bluuebunny/crossref_metadata_2025 huggingface dataset:
Apolipoprotein B mRNA editing is associated with UV crosslinking of proteins to the editing site.
Authors: Navaratnam N , Shah R , Patel D , Fay V , Scott J
Published: May 2006

Apolipoprotein (apo) B100 mRNA undergoes editing of C-6666 to a U residue, which generates a stop-translation codon and defines the carboxyl terminus of apoB48. To aid purification of the editing enzyme we have undertaken UV crosslinking of a 32P-labeled substrate for apoB mRNA editing in vitro to proteins in an enterocyte editing extract. Proteins of 60 (p60) and 43 (p43) kDa, prominent among crosslinking bands, were competed for by unlabeled substrate, but not by nonspecific RNA, and did not crosslink to antisense RNA. Editing in vitro and UV crosslinking were inhibited by NaCl and vanadyl ribonucleoside complexes and by chemical modification of sulfhydryl, imidazolium, and guanidinium groups on the protein. The editing activity copurified predominantly with p60. To define the binding site for p60 on the substrate RNA, a series of scanning and point mutant RNAs, previously used to define nucleotides 6671-6681 as essential for editing, were used in competition studies with wild-type substrate. Results demonstrated that p60 binding is centered on nucleotides 6671-6674. We suggest that p60 contains the RNA-recognition component of the apoB mRNA-editing enzyme.
Spontaneous and radiation-induced renal tumors in the Eker rat model of dominantly inherited cancer.
Authors: Hino O , Klein-Szanto A J , Freed J J , Testa J R , Brown D Q , Vilensky M , Yeung R S , Tartof K D , Knudson A G
Published: May 2006

Hereditary renal carcinoma (RC) in the rat, originally reported by R. Eker in 1954, is an example of a Mendelian dominant predisposition to a specific cancer in an experimental animal. At the histologic level, RCs develop through multiple stages from early preneoplastic lesions (e.g., atypical tubules) to adenomas in virtually all heterozygotes by the age of 1 year. The homozygous mutant condition is lethal at approximately 10 days of fetal life. Ionizing radiation induces additional tumors in a linear dose-response relationship, suggesting that in heterozygotes two events (one inherited, one somatic) are necessary to produce tumors, and that the predisposing gene is a tumor suppressor gene. No genetic linkage has yet been found between the Eker mutation and rat DNA sequences homologous to those in human chromosome 3p, the presumed site of the putative tumor suppressor gene responsible for human RC. Nonrandom loss of rat chromosome 5 in RC-derived cell lines is sometimes associated with homozygous deletion of the interferon gene loci at rat chromosome bands 5q31-q33. Since this locus is not linked with the predisposing inherited gene in the Eker rat, it probably represents a second tumor suppressor gene involved in tumor progression.
Palmitoylation of bovine opsin and its cysteine mutants in COS cells.
Authors: Karnik S S , Ridge K D , Bhattacharya S , Khorana H G
Published: May 2006

Previously, bovine rhodopsin has been shown to be palmitoylated at cysteine residues 322 and 323. Here we report on palmitoylation of bovine opsin in COS-1 cells following expression of the synthetic wild-type opsin gene and several of its cysteine mutants in the presence of [3H]palmitic acid. Two moles of palmitic acid are introduced per wild-type opsin molecule in thioester linkages. Palmitoylation is abolished when both Cys-322 and Cys-323 are replaced by serine residues. Replacement of Cys-322 by serine prevents palmitoylation at Cys-323, whereas replacement of the latter with serine allows palmitoylation at Cys-322. Opsin mutants that evidently do not contain a Cys-110/Cys-187 disulfide bond and presumably remain in the endoplasmic reticulum are not palmitoylated. Replacement of Cys-140 or Cys-185 reduces the extent of palmitoylation of the opsin. Lack of palmitoylation at Cys-322 and/or Cys-323 does not affect 11-cis-retinal binding, absorption maximum or extinction coefficient of the chromophore, the bleaching behavior of the chromophore, or the light-dependent binding and activation of transducin. Mutants containing serine substitutions at Cys-140 or Cys-323 showed reduced light-dependent phosphorylation by rhodopsin kinase.
Hemopoietic stem-cell compartment of the SCID mouse: double-exponential survival curve after gamma irradiation.
Authors: Taniguchi S , Hirabayashi Y , Inoue T , Kanisawa M , Sasaki H , Komatsu K , Mori K J
Published: May 2006

It has been reported that SCID (severe combined immunodeficiency, scid/scid) mice are more radiosensitive than normal mice. In the present studies, graded doses of radiation were given to bone marrow cells from SCID mice, and double-exponential survival curves were observed for day-9 and day-12 colony-forming units in the spleen (CFU-S). Single-exponential curves were found for SCID CFU in in vitro assays for granulocyte/macrophage-CFUs and erythroid burst-forming units, as reported elsewhere. Since the size of this more resistant fraction seems to decrease with stem-cell maturation, the finding implies that this fraction is a primitive subpopulation of the stem-cell compartment. The mean lethal dose (D0), however, of this less sensitive SCID CFU-S is much less than the D0 of regular CFU-S in normal littermates. Spleen colonies produced by SCID bone marrow were relatively small and abortive. The size of these colonies decreased nearly exponentially with increasing doses of radiation. These colonies were believed to be produced by this less sensitive fraction of the stem cells, which carried residual injuries. The colonies produced by the sensitive fraction have disappeared, being killed by a relatively low dose of radiation. This observation might account for the high lymphomagenesis arising from primitive hemopoietic stem cells in SCID mice, because the smallness of the colonies suggests that there is unrepaired or misrepaired damage. Furthermore, this less sensitive fraction might be a source of the "leaky" change of T and B cells, possibly due to the induction of an equivocal repair system which appears in the later stages of life in the SCID mice.
How clonal are bacteria?
Authors: Smith J M , Smith N H , O'Rourke M , Spratt B G
Published: May 2006

