The action spectrum (sensitivity per incident photon as a function of wavelength) for melanoma induction shows appreciable sensitivity at 365, 405, and probably 436 nm, suggesting that wavelengths not absorbed directly in DNA are effective in induction. We interpret the results as indicating that light energy absorbed in melanin is effective in inducing melanomas in this animal model and that, in natural sunlight, 90-95% of melanoma induction may be attributed to wavelengths > 320 nm--the UV-A and visible spectral regions.
Suppression of oncogenic Ras by mutant neurofibromatosis type 1 genes with single amino acid substitutions.
Authors: Nakafuku M , Nagamine M , Ohtoshi A , Tanaka K , Toh-e A , Kaziro Y
Published: May 2006

NF1 was first identified as the gene responsible for the pathogenesis of the human genetic disorder neurofibromatosis type 1. cDNA cloning revealed that its putative protein product has a domain showing significant sequence homology with the mammalian Ras GTPase activating protein and two yeast Saccharomyces cerevisiae proteins, Ira1 and Ira2. The Ras GTPase activating protein-related domain of the NF1 gene product (NF1-GRD) stimulates GTPase activity of normal Ras proteins but not of oncogenic mutant Ras from both mammalian and yeast cells. Thus, in yeast, NF1-GRD can suppress the heat-shock-sensitive phenotype of ira- cells but not the same phenotype of activated RAS such as RAS2Val19 and RAS2Leu68. We have screened a pool of mutagenized NF1 expression plasmids and obtained two mutant NF1 cDNA clones that can suppress the heat-shock-sensitive phenotype of RAS2Val19 cells. One clone (NF201) suppressed RAS2Leu68, RAS2Ser41, and RAS2Val19, whereas another clone (NF204) preferentially suppressed RAS2Val19. When expressed in mammalian cells, these mutant NF1-GRDs were able to induce the morphological reversion of v-ras-transformed NIH 3T3 cells. Both wild-type and mutant NF1-GRDs can stimulate the GTPase activity of normal but not transforming Ras. We suggest that mutant NF1-GRDs may bind tightly to transforming Ras, which stays in GTP-bound conformation, thus preventing the interaction with the putative effector molecule. On the other hand, normal Ras cannot be sequestered since the bound GTP is rapidly hydrolyzed upon interaction with mutant NF1-GRD to yield Ras-GDP, which is readily released from the NF1-GRD and recycled.
Intervening sequence with conserved open reading frame in eubacterial 23S rRNA genes.
Authors: Ralph D , McClelland M
Published: May 2006

An intervening sequence (IVS) occurred in the 23S rRNA genes (rrl) of some, but not all, strains of four species of the spirochete genus Leptospira and was absent from strains in three other species. The IVS varied in size from 485 to 759 base pairs and replaced bases 1224-1245 in both copies of rrl. The two ends of each IVS shared 22-35 bases of complementarity that could form a stable double helix. The presence of an IVS correlated with a cleaved mature 23S rRNA that probably results from removal of the IVS without religation. The 3' site of cleavage was mapped within the inverted repeat of the IVS. An open reading frame of 121-133 amino acids was conserved in the IVS in all four species, oriented so that the sense strand was in the rRNA transcript. When the open reading frames were compared between species, they predicted polypeptides that showed between 51% and 78% amino acid conservation and similar DNA sequence conservation, indicating selection for protein function.
Discrete and integral fourier transforms: analytical examples.
Authors: Thompson W J , Macdonald J R
Published: May 2006

Analytical examples of the discrete Fourier transform (DFT) help in understanding relations between the DFT and the Fourier integral transform (FIT). Such examples enable one to estimate the errors involved when one transform is approximated by the other, and they suggest how such approximation errors might be reduced. We present mathematical and numerical analyses of the time-to-frequency DFT of the complex exponential function and of the frequency-to-time inverse DFT of the relaxation function. The FITs of these functions are exact inverses, and so they serve to clarify the effects of aliasing and truncation on the DFT.
Calsequestrinlike calcium-binding protein is expressed in calcium-accumulating cells of Pistia stratiotes.
Authors: Franceschi V R , Li X , Zhang D , Okita T W
Published: May 2006

To contend with high calcium (Ca) levels in the environment, many plant species contain crystal idioblasts, specialized cells which accumulate large amounts of Ca as oxalate crystals. The biochemical processes involved in the accumulation of Ca in crystal idioblasts are unknown, as these cells constitute only a minor proportion of the total plant tissue. To address how crystal idioblasts buffer cytosolic Ca during crystal formation, we purified these cells from water lettuce and assessed their biochemistry. We show here that crystal idioblast cells contain three Ca-binding proteins not detectable in mesophyll cells. One of the Ca-binding proteins shares antigenicity with rabbit calsequestrin, a high-capacity low-affinity Ca-binding protein, and is encoded by related nucleotide sequences. Immunocytochemical localization studies further demonstrate that a calsequestrinlike protein is present primarily in crystal idioblasts and is preferentially localized in the endoplasmic reticulum, an organelle enriched in Ca as evidenced by vital staining. We thus conclude that crystal idioblasts possess a buffering system involving calsequestrinlike proteins, a process that likely plays an essential role in the bulk control of Ca in plant cells.
Reproductive failure of dominant males in the poeciliid fish Limia perugiae determined by DNA fingerprinting.
Authors: Schartl M , Erbelding-Denk C , Hölter S , Nanda I , Schmid M , Schröder J H , Epplen J T
Published: May 2006

