bluuebunny/crossref_metadata_2025 huggingface dataset:
Activity of the purified mutagenesis proteins UmuC, UmuD', and RecA in replicative bypass of an abasic DNA lesion by DNA polymerase III.
Authors: Rajagopalan M , Lu C , Woodgate R , O'Donnell M , Goodman M F , Echols H
Published: May 2006

The introduction of a replication-inhibiting lesion into the DNA of Escherichia coli generates the induced, multigene SOS response. One component of the SOS response is a marked increase in mutation rate, dependent on RecA protein and the induced mutagenesis proteins UmuC and UmuD. A variety of previous indirect approaches have indicated that SOS mutagenesis results from replicative bypass of the DNA lesion by DNA polymerase III (pol III) holoenzyme in a reaction mediated by RecA, UmuC, and a processed form of UmuD termed UmuD'. To study the biochemistry of SOS mutagenesis, we have reconstituted replicative bypass with a defined in vitro system containing purified protein and a DNA substrate with a single abasic DNA lesion. The replicative bypass reaction requires pol III, UmuC, UmuD', and RecA. The nonprocessed UmuD protein does not replace UmuD' but inhibits the bypass activity of UmuD', perhaps by sequestering UmuD' in a heterodimer. Our experiments demonstrate directly that the UmuC-UmuD' complex and RecA act to rescue an otherwise stalled pol III holoenzyme at a replication-blocking DNA lesion.
Assembly of a hetero-oligomeric membrane protein complex.
Authors: Traxler B , Beckwith J
Published: May 2006

The maltose transporter of Escherichia coli is a hetero-oligomeric complex located in the cytoplasmic membrane of the cell. The in vivo assembly of this complex has been examined by using an assay based on the proteolytic sensitivity of one of its components, MalF. Immediately after synthesis and insertion into the membrane, MalF is sensitive to exogenously added proteases. In a time- and complex assembly-dependent fashion, MalF becomes protease resistant. Using this assay, we show that MalF is inserted into the membrane independently of other components of the transport complex. The assembly of the maltose transport complex occurs subsequently from a pool of freely diffusing protein in the membrane. This assembly process is efficient and occurs with rapid kinetics.
Association of efferent neurons to the compartmental architecture of the superior colliculus.
Authors: Illing R B
Published: May 2006

The superior colliculus is a layered structure in the mammalian midbrain serving multimodal sensorimotor integration. Its intermediate layers are characterized by a compartmental architecture. These compartments are apparent through the clustering of terminals of major collicular afferents, which in many instances match the heterogeneous distribution of tissue components such as acetylcholinesterase, choline acetyltransferase, substance P, and parvalbumin. The present study was undertaken to determine whether efferent cells observe this compartmental architecture. It was found that subpopulations of both descending and ascending collicular efferents originate from perikarya situated in characteristic positions relative to the collicular compartments defined by elevated acetylcholinesterase activity and that their dendrites appear to be specifically coordinated with the heterogeneous environment. With the specific interlocking of afferent and efferent neurons through spatially distinguished neural networks, the compartmental architecture apparently constitutes an essential element for the determination of information flow in the superior colliculus.
Signal-mediated nuclear transport in simian virus 40-transformed cells is regulated by large tumor antigen.
Authors: Feldherr C M , Lanford R E , Akin D
Published: May 2006

Transformation of cultured cells with simian virus 40 (SV40), or transfection with the early region of the SV40 genome, causes a significant increase in both the rate of signal-mediated nuclear transport and the functional size of the transport channels (located in the pore complexes). By microinjecting purified large tumor (T) antigen into the cytoplasm of murine BALB/c 3T3 cells, we have demonstrated that this protein alone can account for the increase in transport capacity. The T antigen-dependent changes can be partially inhibited by cycloheximide and require a functional nuclear localization sequence. Although necessary, the nuclear localization sequence by itself cannot produce the observed variations in nuclear permeability and presumably function in a "helper" capacity, in association with another, as yet unidentified domain.
XY sex reversal associated with a deletion 5' to the SRY "HMG box" in the testis-determining region.
Authors: McElreavy K , Vilain E , Abbas N , Costa J M , Souleyreau N , Kucheria K , Boucekkine C , Thibaud E , Brauner R , Flamant F
Published: May 2006