Data from multilocus enzyme electrophoresis of bacterial populations were analyzed using a statistical test designed to detect associations between genes at different loci. Some species (e.g., Salmonella) were found to be clonal at all levels of analysis. At the other extreme, Neisseria gonorrhoeae is panmictic, with random association between loci. Two intermediate types of population structure were also found. Neisseria meningitidis displays what we have called an "epidemic" structure. There is significant association between loci, but this arises only because of the recent, explosive, increase in particular electrophoretic types; when this effect is eliminated the population is found to be effectively panmictic. In contrast, linkage disequilibrium in a population of Rhizobium meliloti exists because the sample consisted of two genetically isolated divisions, often fixed for different alleles: within each division association between loci was almost random. The method of analysis is appropriate whenever there is doubt about the extent of genetic recombination between members of a population. To illustrate this we analyzed data on protozoan parasites and again found panmictic, epidemic, and clonal population structures.
Association of casein kinase 2 with nuclear chromatin in relation to androgenic regulation of rat prostate.
Authors: Ahmed K , Yenice S , Davis A , Goueli S A
Published: May 2006

Casein kinase 2 (CK-2) is a ubiquitous messenger-independent protein serine/threonine kinase that has been implicated in growth control. We have studied the activity and subcellular location of CK-2 in adult rat ventral prostate in relation to androgen withdrawal and administration. Androgen deprivation by castration results in a faster decline in CK-2 activity associated with prostatic nuclei than that in the cytosol. Nuclear CK-2 associated with chromatin is reduced at an even greater rate than that in the total nucleus. Reversal of these events by administration of a single dose of 5 alpha-dihydrotestosterone to adult rats castrated 144 hr previously was accompanied by a differential early enhancement of chromatin-associated CK-2 activity, with a concomitant decrease in the CK-2 activity present in the cytosol. Changes in the nuclear CK-2 activity correlated with the immunostainable enzyme protein in the nucleus. We propose that androgens evoke translocation of CK-2 from the cytoplasm to the nucleus (nucleoplasm) where its enhanced association with the chromatin constituents takes place. Conversely, withdrawal of circulating androgens due to castration evokes a dissociation of CK-2 from chromatin and eventual translocation of nucleoplasmic CK-2 to the cytoplasm. Modulations in the association of CK-2 with nuclear chromatin may represent an important mechanism of post-transcriptional regulation of nuclear CK-2 in relation to androgen action in the prostate.
A point mutation of the Na+/H+ exchanger gene (NHE1) and amplification of the mutated allele confer amiloride resistance upon chronic acidosis.
Authors: Counillon L , Franchi A , Pouysségur J
Published: May 2006

The diuretic drug amiloride and its 5-amino substitute N5-methyl-N5-propylamiloride (MPA) are potent inhibitors of the growth factor-activatable Na+/H+ exchanger isoform 1 (NHE1). This inhibitor competes with Na+, presumably by interacting with the ion-transport site of the NHE molecule. As an approach to identify this site, we previously reported the use of a specific H(+)-killing selection technique for isolating amiloride-resistant variants of Chinese hamster lung fibroblasts. After long-term selection, two variants, AR40 and AR300, 100- and 1000-fold, respectively, resistant to MPA, were isolated. By comparing NHE1 cDNA sequences of parental and two variant cell lines, we show that the 1000-fold resistance to MPA results from two sequential genetic events. (i) In one AR40 allele a point mutation, Phe-167--> Leu, occurs in the middle of the fourth putative transmembrane segment of NHE1. Producing this mutant protein from human NHE1 cDNA by site-directed mutagenesis increased the Ki for MPA by 30-fold, as seen in AR300 cells. (ii) An approximately 10-fold amplification of the mutated allele, which contributes to the acquired MPA resistance, accounts for the Vmax increase. Mutating a close residue, Phe-165--> Tyr, increased by 40-fold the Ki for amiloride and reduced Na+ transport rate 3- to 4-fold, indicating that we have identified a critical domain of the NHE molecule that controls amiloride binding and Na+ transport. Interestingly, the epithelial amiloride-resistant NHE isoforms that occurred naturally possess some of the amino acid substitutions described here.
Copper-controllable gene expression system for whole plants.
Authors: Mett V L , Lochhead L P , Reynolds P H
Published: May 2006