Hierarchical structures among male individuals in a population are frequently reflected in differences in aggressive and reproductive behavior and access to the females. In general, social dominance requires large investments, which in turn then may have to be compensated for by high reproductive success. However, this hypothesis has so far only been sufficiently tested in small mating groups (one or two males with one or two females) due to the difficulties of determining paternity by conventional methods. DNA fingerprinting overcomes these problems by offering the possibility to determine genetic relationships and mating patterns within larger groups [Burke, T. (1989) Trends Ecol. Evol. 4, 139-144]. We show here that in the poeciliid fish Limia perugiae, in small mating groups the dominant male has a mating success of 100%, whereas in larger groups its contribution to the offspring unexpectedly drops to zero.
Xenopus Ro ribonucleoproteins: members of an evolutionarily conserved class of cytoplasmic ribonucleoproteins.
Authors: O'Brien C A , Margelot K , Wolin S L
Published: May 2006

Ro small ribonucleoproteins consist of a 60-kDa protein and possibly additional proteins complexed with several small RNA molecules. The RNA components of these particles, designated Y RNAs, are about 100 nt long. Although these small ribonucleoproteins are abundant components of a variety of vertebrate species and cell types, their subcellular location is controversial, and their function is completely unknown. We have identified and characterized the Ro RNPs of Xenopus laevis. Three of the four distinct Xenopus Y RNAs appear to be related to the previously sequenced human hY3, hY4, and hY5 RNAs. The fourth Xenopus Y RNA, xY alpha, does not appear to be a homologue of any of the human Y RNAs. Each of the human and Xenopus Y RNAs possesses a conserved stem that contains the binding site for the 60-kDa Ro protein. Xenopus and human 60-kDa Ro proteins are 78% identical in amino acid sequence, with the conservation extending throughout the entire protein. When human hY3 RNA is mixed with Xenopus egg extracts, the human RNA assembles with the Xenopus Ro protein to form chimeric Ro ribonucleoproteins. By analyzing RNA extracted from manually enucleated oocytes and germinal vesicles, we have determined that Y RNAs are located in the oocyte cytoplasm. By examining the distribution of mouse Ro ribonucleoproteins in cytoplast and karyoplast fractions derived from L-929 cells, we have determined that Ro ribonucleoprotein particles also primarily reside in the cytoplasm of mammalian cells.
Transmembrane signaling in human polymorphonuclear neutrophils: 15(S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid modulates receptor agonist-triggered cell activation.
Authors: Smith R J , Justen J M , Nidy E G , Sam L M , Bleasdale J E
Published: May 2006

15(S)-Hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid (15-HETE) exerted a time- and concentration-dependent inhibition of superoxide anion (O2-) production and exocytosis of both azurophil and specific granule constituents from human polymorphonuclear neutrophils (PMN) stimulated with the receptor-specific agonists, N-formylmethionylleucylphenylalanine (FMLP), platelet-activating factor, and leukotriene B4, but not that elicited by phorbol 12-myristate 13-acetate. 15-HETE did not alter the binding of FMLP to its specific receptors on PMN but, rather, appeared to interfere with a subsequent process in signal transduction. Receptor-coupled production of inositol 1,4,5-trisphosphate (InsP3) and increases in cytosolic free calcium elicited with FMLP, platelet-activating factor, and leukotriene B4 were suppressed by 15-HETE. 15-HETE did not, however, inhibit the mobilization of 45Ca from intracellular stores elicited by the addition of InsP3 to permeabilized PMN. 15-HETE suppressed O2- production and increases in intracellular [Ca2+] induced when cell-surface receptors were bypassed and the PMN were activated directly by the guanine nucleotide-binding protein (G protein) activators aluminum fluoride (AlF4-) and mastoparan. 15-HETE, however, did not perturb all G protein functions because cAMP production in FMLP-activated PMN was essentially unaffected by 15-HETE. These data support the proposition that 15-HETE modulates receptor-triggered activation of PMN either by uncoupling G protein stimulation of phospholipase C or by directly inhibiting phospholipase C, thus inhibiting the InsP3-dependent rise in intracellular [Ca2+] that is prerequisite for PMN responsiveness to receptor agonists.
Atomic force microscopy of cloned nicotinic acetylcholine receptor expressed in Xenopus oocytes.
Authors: Lal R , Yu L
Published: May 2006

The nicotinic acetylcholine receptor (AChR) was expressed in Xenopus oocytes from in vitro transcribed mRNA and was imaged by atomic force microscopy. A characteristic pentameric structure of AChR was readily observed on the extracellular face of the cell membrane, with a central pore surrounded by protruding AChR subunits. These structures were seen only in mRNA-injected oocytes that also gave acetylcholine-induced membrane currents. The size of individual AChR channels, the angles between subunits, and the interchannel spacing were all compatible with the current model of AChR. In addition, localized patches of microscopic AChR clustering were observed, with packing density approaching that at the neuromuscular junction. These findings show the potential of studying cloned membrane proteins in oocytes for both their surface topography and their structure-function relationship in native membrane without the need for crystallization.
Pleiotropic effect of the human T-cell leukemia virus Tax protein on the DNA binding activity of eukaryotic transcription factors.
Authors: Armstrong A P , Franklin A A , Uittenbogaard M N , Giebler H A , Nyborg J K
Published: May 2006