The human testis-determining factor resides within a 35-kilobase (kb) region of the Y chromosome immediately adjacent to the pseudoautosomal boundary. A candidate gene for human sex determination (SRY) was isolated in this region. Here, we describe a study of 25 cases of XY females with pure gonadal dysgenesis for mutations on the Y chromosome short arm, including SRY. Southern blotting revealed a sex-reversed female harboring a deletion extending from approximately 8 kb from the pseudoautosomal boundary of the Y chromosome to at least 33 kb and no more than 60 kb upstream, toward the centromere. The deletion begins no more than 1.8 kb upstream from the first ATG of the SRY open reading frame present in the clone pY53.3. To our knowledge, no mutation has been described previously outside the SRY "HMG box" on the short arm of the Y chromosome, which is associated with sex reversal. Since the 5' extent of the SRY transcriptional unit has not been defined, the deletion may remove upstream exons of SRY and/or transcriptional regulatory motifs, either situation resulting in lack of testicular development. It cannot be formally excluded that the mutation removes a second locus, independent of SRY, that is critical for sex determination. Denaturant gradient gel electrophoresis analysis of the SRY open reading frame in the remaining 24 cases revealed de novo single base-pair transitions in the SRY conserved domain in 4 cases.
Hydrolytic elimination of a mutagenic nucleotide, 8-oxodGTP, by human 18-kilodalton protein: sanitization of nucleotide pool.
Authors: Mo J Y , Maki H , Sekiguchi M
Published: May 2006

8-Oxoguanine nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies. Once 8-oxodGTP is formed in the cellular nucleotide pool, this mutagenic nucleotide is incorporated into DNA and would cause transversion mutations. The MutT protein of Escherichia coli possesses enzyme activity to hydrolyze 8-oxodGTP to the corresponding nucleoside monophosphate and thus may be responsible for preventing the occurrence of such mutations. Here we show that the human cell has an enzyme specifically hydrolyzing 8-oxodGTP in a fashion similar to that seen with MutT protein. The human 8-oxodGTPase has been found in cell-free extracts from Jurkat cells and purified > 400-fold. Analyses by gel filtration and gel electrophoresis revealed that the molecular mass of the native form of human 8-oxodGTPase is 18 kDa. Mg2+ ion is required for the enzyme action and the optimum pH for the reaction is pH 8.0. The enzyme hydrolyzes 8-oxodGTP to 8-oxodGMP with a Km value of 12.5 microM. dGTP and dATP are also degraded to dGMP and dAMP, respectively, with Km values 70 times greater than that for 8-oxodGTP. dTTP and dCTP are not hydrolyzed. These properties of the human 8-oxodGTPase are similar to those observed with the E. coli MutT protein, suggesting that the function of protecting the genetic information from the threat of endogenous oxygen radicals is widely distributed in organisms.
Characterization of the precursor for Manduca sexta diuretic hormone Mas-DH.
Authors: Digan M E , Roberts D N , Enderlin F E , Woodworth A R , Kramer S J
Published: May 2006

We have isolated a cDNA clone encoding a precursor form of the diuretic hormone from the tobacco hornworm Manduca sexta (Mas-DH). Translation of the cDNA revealed a 138-amino acid precursor consisting of the Mas-DH amino acid sequence bounded by dibasic amino acid processing sites, a putative signal sequence, and additional peptide sequence on either side of the Mas-DH coding sequence. The region of the precursor upstream of the mature Mas-DH sequence shows limited (28%) homology to the cryptic region of the ovine corticotropin-releasing factor precursor. The Mas-DH RNA is 1.5-1.6 kilobases long; it is present in both the heads and bodies of adult and larval insects. In prewandering fifth stadium larvae, Mas-DH mRNA is expressed in brain, nerve cord, gut, and Malpighian tubules, but not in the fat body. There is a single genomic copy of the Mas-DH gene; the message is multiply spliced.
Casimir energy for dielectrics: spherical geometry.
Authors: Schwinger J
Published: May 2006