We describe a system for gene expression in plants based on the regulation mechanism of the yeast metallothionein (MT) gene. The system consists of two elements: (i) the yeast ace1 (activating copper-MT expression) gene encoding a transcription factor under control of the cauliflower mosaic virus (CaMV) 35S RNA promoter, and (ii) a gene of interest under control of a chimeric promoter consisting of the 90-base-pair domain A of the CaMV 35S RNA promoter linked to the ACE1 transcription factor-binding site. At elevated copper ion concentrations, the ACE1 protein changes conformation, binds to, and activates transcription from the chimeric promoter. To test the functioning of the system in plants, a construct containing the beta-glucuronidase (GUS) reporter gene under control of the chimeric promoter was prepared, and transgenic tobacco plants were produced. It was shown that GUS activity in the leaves of transgenic plants increased up to 50-fold, either after addition of 50 microM CuSO4 to the nutrient solution or after application of 0.5 microM CuSO4 to the plants in a foliar spray. This GUS expression was repressed after the removal of copper ions. The results show that the activity of the described chimeric promoter directly depends on copper ion concentration and that this system can be used in experiments that demand precise timing of expression.
A gene for a low density lipoprotein receptor-related protein in the nematode Caenorhabditis elegans.
Authors: Yochem J , Greenwald I
Published: May 2006

A >23-kb gene that encodes a large integral membrane protein with a predicted structure similar to that of the low density lipoprotein (LDL) receptor-related protein (LRP) of mammals has been isolated and sequenced from the free-living nematode Caenorhabditis elegans. The 4753-amino acid predicted C. elegans product shares a nearly identical number and arrangement of amino acid sequence motifs with human LRP, and several exons of the C. elegans LRP gene correspond to exons of related parts of the human LDL receptor gene. The existence of an apparent homolog of LRP in C. elegans offers the possibility of genetic analysis of the in vivo roles of LRP and of the relationship between protein structure and function in a simple model organism.
Translational regulation by the interferon-induced double-stranded-RNA-activated 68-kDa protein kinase.
Authors: Barber G N , Wambach M , Wong M L , Dever T E , Hinnebusch A G , Katze M G
Published: May 2006

Activation of the interferon-inducible 68-kDa protein kinase (referred to as P68) by double-stranded RNA catalyzes phosphorylation of the alpha subunit of eukaryotic protein synthesis initiation factor 2. We have analyzed the transient expression of mutant and wild-type kinase molecules in transfected COS cells to examine the effects of the kinase on gene expression in the absence of other interferon-induced gene products. The wild-type P68 kinase was expressed inefficiently whereas a catalytically inactive P68 was expressed at 30- to 40-fold higher levels. Protein stability measurements and primer-extension analysis of human kinase-specific mRNA levels provided evidence that kinase expression was regulated at the level of mRNA translation. Further, cotransfection experiments revealed that the domain II catalytically inactive mutant could stimulate reporter gene protein synthesis in a transdominant manner. We also examined the expression of mutants with deletions in the N-terminal double-stranded RNA binding domains and found that a kinase construct lacking aa 156-243 was expressed at levels comparable to the wild type whereas a P68 construct lacking aa 91-243 was expressed at levels 70-fold higher. Both the inactive domain II P68 mutant and the deletion mutant lacking aa 91-243 were less inhibitory to growth in yeast due to the reduced ability to phosphorylate initiation factor 2 alpha in vivo. In conclusion we have demonstrated that the P68 kinase can regulate mRNA translation primarily of its own mRNA and to a lesser extent of a heterologous mRNA and that this regulation is notably affected by mutations in either the catalytic or N-terminal regulatory domains.
Postcopulatory sexual selection in an arctiid moth (Utetheisa ornatrix).
Authors: LaMunyon C W , Eisner T
Published: May 2006

The offspring of twice-mated female Utetheisa ornatrix show low incidence of mixed paternity. Most progeny are sired exclusively by one male, the larger one, irrespective of parental age, male pyrrolizidine alkaloid content, mating order, between-mating interval, or duration of copulation. Data are presented suggesting that the female herself may exert control over the process by which one set of sperm is utilized at the expense of the other. Evidence for such postcopulatory female choice of sperm had not previously been obtained for an insect. Promiscuity provides the female with a means for accruing nuptial gifts (nutrients, defensive alkaloids). Sperm selection provides her with the option of utilizing sperm from larger, potentially more fit, males.
A mutation in reverse transcriptase of bis(heteroaryl)piperazine-resistant human immunodeficiency virus type 1 that confers increased sensitivity to other nonnucleoside inhibitors.
Authors: Dueweke T J , Pushkarskaya T , Poppe S M , Swaney S M , Zhao J Q , Chen I S , Stevenson M , Tarpley W G
Published: May 2006