The Tax protein, encoded by the human T-cell leukemia virus type I, is a potent activator of viral and cellular gene transcription. Tax does not bind DNA directly but appears to trans-activate through an interaction with host-cell transcription factors that recognize sequences within the promoters of Tax-responsive genes. Cellular transcriptional activators implicated in mediating Tax trans-activation include members of the activating transcription factor/cAMP response element binding protein (ATF/CREB) family of proteins, serum response factor, Fos-Jun, and NF-kappa B. Recent evidence suggests that Tax may stimulate human T-cell leukemia virus type I transcription, at least in part, through enhanced binding of ATF/CREB proteins to their recognition elements within the Tax-responsive 21-bp repeats of the viral promoter. In this report, we demonstrate that Tax also enhances the site-specific DNA binding activity of serum response factor and Fos-Jun and modestly enhances the binding of the NF-kappa B subunits, p50 and p65. We also show that Tax increases the DNA binding activity of the eukaryotic transcription factors ATF-1, Sp1, and GAL4. These results are consistent with the finding that Tax is highly pleiotropic and suggest that Tax trans-activation may involve enhancement in the DNA binding activity of target transcriptional regulatory proteins. In addition, we show that the mechanism of Tax-enhanced DNA binding activity does not involve an alteration in the redox state of the target protein.
Three-dimensional waves of excitation during Dictyostelium morphogenesis.
Authors: Steinbock O , Siegert F , Müller S C , Weijer C J
Published: May 2006

Cells in Dictyostelium slugs follow well-defined patterns of motion. We found that the chemotactic cell response is controlled by a scroll wave of messenger concentration in the highly excitable prestalk zone of the slug that decays in the less-excitable prespore region into planar wave fronts. This phenomenon is investigated by numerical solutions of partial differential equations that couple local nonlinear kinetics and diffusive transport of the chemotactic signal. In the interface of both regions a complex twisted scroll wave is formed that reduces the wave frequency in the prespore zone. The spatio-temporal dynamics of waves and filaments are followed over 33 periods of rotation. These results yield an explanation of collective self-organized cell motion in a multicellular organism.
Vpu protein of human immunodeficiency virus type 1 enhances the release of capsids produced by gag gene constructs of widely divergent retroviruses.
Authors: Göttlinger H G , Dorfman T , Cohen E A , Haseltine W A
Published: May 2006

The Vpu protein of human immunodeficiency virus type 1 facilitates the release of virus particles from the surface of infected cells. The ability of the Vpu protein to facilitate release of Gag proteins from retroviruses that lack a Vpu-like protein was examined. The results of these experiments show that Vpu significantly increases the release of the Gag proteins of human immunodeficiency virus type 2, visna virus, and Moloney murine leukemia virus from HeLa cells. The results indicate that Vpu-mediated enhancement of particle release requires neither amino-terminal myristoylation of the Gag precursor nor cleavage of the Gag precursor by the viral protease. The results raise the possibility that Vpu modifies a cellular pathway common to the release of all retroviruses from the cell surface.
Long-term depression of glutamate-induced gamma-aminobutyric acid release in cerebellum by insulin-like growth factor I.
Authors: Castro-Alamancos M A , Torres-Aleman I
Published: May 2006

We tested the possibility that insulin-like growth factor I (IGF-I) acts as a neuromodulator in the adult cerebellar cortex since previous observations indicated that IGF-I is located in the olivo-cerebellar system encompassing the inferior olive and Purkinje cells. We found that conjoint administration of IGF-I and glutamate through a microdialysis probe stereotaxically implanted into the cerebellar cortex and deep cerebellar nuclei greatly depressed the release of gamma-aminobutyric acid (GABA), which normally follows a glutamate pulse. This inhibition was dose-dependent and long-lasting. Moreover, the effect was specific for glutamate since KCl-induced GABA release was not modified by IGF-I. Basic fibroblast growth factor, another growth-related peptide present in the cerebellum, did not alter the response of GABA to glutamate stimulation. In addition, electrical stimulation of the inferior olivary complex significantly raised IGF-I levels in the cerebellar cortex. Interestingly, when the inferior olive was stimulated in conjunction with glutamate administration, GABA release by cerebellar cells in response to subsequent glutamate pulses was depressed in a manner reminiscent of that seen after IGF-I. These findings indicate that IGF-I produces a long-lasting depression of GABA release by Purkinje cells in response to glutamate. IGF-I might be present in climbing fiber terminals and/or cells within the cerebellar cortex and thereby might affect Purkinje cell function. Whether this IGF-I-induced impairment of glutamate stimulation of Purkinje cells underlies functionally plastic processes such as long-term depression is open to question.
Individual variation in contractile cost and recovery in a human skeletal muscle.
Authors: Blei M L , Conley K E , Odderson I B , Esselman P C , Kushmerick M J
Published: May 2006