An expression is produced for the Casimir energy of a dielectric medium with a spherically symmetrical dielectric discontinuity. Tests based on dielectric and geometrical limits are successful.
A putative ATP-dependent RNA helicase involved in Saccharomyces cerevisiae ribosome assembly.
Authors: Ripmaster T L , Vaughn G P , Woolford J L
Published: May 2006

We have isolated a cold-sensitive mutant of Saccharomyces cerevisiae in which there is a deficit of 60S ribosomal subunits. Cold sensitivity and the assembly defect are recessive and cosegregate, defining a single essential gene that we designated DRS1 (deficiency of ribosomal subunits). The wild-type DRS1 gene was cloned by complementation of the cold-sensitive phenotype of drs1. Sequence analysis reveals a high degree of similarity to a family of proteins that are thought to function as ATP-dependent RNA helicases. Pulse-chase analysis of ribosomal RNA synthesis and processing indicates that the drs1 mutant accumulates the 27S precursor of the mature 25S rRNA. These results suggest that, as in pre-mRNA splicing, RNA helicase activities are involved in ribosomal RNA processing.
Control of gal transcription through DNA looping: inhibition of the initial transcribing complex.
Authors: Choy H E , Adhya S
Published: May 2006

Involvement of DNA looping between two spatially separated gal operators, OE and OI, in repression of the gal operon has been demonstrated in vivo. An in vitro transcription assay using a minicircle DNA containing the gal promoter region with lac operators was employed to elucidate the molecular mechanism of repression. Wild-type lac repressors (LacI+ protein molecules), which are capable of associating into a tetramer and forming a DNA loop, repressed transcription from promoter sites P1 and P2, whereas a non-looping lac repressor mutant (LacI(adi)) failed to show normal repression of both of the gal promoters. Thus a DNA loop is also required for repression of transcription in vitro. Repression mediated by DNA looping resulted in the inhibition of the synthesis of complete as well as aborted transcripts, demonstrating that the repressive action was on the formation or activity of the initial transcribing complex. Under similar conditions, the gal repressor (GalR protein) did not repress the gal promoters effectively, apparently because it failed to loop DNA containing gal operators in the purified system. The component(s) or conditions that aid GalR in DNA looping remain to be identified.
Probucol inhibits neointimal thickening and macrophage accumulation after balloon injury in the cholesterol-fed rabbit.
Authors: Ferns G A , Forster L , Stewart-Lee A , Konneh M , Nourooz-Zadeh J , Anggård E E
Published: May 2006

Restenosis is a frequent long-term complication after balloon angioplasty. Although smooth muscle cells form the major constituent of the occluding lesion, macrophage-derived foam cells are usually also present in high abundance. The latter have the potential to accelerate the rate of reocclusion because they elaborate many potent cytokines and growth factors, which may act to either recruit cells into the neointima or cause neointimal cell proliferation. Macrophage-derived foam-cell formation depends upon the uptake of modified low density lipoprotein via a scavenger receptor-mediated pathway. Foam-cell formation is accompanied by the release of smooth muscle cell mitogens and chemoattractants. We have examined the effects of probucol, a lipid-soluble antioxidant, in the balloon-catheterized carotid artery of the cholesterol-fed rabbit to evaluate the importance of oxidative processes in restenosis. After 5 weeks, serum cholesterol levels were 32% lower (P < 0.05) in rabbits fed 1% probucol with 2% cholesterol, compared with those receiving cholesterol alone. Probucol inhibited neointimal macrophage accumulation by 68% (P < 0.001), reduced absolute intimal size by 51% (P < 0.05), and reduced the intima/media thickness ratio by 51%. These inhibitory effects were directly related to serum probucol concentrations and appeared to be unrelated to probucol's hypocholesterolemic activity. These data suggest that reactive oxygen species may be involved in the intimal response to injury and that antioxidants, such as probucol, may be therapeutically useful as inhibitors of restenosis.
Drug design by machine learning: the use of inductive logic programming to model the structure-activity relationships of trimethoprim analogues binding to dihydrofolate reductase.
Authors: King R D , Muggleton S , Lewis R A , Sternberg M J
Published: May 2006