Several nonnucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been described, including Nevirapine, thiobenzimidazolone (TIBO) derivatives, pyridinone derivatives such as L-697,661, and the bis(heteroaryl)piperazines (BHAPs). HIV-1 resistant to L-697,661 or Nevirapine emerges rapidly in infected patients treated with these drugs, and the resistance is caused primarily by substitutions at amino acids 181 and 103 of RT that also confer cross resistance to the other nonnucleoside inhibitors. We describe derivation and characterization of two BHAP-resistant HIV-1 variants that differ from this pattern of cross resistance. With both variants, HIV-1 resistance to BHAP RT inhibitors was caused by a RT mutation that results in a proline-to-leucine substitution at amino acid 236 (P236L). Rather than conferring cross resistance to other RT inhibitors, this substitution sensitized RT 7- to 10-fold to Nevirapine, TIBO R82913, and L-697,661 without influencing sensitivity to nucleoside analogue RT inhibitors. This sensitization caused by P236L was also observed in cell culture with BHAP-resistant HIV-1. The effects of the P236L RT substitution suggest that emergence of BHAP-resistant virus in vivo could produce a viral population sensitized to inhibition by these other nonnucleoside RT inhibitors.
Abundances of light elements.
Authors: Pagel B E
Published: May 2006

Recent developments in the study of abundances of light elements and their relevance to cosmological nucleosynthesis are briefly reviewed. The simplest model, based on standard cosmology and particle physics and assuming homogeneous baryon density at the relevant times, continues to stand up well.
Dissipative processes in galaxy formation.
Authors: Silk J
Published: May 2006

A galaxy commences its life in a diffuse gas cloud that evolves into a predominantly stellar aggregation. Considerable dissipation of gravitational binding energy occurs during this transition. I review here the dissipative processes that determine the critical scales of luminous galaxies and the generation of their morphology. The universal scaling relations for spirals and ellipticals are shown to be sensitive to the history of star formation. Semiphenomenological expressions are given for star-formation rates in protogalaxies and in starbursts. Implications are described for elliptical galaxy formation and for the evolution of disk galaxies.
Echo-planar magnetic resonance imaging studies of frontal cortex activation during word generation in humans.
Authors: McCarthy G , Blamire A M , Rothman D L , Gruetter R , Shulman R G
Published: May 2006

Nine subjects were studied by high-speed magnetic resonance imaging while performing language-based tasks. Subjects were asked either to repeat or to generate verbs associated with nouns read by an experimenter while magnetic resonance images were obtained of the left inferior frontal lobe. The echo-planar imaging sequence was used with a gradient echo time of 70 ms to give an apparent transverse relaxation time weighting (T2* that is sensitive to local hemoglobin levels. Images were acquired every 3 s (repetition time) in series of 32. In plane resolution was 6 x 4.5 mm and slice thickness was 10 mm. An increase in signal accompanied performance of the tasks, with significantly more activation for verb generation than for repeating. The activation effect occurred within 3 s after task onset and could be observed in single images from individual subjects. The primary focus of activation appeared in gray matter along a sulcus anterior to the lateral sulcus that included the anterior insula, Brodmann's area 47, and extending to area 10. Little or no activation of this region was found for a passive listening, covert generation, or mouth-movement control tasks. Significant activation was also found for a homologous region in the right frontal cortex but not for control regions in calcarine cortex. These results are consistent with prior studies that have used positron emission tomography imaging with 15O-labeled water as a blood flow tracer.
p53 gene mutations and protein accumulation in human ovarian cancer.
Authors: Kupryjańczyk J , Thor A D , Beauchamp R , Merritt V , Edgerton S M , Bell D A , Yandell D W
Published: May 2006

Mutations of the p53 gene on chromosome 17p are a common genetic change in the malignant progression of many cancers. We have analyzed 38 malignant tumors of ovarian or peritoneal müllerian type for evidence of p53 variations at either the DNA or protein levels. Genetic studies were based on single-strand conformation polymorphism analysis and DNA sequencing of exons 2 through 11 of the p53 gene; mutations were detected in 79% of the tumors. These data show a statistically significant association between mutations at C.G pairs and a history of estrogen therapy. Two of 20 patients whose normal tissue could be studied carried germ-line mutations of p53. Immunohistochemical analysis of the p53 protein was carried out using monoclonal antibody PAb1801. Ninety-six percent of the missense mutations were associated with abnormal accumulation of p53 protein, but nonsense mutations, a splicing mutation, and most deletions did not result in p53 protein accumulation. A statistically significant association between p53 protein accumulation in poorly differentiated stage III serous carcinomas and small primary tumor size at diagnosis was found, perhaps suggesting that p53 protein accumulation accelerates the metastatic spread from a primary tumor. Overall, our findings indicate that alterations of p53 play a major role in ovarian cancer, including predisposition to the disease in some patients, and suggest a possible mechanism for somatic mutations leading to this cancer.
Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases.
Authors: Henzel W J , Billeci T M , Stults J T , Wong S C , Grimley C , Watanabe C
Published: May 2006

A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database. The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from two-dimensional gels. Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest. A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses. To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed. This method facilitates simultaneous processing of a large number of two-dimensional gel spots. The method was applied to a two-dimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane. Ten randomly chosen spots were analyzed. With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences. All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot. One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identity.
Mutation of Asp74 of the rat angiotensin II receptor confers changes in antagonist affinities and abolishes G-protein coupling.
Authors: Bihoreau C , Monnot C , Davies E , Teutsch B , Bernstein K E , Corvol P , Clauser E
Published: May 2006