This study determined the variation among individuals in ATP use during contraction and ATP synthesis after stimulation in a human limb muscle. Muscle energetics were evaluated using a metabolic stress test that separates ATP utilization from synthesis by 31P NMR spectroscopy. Epicutaneous supramaximal twitch stimulation (1 Hz) of the median and ulnar nerves was applied in combination with ischemia of the finger and wrist flexors in eight normal subjects. The linear creatine phosphate (PCr) breakdown during ischemic stimulation defined ATP use (delta PCr per twitch or approximately P/twitch) and was highly reproducible as shown by the relative standard deviation [(standard deviation/mean) x 100] of 11% in three repeated measures. The time constant of the monoexponential PCr change during aerobic recovery represented ATP synthesis rate and also showed a low relative standard deviation (9%). Individuals were found to differ significantly in both mean approximately P/twitch (PCr breakdown rates, 0.29-0.45% PCr per sec or % PCr per twitch; ANOVA, p < 0.001) and in mean recovery time constants (41-74 sec; ANOVA, P < 0.001). This range of approximately P/twitch corresponded with the range of fiber types reported for a flexor muscle. In addition, approximately P/twitch was negatively correlated with a metabolite marker of slow-twitch fiber composition (Pi/ATP). The nearly 2-fold range of recovery time constants agreed with the range of mitochondrial volume densities found in human muscle biopsies. These results indicate that both components involved in the muscle energy balance--oxidative capacity and contractile costs--vary among individuals in human muscle and can be measured noninvasively by 31 P NMR.
Peroxisome biogenesis in the yeast Hansenula polymorpha is controlled by a complex set of interacting gene products.
Authors: Titorenko V I , Waterham H R , Cregg J M , Harder W , Veenhuis M
Published: May 2006

We have studied the genetic interactions between mutant alleles in 12 genes, designated PER1-PER12, which are essential for peroxisome biogenesis in the yeast Hansenula polymorpha. Recessive mutations in any of these genes determined three different morphological phenotypes: (i) complete absence of peroxisomes (Per-); (ii) presence of small peroxisomes in conjunction with a major fraction of peroxisomal matrix proteins in the cytosol (Pim-); and (iii) presence of peroxisomes with aberrant crystalline matrix substructure (Pss-). Extensive complementation analysis showed many cases of noncomplementation--that is, diploids that contained both wild-type and mutant alleles of two different PER genes were unable to grow on methanol and showed peroxisomal defects. The observed cases of unlinked noncomplementation appeared to be gene and allele specific and were predominantly observed at lower temperatures (cold sensitive). The genetic results obtained were used to formulate a model of PER gene product interactions. In this model, five PER gene products are key or core components of the complex. Other PER gene products appear to play a more peripheral role.
A transmissible Creutzfeldt-Jakob disease-like agent is prevalent in the human population.
Authors: Manuelidis E E , Manuelidis L
Published: May 2006

The etiology of most human dementias is unknown. Creutzfeldt-Jakob disease (CJD), a relatively uncommon human dementia, is caused by a transmissible virus-like agent. Molecular markers that are specific for the agent have not yet been defined. However, the infectious disease can be transmitted to rodents from both brain and infected buffy coat (blood) samples. To determine whether human CJD infections are more widespread than is apparent from the low incidence of neurological disease, we attempted to transmit CJD from buffy coat samples of 30 healthy volunteers who had no family history of dementing illness. Primary transmissions from 26 of 30 individuals produced CJD-like spongiform changes in the brains of recipient hamsters at 200-500 days postinoculation. This positive evidence of viremia was found for individuals in all age groups (20-30, 40-50, and 61-71 years old), whereas 12 negatively scored brain samples failed to produce similar changes in hamsters observed for > 900 days in the same setting. We suggest that a CJD agent endemically infects humans but only infrequently produces an infectious dementia. Disease expression is likely to be influenced by several host factors in combination with viral variants that have altered neurovirulence.
Plasma arginine and citrulline kinetics in adults given adequate and arginine-free diets.
Authors: Castillo L , Chapman T E , Sanchez M , Yu Y M , Burke J F , Ajami A M , Vogt J , Young V R
Published: May 2006

The fluxes of arginine and citrulline through plasma and the rate of conversion of labeled citrulline to arginine were estimated in two pilot studies (with a total of six adult subjects) and in a dietary study with five healthy young men. These latter subjects received an L-amino acid-based diet that was arginine-rich or arginine-free each for 6 days prior to conduct, on day 7, of an 8-hr (first 3 hr, fast; final 5 hr, fed) primed continuous intravenous infusion protocol using L-[guanidino-13C]arginine, L-[5,5-2H2]citrulline, and L-[5,5,5-2H3]leucine, as tracers. A pilot study indicated that citrulline flux was about 20% higher (P < 0.05) when determined with [ureido-13C]citrulline compared with [2H2]citrulline, indicating recycling of the latter tracer. Mean citrulline fluxes were about 8-11 mumol.kg-1.hr-1 for the various metabolic/diet groups and did not differ significantly between fast and fed states or arginine-rich and arginine-free periods. Arginine fluxes (mean +/- SD) were 60.2 +/- 5.4 and 73.3 +/- 13.9 mumol.kg-1.hr-1 for fast and fed states during the arginine-rich period, respectively, and were significantly lowered (P < 0.05), by 20-40%, during the arginine-free period, especially for the fed state, where this was due largely to reduced entry of dietary arginine into plasma. The conversion of plasma citrulline to arginine approximated 5.5 mumol.kg-1.hr-1 for the various groups and also was unaffected by arginine intake. Thus, endogenous arginine synthesis is not markedly responsive to acute alterations in arginine intake in healthy adults. We propose that arginine homeostasis is achieved largely via modulating arginine intake and/or the net rate of arginine degradation.
Time-resolved EPR studies with DNA photolyase: excited-state FADH0 abstracts an electron from Trp-306 to generate FADH-, the catalytically active form of the cofactor.
Authors: Kim S T , Sancar A , Essenmacher C , Babcock G T
Published: May 2006