The machine learning program GOLEM from the field of inductive logic programming was applied to the drug design problem of modeling structure-activity relationships. The training data for the program were 44 trimethoprim analogues and their observed inhibition of Escherichia coli dihydrofolate reductase. A further 11 compounds were used as unseen test data. GOLEM obtained rules that were statistically more accurate on the training data and also better on the test data than a Hansch linear regression model. Importantly machine learning yields understandable rules that characterized the chemistry of favored inhibitors in terms of polarity, flexibility, and hydrogen-bonding character. These rules agree with the stereochemistry of the interaction observed crystallographically.
Major histocompatibility complex class I-specific and -restricted killing of beta 2-microglobulin-deficient cells by CD8+ cytotoxic T lymphocytes.
Authors: Glas R , Franksson L , Ohlén C , Höglund P , Koller B , Ljunggren H G , Kärre K
Published: May 2006

Cytotoxic T lymphocytes (CTLs) recognize major histocompatibility complex (MHC) class I molecules, normally composed of a heavy chain, a beta 2-microglobulin (beta 2m), and peptide antigens. beta 2m is considered essential for the assembly and intracellular transport of MHC class I molecules as well as their peptide presentation to CTLs. Contrary to this dogma, we now report the generation of allospecific and restricted CD8+ and TCR alpha beta+ CTLs (where TCR is T-cell receptor) capable of killing beta 2m-deficient cells. Such CTLs were obtained by priming mice with live allogeneic beta 2m- spleen cells or mutant lymphoma cells producing MHC class I protein but no detectable beta 2m. Although both beta 2m- and beta 2m-expressing lymphoma cells were rejected in allogeneic mice, only the former were efficient inducers of CTLs recognizing beta 2m- cells. These CTLs were MHC class I (H-2Kb or Db)-specific and CD8-dependent and did not require serum as a source of external beta 2m in the culture. They could be induced across major and minor histocompatibility barriers. The H-2-restricted CTLs generated in the latter case failed to kill the antigen-processing-deficient target RMA-S cells. The results show that MHC class I heavy chains in beta 2m- cells can be transported to the cell surface and act as antigens or antigen-presenting molecules to allospecific and MHC-restricted CTLs.
Positive supercoiling of DNA greatly diminishes mRNA synthesis in yeast.
Authors: Gartenberg M R , Wang J C
Published: May 2006

In Saccharomyces cerevisiae cells harboring a GAL1 promoter-linked beta-galactosidase gene, the simultaneous expression of Escherichia coli DNA topoisomerase I and inactivation of yeast DNA topoisomerases I and II reduces the cellular level of beta-galactosidase to an undetectable level. Analysis of intracellular mRNA level and the density of RNA polymerase along DNA indicates that this reduction is due to the suppression of transcription and that both plasmid-borne and chromosomally located genes are affected. These results are interpreted in terms of inhibition of transcription in vivo due to positive supercoiling of the DNA template: preferential removal of transcription-generated negative supercoils by E. coli DNA topoisomerase I in the absence of both yeast DNA topoisomerases I and II results in the accumulation of positive supercoils in intracellular DNA. In normal prokaryotic or eukaryotic cells, accumulation of positive supercoils is presumably avoided through the balanced actions of DNA topoisomerases.
Methodologic aspects of a population pharmacodynamic model for cognitive effects in Alzheimer patients treated with tacrine.
Authors: Holford N H , Peace K E
Published: May 2006

Tacrine is a cholinesterase inhibitor with activity in the central nervous system originally marketed for the reversal of competitive neuromuscular blockade. Because a marked reduction in cholinergic neurons is a hallmark of brain changes in Alzheimer disease, tacrine has been studied in two placebo-controlled clinical trials of patients with probable Alzheimer disease. Standard analysis of variance (ANOVA) and analysis of covariance (ANCOVA) have shown a difference between the tacrine group and the placebo group in terms of the cognitive component of the Alzheimer disease assessment scale at the end of the placebo-controlled phase. Due to limitations of ANOVA and ANCOVA, only a selected group of patients could be analyzed by those methods. A population pharmacodynamic model has been developed that allows the use of all observations from one or more trials to be combined. It can incorporate any sequence of active or placebo treatments and account for carryover effects of both placebo and active drug. The time courses of active or placebo treatment response and the development of tolerance to active drug or placebo can be defined. The model describes disease progression without treatment, the placebo effect, and the effect of tacrine as a function of daily dose. Placebo effect and active drug effects are modeled by effect site concentration components.
Identification of a functionally important sequence in the C terminus of the interferon-gamma receptor.
Authors: Farrar M A , Campbell J D , Schreiber R D
Published: May 2006