Aspartic acid in the second transmembrane domain is a highly conserved amino acid among the G protein-coupled receptors and is functionally important for agonist binding and G-protein coupling in beta 2-adrenergic and luteinizing hormone receptors. To determine whether this aspartic acid is also involved in the function of the rat vascular angiotensin II receptor subtype 1 (AT1a), Asp74 was replaced either by asparagine or by glutamic acid. When expressed in CHO cells, the two mutants and the wild-type receptor displayed similarly high affinities for the agonist [Sar1, Tyr(125I)4]angiotensin II [where Sar is sarcosine and Tyr(125I) is monoiodinated tyrosine] and the other agonists: ([Sar1]angiotensin II > angiotensin II > angiotensin III >> angiotensin I). However, the Asn74 mutant shows striking differences in its affinity for some antagonists when compared with the wild-type receptor: the affinity for DUP753 was decreased 10-fold, whereas it was increased 6-fold for [Sar1,Ala8]angiotensin II and 20-fold for CGP42112A. These pharmacological changes were associated with a major defect in transmembrane signaling, since angiotensin II was unable to stimulate inositol phosphate production and increase cytosolic Ca2+ concentration through the two mutated receptors, whereas a clear dose-dependent stimulation was observed in cells expressing the wild-type receptor. Angiotensin II was able to promote DNA synthesis through the wild type but not through the mutated receptors. These data indicate that the conserved Asp74 residue of the AT1a receptor is important for the binding of angiotensin II antagonists and is essential for the transmembrane signaling cascade.
On the regeneration of the actin-myosin power stroke in contracting muscle.
Authors: Chen Y D , Brenner B
Published: May 2006

The transient behavior of muscle in double-or multiple-step length perturbations [Lombardi, V., Piazzesi, G. & Linari, M. (1992) Nature (London) 355, 638-641] is simulated with a "conventional" cross-bridge model, which has been reported [Eisenberg, E., Hill, T. L. & Chen, Y. (1980) Biophys. J. 29, 195-227] to account for many mechanical, as well as biochemical, muscle data. The quick recovery of tension after double- or multiple-length perturbations was calculated for the model without any readjustment of its original parameters. The regeneration rate of the quick tension recovery of the model is fast and comparable to that measured experimentally by Lombardi et al. For multiple-step "stair-case"-type length releases, the tension response reaches a steady-state shape after three or four steps, and the average ATP turnover is much slower than the regeneration of the quick tension recovery. Our simulation shows that the experimental findings of Lombardi et al. can easily be reproduced by this simple conventional cross-bridge model, in which the completion of one work-producing power stroke is coupled to the hydrolysis of one ATP molecule. Thus, to account for the data of Lombardi et al., there is no need to assume that cross-bridges can execute multiple power strokes per ATPase cycle, although cross-bridges may well be able to do so. The mechanism that underlies the fast regeneration of the quick tension recovery in the conventional model used here is discussed.
Phosphorylation of Ser-42 and Ser-59 in the N-terminal region of the tyrosine kinase p56lck.
Authors: Winkler D G , Park I , Kim T , Payne N S , Walsh C T , Strominger J L , Shin J
Published: May 2006

Ser-42 and Ser-59 in the N-terminal region have been identified as the major phorbol ester-induced phosphorylation sites of p56lck. Phosphorylation of Ser-59 results in a gel shift from 56 kDa to 61 kDa. Simultaneous phosphorylation of Ser-42 and Ser-59 results in a further gel shift to 63 kDa. In vitro kinase assays show that Ser-59 can be uniquely phosphorylated by mitogen-activated protein kinase and that Ser-42 can be phosphorylated by either protein kinase A or protein kinase C.
Direction of microtubule movement is an intrinsic property of the motor domains of kinesin heavy chain and Drosophila ncd protein.
Authors: Stewart R J , Thaler J P , Goldstein L S
Published: May 2006

The kinesin heavy chain and the ncd (non-claret disjunctional) gene product of Drosophila are microtubule-associated motor proteins related by sequence similarity within an approximately 340-aa domain. Despite the sequence similarity, the kinesin heavy chain and ncd protein move in opposite directions on microtubules. To investigate the molecular basis for direction of movement, we created a series of truncated kinesin heavy chain and ncd proteins. We found that the conserved domain of both proteins has microtubule motor activity, although the efficiency with which ATP hydrolysis is coupled to microtubule movement declines dramatically with increasing truncation. Further, the direction of movement is intrinsic to the conserved motor domains, rather than being a consequence of domain organization or adjacent sequences.
Control of dorsoventral patterning of somitic derivatives by notochord and floor plate.
Authors: Pourquié O , Coltey M , Teillet M A , Ordahl C , Le Douarin N M
Published: May 2006

We have examined the effect of implantation of a supernumerary notochord or floor plate on dorsoventral somitic organization. We show that notochord and floor plate are able to inhibit the differentiation of the dorsal somitic derivatives--i.e., axial muscles and dermis--thus converting the entire somite into cartilage, which normally arises only from its ventral part. We infer from these results that the dorsoventral patterning of somitic derivatives is controlled by signals provided by ventral axial structures.
Prodrug activation via catalytic antibodies.
Authors: Miyashita H , Karaki Y , Kikuchi M , Fujii I
Published: May 2006