Photolyase repairs UV-induced cyclobutane-pyrimidine dimers in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains 5,10-methenyltetrahydrofolate, which functions as the light-harvesting chromophore, and fully reduced flavin adenine dinucleotide (FAD), which functions as the redox catalyst. During enzyme preparation, the flavin is oxidized to FADH0, which is catalytically inert. Illumination of the enzyme with 300- to 600-nm light converts the flavin to the fully reduced form in a reaction that involves photooxidation of an amino acid in the apoenzyme. The results of earlier optical studies had indicated that the redox-active amino acid in this photoactivation process was tryptophan. We have now used time-resolved electron paramagnetic resonance (EPR) spectroscopy to investigate the photoactivation reaction. Excitation of the flavin-radical-containing inactive enzyme produces a spin-polarized radical that we identify by 2H and 15N labeling as originating from a tryptophan residue, confirming the inferences from the optical work. These results and Trp-->Phe replacement by site-directed mutagenesis reveal that flavin radical photoreduction is achieved by electron abstraction from Trp-306 by the excited-state FADH0. Analysis of the hyperfine couplings and spin density distribution deduced from the isotopic-labeling results shows that the product of the light-driven redox chemistry is the Trp-306 cation radical. The results strongly suggest that the active form of photolyase contains FADH- and not FADH2.
Comparison of the P2 specificity pocket in three human histocompatibility antigens: HLA-A*6801, HLA-A*0201, and HLA-B*2705.
Authors: Guo H C , Madden D R , Silver M L , Jardetzky T S , Gorga J C , Strominger J L , Wiley D C
Published: May 2006

Coordinates from x-ray structures of HLA-A*6801, HLA-A*0201, and HLA-B*2705 were analyzed to examine the basis for their selectivity in peptide binding. The pocket that binds the side chain of the peptide's second amino acid residue (P2 residue) shows a preference for Val, Leu, and Arg in these three HLA subtypes, respectively. The Arg-specific pocket of HLA-B*2705 differs markedly from those of HLA-A*0201 and HLA-A*6801, as a result of numerous differences in the side chains that form the pocket's surface. The cause of the specificity differences between HLA-A*0201 and HLA-A*6801 is more subtle and depends both on a change in conformation of pocket residue Val-67 and on a sequence difference at residue 9. The Val-67 conformational change appears to be caused by a shift in the position of the alpha 1-domain alpha-helix relative to the beta-sheet in the cleft and may, in fact, depend on amino acid differences remote from the P2 pocket. Analysis of the stereochemistry of the P2 side chain interacting with its binding pocket permits an estimate to be made of its contribution to the free-energy change of peptide binding.
Isoproterenol stimulates rapid extrusion of sodium from isolated smooth muscle cells.
Authors: Moore E D , Fay F S
Published: May 2006

beta-Agonists cause an inhibition of contractility and a transient stimulation of Na+/K+ pumping in smooth muscle cells of the stomach from the toad Bufo marinus. To determine if the stimulation of Na+/K+ pumping causes changes in intracellular [Na+] ([Na+]i) that might link Na+ pump stimulation to decrease Ca2+ availability for contraction, [Na+]i was measured in these cells with SBFI, a Na(+)-sensitive fluorescent indicator. Basal [Na+]i was 12.8 +/- 4.2 mM (n = 32) and was uniform throughout the cell. In response to isoproterenol, [Na+]i decreased an average of 7.1 +/- 1.1 mM in 3 sec. Since this decrease in [Na+]i could be completely blocked by inhibition of the Na+ pump, or by blockade of the beta-receptor, [Na+]i reduction is the result of occupation of the beta-receptor by isoproterenol and subsequent stimulation of the Na+ pump. 8-Bromoadenosine 3',5'-cyclic monophosphate and forskolin mimicked the effect of isoproterenol, indicating that the sequence of events linking beta-receptor occupation to Na+ pump stimulation most likely includes activation of adenylate cyclase, production of cAMP, and stimulation of cAMP-dependent protein kinase. The decrease in [Na+]i is sufficiently large and fast that it is expected to stimulate turnover of the Na+/Ca2+ exchanger in the Ca2+ extrusion mode, thereby accounting for the observed linkage between stimulation of the Na+/K+ pump and inhibition of contractility in response to beta-adrenergic agonists.
Major histocompatibility complex monomorphism and low levels of DNA fingerprinting variability in a reintroduced and rapidly expanding population of beavers.
Authors: Ellegren H , Hartman G , Johansson M , Andersson L
Published: May 2006