We have previously shown that the intracellular domain of the interferon-gamma (IFN-gamma) receptor plays an obligate role in receptor-mediated signal transduction. Moreover, we have specifically identified two regions within the human IFN-gamma receptor's intracellular domain required for functional activity: the membrane-proximal 48 amino acids required for both functional activity and receptor-mediated ligand internalization and the C-terminal 39 amino acids required exclusively for biologic response induction. Herein we report the identification of the 3 amino acids within the C-terminal region of the receptor that are obligatorily required for receptor function. By using a set of overlapping truncation mutants, the minimal functional sequence within the C-terminal region was localized to residues 434-444 (APTSFGYD-KPH). By mutating each individual residue within this sequence to alanine, three residues (Tyr-440, Asp-441, and His-444) were identified as being critical for IFN-gamma-dependent (i) upregulation of major histocompatibility complex class I proteins, (ii) activation of the IFN regulatory factor 1 gene, and (iii) stimulation of cells to produce nitric oxide. The more conservative Tyr-440-->Phe substitution also resulted in a nonfunctional receptor. Subsequent mutational analysis of all five of the IFN-gamma receptor's intracellular tyrosine residues revealed that Tyr-440 was the sole tyrosine required for receptor activity. These results thus identify a unique sequence in the IFN-gamma receptor that is required for initiation of IFN-gamma-dependent biologic responses and highlight the importance of the hydroxyl side chain of Tyr-440 in this process.
Ligand-dependent conformational changes in the progesterone receptor are necessary for events that follow DNA binding.
Authors: Allan G F , Tsai S Y , Tsai M J , O'Malley B W
Published: May 2006

Hormones and antihormones induce related, but distinct, conformational changes in the progesterone receptor [Allan, G. F., Leng, X., Tsai, S. Y., Weigel, N. L., Edwards, D. P., Tsai, M.-J. & O'Malley, B. W. (1992) J. Biol. Chem. 267, 19513-19520]. In both cases the conformational change precedes the dissociation of heat shock proteins and binding to DNA. We have now investigated the steps in hormone action which are dependent upon this conformational change. We show that in the absence of ligand, monoclonal antibodies directed against different regions of the progesterone receptor can induce high-affinity binding to its response element in vitro. This antibody-induced DNA binding is presumably facilitated by enhanced dimerization of receptor monomers. However, antibodies do not induce the hormone-specific conformational change in the progesterone receptor and do not induce in vitro transcription by the receptor. In contrast, the antiprogestin ZK98299, which inhibits receptor binding to DNA, fully induces the antihormone-specific conformational change. Thus, our data imply that steroids induce a conformational change in their receptors which is necessary for events subsequent to DNA binding, most likely for transactivation.
Molecular cloning of a gene encoding a seed-specific protein with nutritionally balanced amino acid composition from Amaranthus.
Authors: Raina A , Datta A
Published: May 2006

An albumin with a well-balanced amino acid composition and high levels of the essential amino acids was purified to homogeneity from the mature seeds of Amaranthus hypochondriacus. The amino acid composition of this protein is comparable to the World Health Organization recommended values for a highly nutritional protein. The protein is a 35-kDa monomer with four isoforms that can be separated by chromatofocusing. Antibodies raised against one of the isoforms, AmA1, cross-reacted with the other three isoforms. Affinity-purified AmA1 antibodies were used to isolate cDNA clones from a developing-seed expression library. The six immunopositive recombinants obtained were found to be related. The cDNA of the largest clone (1.2 kilobases) has a single major open reading frame corresponding to a 304-amino acid polypeptide. The clone was confirmed by hybrid-selected translation and immunoprecipitation. The size of the immunoprecipitated product was identical to the mature protein. Analysis of RNA and protein in developing seeds showed that AmA1 is synthesized during early embryogenesis, reaching a maximum by midmaturation. No RNA was detected in 1-day-old seedlings although the protein showed delayed breakdown on germination. Expression of the AmA1 gene was found to be seed-specific, as no protein or RNA was detected in other plant tissues.
Tumor necrosis factor alpha stimulates expression of adenovirus early region 3 proteins: implications for viral persistence.
Authors: Körner H , Fritzsche U , Burgert H G
Published: May 2006