Prodrug activation via antibodies was examined by using the antibiotic chloramphenicol as a model drug. Based on the conformational change between substrate and product, this antibody-catalyzed reaction was designed to prevent product inhibition, thus enhancing turnover. Antibodies elicited against a phosphonate transition-state analogue were found to catalyze hydrolysis of a nonbioactive chloramphenicol monoester as a prodrug at a significantly higher rate above the uncatalyzed background reaction to regenerate chloramphenicol as a parent molecule. The antibody-catalyzed prodrug activation was tested by the paper-disc diffusion method using Bacillus subtilis as an indicator strain. The antibody 6D9 catalyzes the reaction with multiple turnover to generate enough chloramphenicol to inhibit bacterial growth, as indicated by a clear inhibitory zone after incubation with monoester. Using the same method, no inhibition was detected by incubation of either the monoester or the antibody alone. This result reveals that only the antibody hydrolytically activates the monoester, which can be expected to be a suitable prodrug, as it is resistant to the action of bacterial hydrolytic enzymes. The approach in this study demonstrates the use of catalytic antibody technology in medicine and may be applicable to drugs with undesirable effects, particularly in the field of cancer therapy.
Hydrogenase of the hyperthermophile Pyrococcus furiosus is an elemental sulfur reductase or sulfhydrogenase: evidence for a sulfur-reducing hydrogenase ancestor.
Authors: Ma K , Schicho R N , Kelly R M , Adams M W
Published: May 2006

Microorganisms growing near and above 100 degrees C have recently been discovered near shallow and deep sea hydrothermal vents. Most are obligately dependent upon the reduction of elemental sulfur (S0) to hydrogen sulfide (H2S) for optimal growth, even though S0 reduction readily occurs abiotically at their growth temperatures. The sulfur reductase activity of the anaerobic archaeon Pyrococcus furiosus, which grows optimally at 100 degrees C by a metabolism that produces H2S if S0 is present, was found in the cytoplasm. It was purified anaerobically and was shown to be identical to the hydrogenase that had been previously purified from this organism. Both S0 and polysulfide served as substrates for H2S production, and the S0 reduction activity but not the H2-oxidation activity was enhanced by the redox protein rubredoxin. The H2-oxidizing and S0-reduction activities of the enzyme also showed different responses to pH, temperature, and inhibitors. This bifunctional "sulfhydrogenase" enzyme can, therefore, dispose of the excess reductant generated during fermentation using either protons or polysulfides as the electron acceptor. In addition, purified hydrogenases from both hyperthermophilic and mesophilic representatives of the archaeal and bacterial domains were shown to reduce S0 to H2S. It is suggested that the function of some form of ancestral hydrogenase was S0 reduction rather than, or in addition to, the reduction of protons.
Gaucher disease as a paradigm of current issues regarding single gene mutations of humans.
Authors: Beutler E
Published: May 2006

Gaucher disease is a glycolytic storage disease caused by a deficiency in activity of the catabolic enzyme glucocerebrosidase. Over 35 different mutations have been documented, including missense and nonsense point mutations, splicing mutations, deletions and insertions, a fusion gene, and examples of gene conversion. Gaucher disease is most common in the Ashkenazi Jewish population, in which just five of the mutations in this population account for 98% of the disease-producing alleles. Each of these mutations is found in the context of a single haplotype, a finding consistent with a single origin of each mutation. Although it is clear that these mutations provide a selective advantage in the Jewish population and thus constitute a balanced polymorphism, the nature of the advantage is unknown. Gaucher disease can be treated symptomatically, by administration of the missing enzyme, by allogeneic bone marrow transplantation, and potentially by gene transfer into hematopoietic stem cells. Increasing understanding of this disease has, as in other genetic disorders, created a host of social and ethical dilemmas regarding matters such as the cost of treatment for rare diseases and the advantages and disadvantages of population-targeted genetic screening.
Model system for DNA replication of a plasmid DNA containing the autonomously replicating sequence from Saccharomyces cerevisiae.
Authors: Ishimi Y , Matsumoto K
Published: May 2006

A negatively supercoiled plasmid DNA containing autonomously replicating sequence (ARS) 1 from Saccharomyces cerevisiae was replicated with the proteins required for simian virus 40 DNA replication. The proteins included simian virus 40 large tumor antigen as a DNA helicase, DNA polymerase alpha.primase, and the multisubunit human single-stranded DNA-binding protein from HeLa cells; DNA gyrase from Escherichia coli, which relaxes positive but not negative supercoils, was included as a "swivelase." DNA replication started from the ARS region, proceeded bidirectionally with the synthesis of leading and lagging strands, and resulted in the synthesis of up to 10% of the input DNA in 1 h. The addition of HeLa DNA topoisomerase I, which relaxes both positive and negative supercoils, to this system inhibited DNA replication, suggesting that negative supercoiling of the template DNA is required for initiation. These results suggest that DNA replication starts from the ARS region where the DNA duplex is unwound by torsional stress; this unwound region can be recognized by a DNA helicase with the assistance of the multisubunit human single-stranded DNA-binding protein.
A site-specific endonuclease encoded by a typical archaeal intron.
Authors: Dalgaard J Z , Garrett R A , Belfort M
Published: May 2006