Loss of genetic variation due to population bottlenecks may be a severe threat for the survival of endangered species. Assessment and maintenance of genetic variability are thus crucial for conservation programs related to endangered populations. Scandinavian beavers went through an extensive bottleneck during the last century due to overhunting. In Sweden the species became extirpated but in Norway extinction was avoided by legal protection. Following reintroductions of small numbers of remaining Norwegian animals in 1922-1939, the Swedish population has increased tremendously, now harboring 100,000 animals. We show here that this viable population of beavers possesses extremely low levels of genetic variability at DNA fingerprinting loci and monomorphism at major histocompatibility complex (MHC) class I and class II loci. A similar pattern was also evident among Norwegian beavers but low levels of genetic variability were not a characteristic of the species since Russian conspecifics displayed substantial DNA fingerprinting polymorphism. However, the Russian animals were monomorphic at MHC loci, indicating that the European beaver is exceptional in its low level of MHC variability. The results demonstrate that a conservation program can be successful despite low levels of genetic variation in the founder population.
Cytotoxic and interferon gamma-producing activities of gamma delta T cells in the mouse intestinal epithelium are strain dependent.
Authors: Ishikawa H , Li Y , Abeliovich A , Yamamoto S , Kaufmann S H , Tonegawa S
Published: May 2006

We have analyzed the cytolytic activity of freshly isolated intraepithelial T cells (i-IEL) from the intestines of several different mouse strains in an anti-T-cell receptor monoclonal antibody-mediated redirected lysis assay. The cytolytic activity of gamma delta i-IEL but not that of alpha beta i-IEL was strain dependent. Mouse strains could be divided into high (H), marginal (M), and null (N) strains. The anti-gamma delta T-cell receptor monoclonal antibody-induced interferon gamma production showed the same strain-dependent variability, but the proliferative responses to gamma delta T-cell receptor crosslinking did not show this variability. The N phenotype of gamma delta i-IEL was found to be dominant in (H x N)F1 mice. In radiation bone-marrow chimeras the H/N phenotype was determined by the genotype of the reconstituting bone-marrow-derived cells but was not determined by the genotype of the radioresistant host cells. Analysis of (H x N)F1 backcross animals indicated that at least two genes are involved in determination of the H/N phenotype. One of these genes is major-histocompatibility-complex linked. No difference in the use of the variable region segment of the gamma-chain or delta-chain was seen between the gamma delta i-IEL from H and N strains. Various models that might explain the strain-dependent gamma delta i-IEL phenotypes are discussed.
Alterations in phenotypic biochemical markers in bladder epithelium during tumorigenesis.
Authors: Rao J Y , Hemstreet G P , Hurst R E , Bonner R B , Jones P L , Min K W , Fradet Y
Published: May 2006

Phenotypic biochemical markers of oncogenesis and differentiation were mapped in bladder biopsies to investigate changes that occur in bladder tumorigenesis and to identify markers for increased bladder cancer risk. Touch preparations from biopsy specimens from 30 patients were obtained from tumors, the adjacent bladder epithelium, and random distant bladder epithelium. Markers, including DNA ploidy, epidermal growth factor receptor (EGFR), and oncoproteins, were quantified in individual cells by using quantitative fluorescence image analysis. Cluster analysis revealed the markers fell into three independent groups: (i) G-actin and EGFR; (ii) ploidy, cytology, and p185 (HER-2/neu oncoprotein) (ERBB2); and (iii) p300, a low-grade tumor antigen. Each marker displayed a gradient of abnormality from distant field to adjacent field to tumor. Different patterns for each marker suggested a developmental sequence of bladder cancer oncogenesis; G-actin was altered in 58% of distant biopsies (vs. 0/6 normals, P < 0.001), ploidy and cytology were altered in < 20% of distant fields and approximately 80% of tumors, and the other markers were intermediate. Patterns of EGFR and p185 suggest low-and high-grade tracks diverge early (P < 0.05 by Mann-Whitney U test for EGFR and ANOVA for p185). In conclusion, this study shows that a sequence of phenotypic changes accompanies development and progression of bladder cancers. Biochemical alterations in cells of the bladder field are often detectable before abnormal pathology, and markers previously thought to be limited to tumors were found in the field. The hierarchy of expression may be useful in identifying high-risk patients, assessing completeness of response to therapy, and monitoring and predicting recurrence.
Mechanism of conversion to mucoidy in Pseudomonas aeruginosa infecting cystic fibrosis patients.
Authors: Martin D W , Schurr M J , Mudd M H , Govan J R , Holloway B W , Deretic V
Published: May 2006

Chronic respiratory infections with mucoid Pseudomonas aeruginosa are the leading cause of high mortality and morbidity in cystic fibrosis (CF). The initially colonizing strains are nonmucoid, but in the CF lung they invariably convert into the mucoid, exopolysaccharide alginate-overproducing form causing further deterioration and poor prognosis. Here we report the molecular basis of conversion to mucoidy. The algU gene is required for expression of the key alginate biosynthetic gene algD and encodes a protein homologous to sigma H, an alternative sigma factor regulating sporulation and other post-exponential-phase processes in Bacillus. The algU gene and the negative regulators mucA and mucB constitute the gene cluster controlling conversion to mucoidy. We demonstrate a critical role of mucA in this process based on (i) the presence of frameshift mutations disrupting the mucA coding region in mucoid cells that were absent in nonmucoid parental strains, (ii) genetic complementation of mucA mutations with the mucA+ gene, (iii) allelic replacements with specific mutant mucA genes causing conversion to mucoidy in previously nonmucoid cells, and (iv) detection of identical and additional mucA mutations in clinical mucoid strains isolated from the lungs of CF patients. These results suggest that the switch from the nonmucoid to mucoid state can be caused by inactivation of mucA, resulting in constitutive expression of alginate biosynthetic genes dependent on algU for transcription and that such mutants may be selected in vivo during chronic infections in CF.
What determines the strength of noncovalent association of ligands to proteins in aqueous solution?
Authors: Miyamoto S , Kollman P A
Published: May 2006