The protein encoded by the archaeal intron in the 23S rRNA gene of the hyperthermophile Desulfurococcus mobilis is a double-strand DNase that, like group I intron homing endonucleases, is capable of cleaving an intronless allele of the gene. This enzyme, I-Dmo I, is unusual among the intron endonucleases in that it is thermostable and is expressed only from linear and cyclized intron species and not from the precursor RNA. However, in analogy to its eukaryotic counterparts, but unlike the bacteriophage enzymes, I-Dmo I makes a staggered double-strand cut that generates 4-nt 3' extensions. Additionally, although the archaeal and group I introns have entirely different structural properties and splicing pathways, I-Dmo I shares sequence similarity, in the form of the LAGLI-DADG motif, with group I intron endonucleases of eukaryotes. These observations support the independent evolutionary origin of endonucleases and intron core elements and are consistent with the invasive potential of endonuclease genes.
Genetic evidence for replication enhancement from a distance.
Authors: Gennaro M L
Published: May 2006

Rolling circle replication of the Staphylococcus aureus plasmid pT181 requires interaction of the RepC initiator protein with the origin of replication (the ori site). A second site named cmp, which is distant from ori, is thought to stimulate replication, since a mutant pT181 plasmid lacking cmp cannot coexist with a cmp+ wild-type plasmid. Second-site mutations compensating for the loss of cmp were shown to map in repC. The compensatory mutations produced RepC proteins that, unlike the wild-type, either failed to discriminate between cmp+ and cmp- plasmids or preferred cmp- to cmp+ plasmids. These studies demonstrate that cmp stimulates the interaction of the replication initiator protein with the origin and therefore enhances DNA replication from a distance.
Localized 1H NMR measurements of gamma-aminobutyric acid in human brain in vivo.
Authors: Rothman D L , Petroff O A , Behar K L , Mattson R H
Published: May 2006

Localized 1H NMR spectroscopy in conjunction with J editing was used to measure the concentration of gamma-aminobutyric acid (GABA) in the occipital lobe of four control human volunteers and four epileptic volunteers who were receiving the drug vigabatrin. The GABA concentration measured in four nonepileptic subjects was 1.1 +/- 0.1 mumol/cm3 of brain, which is in good agreement with previous values measured in surgically removed human cortex. A dose-dependent elevation of GABA concentration was measured in patients receiving the GABA transaminase inhibitor vigabatrin, with the maximum measured level of 3.7 mumol/cm3 of brain measured at the highest dose (6 g per day) studied. 1H NMR measurements of GABA in those patients receiving GABA-elevating agents such as vigabatrin will be of importance in establishing the relationship between seizure suppression and the concentration of brain GABA.
Independently gated multiple substates of an epithelial chloride-channel protein.
Authors: Finn A L , Dillard M , Gaido M
Published: May 2006

We have purified a protein from Necturus maculosus gallbladder cells that forms chloride channels in an artificial membrane. The same protein apparently can form channels that are highly selective for chloride but can have conductances varying from 9 to about 150 pS. The high-conductance channels are blocked by the monoclonal antibody used to purify the protein, but this antibody has no effect on the 9-pS channels. The observation that gating of the low- and high-conductance states is independent and that the antibody affects only the latter has implications regarding the control of chloride conductance in cell membranes and the different types of channels described in those cells.
Diverse peroxisome proliferator-activated receptors bind to the peroxisome proliferator-responsive elements of the rat hydratase/dehydrogenase and fatty acyl-CoA oxidase genes but differentially induce expression.
Authors: Marcus S L , Miyata K S , Zhang B , Subramani S , Rachubinski R A , Capone J P
Published: May 2006

The ability of peroxisome proliferator-activated receptors (PPARs) to induce expression of a reporter gene linked to a peroxisome proliferator-responsive element (PPRE) from either the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase gene or acyl-CoA oxidase [acyl-CoA:oxygen 2-oxidoreductase, EC 1.3.3.6] gene was examined by transient transfection assays in COS cells. Mouse and rat PPARs, as well as Xenopus PPAR alpha (xPPAR alpha) could induce expression of a reporter gene linked to the hydratase/dehydrogenase PPRE in the presence of the peroxisome proliferators ciprofibrate or Wy-14,643, whereas xPPAR beta and xPPAR gamma were ineffective. A similar induction of expression of a reporter gene linked to the acyl-CoA oxidase PPRE was observed with all PPARs except xPPAR beta. Extracts from cells transfected with PPAR-encoding genes contained factors that bound to both PPREs. In vitro synthesized PPARs could interact weakly with both PPREs; however, binding of each PPAR to both PPREs was significantly increased by the addition of COS cell nuclear extracts, demonstrating that efficient PPAR/DNA binding requires auxiliary cofactors. One cofactor was identified as the 9-cis-retinoic acid receptor, RXR alpha (retinoid X receptor alpha). Cooperative DNA binding and heteromerization between RXR alpha and each of the PPARs could be seen with both PPREs. Our results demonstrate that PPAR/PPRE binding and cooperativity with RXR alpha (and other cofactors) are obligatory but not necessarily sufficient for peroxisome proliferator-dependent transcription induction and that distinct PPREs can selectively mediate induction by particular PPARs.
A tumor necrosis factor-responsive long-term-culture-initiating cell is associated with the stromal layer of mouse long-term bone marrow cultures.
Authors: Rogers J A , Berman J W
Published: May 2006

Long-term bone marrow cultures provide a model for the study of hematopoiesis. Both an intact, adherent stromal layer and hematopoietic stem cells are necessary components in these cultures. Mycophenolic acid treatment of mouse long-term bone marrow cultures depletes them of all assayable hematopoietic precursors. The residual stromal cells are functional and support hematopoiesis if new progenitor cells are supplied. We now show that these mycophenolic acid-treated stromal cell cultures contain cells capable of hematopoietic differentiation without the addition of new progenitors. When treated with tumor necrosis factor alpha (20-200 units/ml), the apparently pure stromal cultures undergo an intense burst of hematopoietic activity. After 4 days such cultures contain approximately 2 x 10(6) hematopoietic cells and, by 1 week, they are indistinguishable from control long-term cultures that were not treated with mycophenolic acid. These results suggest that the stromal cultures either contain hematopoietic stem cells that are maintained quiescent and mycophenolic acid-resistant, perhaps by intimate contact with the stroma, or contain adherent cells that can be induced to differentiate into hematopoietic stem cells. These stem cells are primitive, in that they are capable of multilineage development in the long-term cultures, but are unable to form spleen colonies or myeloid colonies in semisolid medium. These data demonstrate that the adherent fraction of cultured bone marrow contains very primitive hematopoietic cells and that tumor necrosis factor alpha activates their proliferation and differentiation. They also suggest a strategy for obtaining the earliest progenitors free of other, more mature cell types.
The androgen-dependent C4-Slp gene is driven by a constitutively competent promoter.
Authors: Miyagoe Y , Georgatsou E , Varin-Blank N , Meo T
Published: May 2006

The androgen-dependent liver protein Slp, together with its constitutively expressed closely related isoform C4, provides a model to address the question of which minimal alteration in DNA can shut off the expression of a gene in a manner reversible by testosterone or by trans-acting mutations. Previous work indicated that sequences located at -1.9, -0.45, and -0.25 kb from the transcription start site of the C4-Slp gene played a critical role in determining its unusual functional divergence from C4. Now, using quantitatively and qualitatively controlled transfection assays in HepG2 human hepatoma cells and mouse L fibroblasts, we have observed that the C4-Slp promoter is fully effective and unhindered by upstream sequences and that the C4 promoter has a consistent albeit modest superiority. The determinant of this nearly 2-fold difference does not coincide with the sites highlighted in previous studies but lies within the most cap-site-proximal nucleotides, at positions -189 to +48. We have also established conditions for cell-free transcription of C4 and C4-Slp from plasmid and cosmid templates by using nuclear extracts from rat and mouse liver of both sexes as well as from L cells. At variance with the rat alpha 2u-globulin gene, C4-Slp transcription in vitro does not require male factors, for it is expressed as efficiently as C4 by all nuclear extracts. Further, the minimal promoter sequences required to direct accurate initiation extend not farther than the most proximal 19 nucleotides. Because L cells efficiently express transfected cosmids covering the whole C4 gene or C4/C4-Slp recombinants, as well as plasmids carrying the C4-Slp promoter, but fail to express the full C4-Slp gene, we favor a model in which the expression of the gene is modulated intragenically.
Association of the high-affinity receptor for IgG (Fc gamma RI) with the gamma subunit of the IgE receptor.
Authors: Ernst L K , Duchemin A M , Anderson C L
Published: May 2006

How receptors for the Fc portion of IgG antibodies (Fc gamma R) trigger a variety of immune functions by clustering upon engagement of ligand is largely unknown. Of the three distinct classes of human Fc gamma R, only Fc gamma RIIIA has been shown to associate with a potential signal-generating subunit, either the gamma chain or the zeta chain. With these studies we show that Fc gamma RI, the high-affinity receptor for IgG, also is found in association with gamma chain. This association can be demonstrated by using anti-Fc gamma RI antibodies, Fc gamma RI ligand, and anti-gamma-chain antibodies that coadsorb the proteins from detergent lysates of Fc gamma RI-expressing cells. The association of Fc gamma RI and gamma chain can be reconstituted by cotransfection of cDNAs for both proteins into COS cells.
Pbx1 is converted into a transcriptional activator upon acquiring the N-terminal region of E2A in pre-B-cell acute lymphoblastoid leukemia.
Authors: Van Dijk M A , Voorhoeve P M , Murre C
Published: May 2006

Twenty-five percent of human pediatric pre-B-cell acute lymphoblastic leukemias (ALLs) are characterized by the t(1;19)(q23;p13.3) chromosomal translocation. This translocation joins the 5' region of the E2A gene to the 3' region of the Pbx1 gene. The protein encoded by this chimeric gene contains the N-terminal transcriptional activation domain of E2A fused to the C-terminal region of Pbx1, which contains a putative homeodomain. Here we show that the Pbx1 homeodomain preferentially binds the sequence ATCAATCAA. We further show that promoters containing Pbx1-binding sites are activated by the chimeric E2A-Pbx1 protein but not by Pbx1. These results indicate that the t(1;19) translocation converts a nonactivating DNA-binding protein into a potent transcriptional activator, suggesting an unusual mechanism for oncogenic transformation.