Free energy perturbation methods using molecular dynamics have been used to calculate the absolute free energy of association of two ligand-protein complexes. The calculations reproduce the significantly more negative free energy of association of biotin to streptavidin, compared to N-L-acetyltryptophanamide/alpha-chymotrypsin. This difference in free energy of association is due to van der Waals/dispersion effects in the nearly ideally performed cavity that streptavidin presents to biotin, which involves four tryptophan residues.
Transcription factor TFIID recognizes DNA sequences downstream of the TATA element in the Hsp70 heat shock gene.
Authors: Emanuel P A , Gilmour D S
Published: May 2006

The interaction between the Hsp70 heat shock gene promoter and a Drosophila protein complex which contains the TATA-binding protein depends on sequence-specific interactions located in the region downstream of the transcription start site. Immunopurification of the complex through the use of antibodies against the TATA-binding protein reveals that the complex is transcription factor TFIID. Binding assays with the immunopurified TFIID confirm that sequence-specific contacts are made in the region between nucleotides +18 and +33 relative to the transcription start site. These sequence-specific interactions could play key roles in recognition of TATA-containing and TATA-less promoters.
Positional cloning of the hereditary renal carcinoma 3;8 chromosome translocation breakpoint.
Authors: Boldog F L , Gemmill R M , Wilke C M , Glover T W , Nilsson A S , Chandrasekharappa S C , Brown R S , Li F P , Drabkin H A
Published: May 2006

The chromosome (p14.2;q24.1) translocation t(3;8) has been associated with hereditary renal cancer in one family. Based on cytogenetic analyses and loss-of-heterozygosity experiments, the 3p14 region has been independently implicated as harboring a tumor suppressor gene critical to kidney and lung cancer development. The 3p14.2 region also contains FRA3B, the most sensitive fragile site induced by aphidicolin. A chromosome 3 probe, R7K145, derived from a radiation-reduced hybrid was positioned between the t(3;8) breakpoint and an aphidicolin-induced 3p14 breakpoint. A yeast artificial chromosome (YAC) contig containing R7K145 was developed that crossed the aphidicolin-induced breakpoint on its telomeric side. A subsequent chromosome walk identified a YAC that crossed the 3;8 translocation breakpoint. A lambda sublibrary allowed isolation of clones spanning the rearrangement. Unique and evolutionarily conserved DNA sequences were used to screen a kidney cDNA library. We have identified a gene, referred to as HRCA1 (hereditary renal cancer associated 1), that maps immediately adjacent to the breakpoint. On the basis of its chromosomal position, HRCA1 may be a candidate tumor suppressor gene.
Platelet-activating factor induces collagenase expression in corneal epithelial cells.
Authors: Bazan H E , Tao Y , Bazan N G
Published: May 2006

Platelet-activating factor (PAF), a potent lipid mediator involved in inflammatory and immune responses, accumulates rapidly in response to injury in a variety of tissues, including the corneal epithelium. However, the precise role of this compound in the cascade of events following insult has not been defined. Here we examined the effect of PAF on gene expression in the epithelial cells of rabbit corneas in organ culture. We found that incubation with 100 nM methylcarbamoyl PAF, a nonhydrolyzable analog of PAF, produced rapid transient 2.8- and 3.5-fold increases in the expression of c-fos and c-jun, respectively, at 1 hr, followed by increased expression of the collagenase type I gene beginning at 3 hr and peaking at 14-fold by 8 hr. Addition of the protein-synthesis-inhibitor cycloheximide superinduced c-fos and c-jun, strongly potentiating the PAF effect, but inhibited the induction of collagenase type I expression, suggesting the existence of a transcriptional factor linking the two events. BN-50730, a selective antagonist of intracellular PAF-binding sites, blocked the expression of the immediate-early genes as well as the increase in collagenase type I mRNA. Our results suggest that one of the functions of PAF may be to enhance the breakdown of the extracellular matrix as a part of the remodeling process during corneal wound healing after injury. Pathologically, a PAF-induced overproduction of collagenase may be a factor in the development of corneal ulcers, as well as other pathophysiological conditions such as cartilage destruction in arthritis. If so, inhibitors of this signal-transduction pathway may be useful as tools for further investigation and, eventually, as therapeutic agents to treat such disorders.
Histopathogenesis of malignant skin melanoma induced in genetically susceptible transgenic mice.
Authors: Mintz B , Silvers W K , Klein-Szanto A J
Published: May 2006

Animal models of human malignant skin melanoma were created in melanoma-susceptible inbred-strain transgenic mice by grafting skin from donors of high-susceptibility lines to hosts of a low-susceptibility line, thereby overcoming the problem of early death of the more susceptible animals from eye melanomas. As already described [Mintz, B. & Silvers, W. K. (1993) Proc. Natl. Acad. Sci. USA 90, 8817-8821], melanocytes within the grafts selectively proliferated in close proximity to areas of greatest wound healing, presumably in response to mitogenic factors from cells contributing to wound repair. An orderly sequence of externally visible events culminated in malignant melanoma. We examine here the histogenetic concomitants of these changes and find that they define a stepwise sequence strikingly comparable to that leading to human cutaneous melanoma. Moreover, the histological details suggest some of the underlying mechanisms. While the early lesions are first seen in the superficial dermis in the mouse, and in the basal layer of the epidermis in the human, both progress by radial growth followed by vertical growth. Melanocytic hyperplasia resulted in nests of densely melanized fusiform cells which were losing their dendrites. Some discrete lesions in the deep dermis appeared as blue nevi. As radial proliferation advanced, cellular atypia increased and the previously independent melanocytes cohered closely and formed a small solid tumor; the cells were usually then hypomelanotic or amelanotic. Ulceration of tumor through the epidermis occurred early. The tumor mass grew rapidly in the deep dermis and invaded and destroyed subcutaneous tissue and muscle. Primary tumors in the skin were often heterogeneous, with lobules or regions differing in pigmentation or atypia. However, the cells in circulating emboli, or in metastases in lymph nodes and lungs, appeared relatively homogeneous. These genetically uniform transgenic mouse models provide experimental access to the multistage genesis of melanoma.
Monoclonal anti-idiotypic antibodies as functional internal images of enzyme active sites: production of a catalytic antibody with a cholinesterase activity.
Authors: Izadyar L , Friboulet A , Remy M H , Roseto A , Thomas D
Published: May 2006

Monoclonal antibody 9A8 was selected by immunizing mice with AE-2, a monoclonal antibody directed against the active site of acetylcholinesterase. In accordance with the idiotypic network theory, monoclonal anti-idiotypic antibody 9A8 displayed internal-image properties of the original immunogen, the acetylcholinesterase active site. Hydrolysis of acetylthiocholine and related esters of thiocholine by 9A8 follows saturation kinetics and kinetic parameters were determined. The hydrolytic activity is characterized by a lowered kcat value (81 s-1) and an increased Km value (0.6 mM) when compared with the original enzyme. However, the rate acceleration (kcat/kuncat = 4.15 x 10(8) remains higher than for the esterase activities usually described for catalytic antibodies directed against transition-state analogs. The 9A8 activity exhibits a relaxation of specificity toward both substrates and inhibitors. This specificity does not correspond to a known enzymatic activity. The anti-idiotypic approach should be valuable for producing different structural and functional copies of the same enzyme active site. This should allow further insights into structure-activity relationships. Furthermore, use of chemically modified enzymes as immunogens may result in anti-idiotypic antibodies with catalytic activities not found in the native enzymes.
Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells.
Authors: Wu J , Issa J P , Herman J , Bassett D E , Nelkin B D , Baylin S B
Published: May 2006

Abnormal regional increases in DNA methylation, which have potential for causing gene inactivation and chromosomal instability, are consistently found in immortalized and tumorigenic cells. Increased DNA methyltransferase activity, which is also a characteristic of such cells, is a candidate to mediate these abnormal DNA methylation patterns. We now show that, in NIH 3T3 mouse fibroblasts, constitutive overexpression of an exogenous mouse DNA methyltransferase gene results in a marked increase in overall DNA methylation which is accompanied by tumorigenic transformation. These transformation changes can also be elicited by dexamethasone-inducible expression of an exogenous DNA methyltransferase gene. Our findings provide strong evidence that the increase in DNA methyltransferase activity associated with tumor progression could be a key step in carcinogenesis and provide a model system that can be used to further study this possibility.
Structure of hydrated oligonucleotides studied by in situ scanning tunneling microscopy.
Authors: Jing T W , Jeffrey A M , DeRose J A , Lyubchenko Y L , Shlyakhtenko L S , Harrington R E , Appella E , Larsen J , Vaught A , Rekesh D
Published: May 2006

We have used the scanning tunneling microscope (STM) to image several synthetic oligonucleotides adsorbed onto a positively charged Au(111) electrode. The molecules were deposited and imaged in aqueous electrolyte under potential control, a procedure that eliminated the problem of the substrate artifacts that had limited some previous STM studies. Experiments were carried out with two types of single-stranded molecules (11 and 20 bases long) and three types of double-stranded molecules (20 and 61 base pairs and 31 bases with 25 bases paired and 6-base "sticky" ends). The molecules lie along symmetry directions on the reconstructed (23 x square root of 3) gold surface, and length measurements indicate that they adopt simple base-stacked structures. The base stacking distances are, within experimental uncertainty, equal to the 0.33 nm measured for polymeric aggregates of stacked purines by direct imaging in identical conditions. The images show features consistent with helical structures. Double helices have a major-groove periodicity that is consistent with a 36 degrees twist. The single helices appear to be more tightly twisted. A simple tunneling model of STM contrast generates good agreement between measured and calculated images.