Human adenovirus (Ad) can cause persistent infections in humans. Early region 3 (E3) of the virus appears to be implicated in this phenomenon. This transcription unit encodes proteins that interfere in various ways with host cell functions, including (i) cell-surface expression of histocompatibility class I antigens (HLA), (ii) cell-surface expression of the epidermal growth factor receptor (EGF-R), and (iii) the biological activity of tumor necrosis factor alpha (TNF-alpha). We transfected the human cell line 293 with the entire E3 region of Ad2 and investigated the influence of the cytokines TNF-alpha and interferon gamma (IFN-gamma) on cell-surface expression of HLA class I and the EGF-R. Whereas IFN-gamma treatment induced expression of HLA to some extent but not that of the EGF-R, TNF-alpha treatment augmented the reduction of these cell-surface molecules. Subsequent studies on the mechanism of this effect showed a TNF-alpha-dependent upregulation of E3 protein (E3/19K) and mRNA. The significance of this phenomenon was confirmed in infection experiments. A dramatic increase in the amount of E3/19K, even after short induction with low doses of TNF-alpha could be demonstrated. The study provides evidence for an interaction between the immune system and Ad in which the virus takes advantage of an immune mediator to escape immunosurveillance of the host.
A 69-kDa RNA-binding protein from Xenopus oocytes recognizes a common motif in two vegetally localized maternal mRNAs.
Authors: Schwartz S P , Aisenthal L , Elisha Z , Oberman F , Yisraeli J K
Published: May 2006

Vg1 mRNA, a maternal message encoding a member of the transforming growth factor beta superfamily, undergoes localization to the vegetal cortex of Xenopus laevis oocytes during a narrow period of oogenesis. A 340-nucleotide sequence has been identified in Vg1 RNA that directs its vegetal localization [Mowry, K. L. & Melton, D. A. (1992) Science 255, 991-994]. To understand how cis- and trans-acting factors are involved in Vg1 mRNA localization, we have looked for specific interactions in vitro between oocyte proteins and Vg1 mRNA. S100 extracts of late-stage oocytes contain a protein-binding activity that protects specific regions of labeled Vg1 mRNA from degradation by RNase T1. The use of different regions of Vg1 RNA in competition reactions reveals two binding sites, both in the first half of the 3' untranslated region of Vg1 message. UV crosslinking predominantly labels a 69-kDa protein; saturation analysis and competitor studies indicate that this protein binds with a high affinity to the down-stream site, which corresponds to the 340-nucleotide vegetal localization sequence. Binding to this region is inhibited by another vegetally localized message, transforming growth factor beta 5 but is not inhibited by an animally localized RNA, An2. These data indicate that vegetally localized mRNAs share a binding motif that helps them achieve their intracellular distribution through specific RNA-protein interactions.
Borocaptate sodium: a potential boron delivery compound for boron neutron capture therapy evaluated in dogs with spontaneous intracranial tumors.
Authors: Kraft S L , Gavin P R , DeHaan C E , Leathers C W , Bauer W F , Miller D L , Dorn R V
Published: May 2006

Borocaptate sodium (Na2B12H11SH) is a boron-carrying compound under consideration for use in boron neutron capture therapy. The biodistribution of boron from borocaptate sodium administration will partly determine boron neutron capture therapy efficacy and normal tissue radiation tolerance. The biodistribution of boron was determined in 30 dogs with spontaneous intracranial tumors at 2, 6, or 12 hr after intravenous borocaptate sodium infusion. Blood and tissue boron concentrations were measured using inductively coupled plasma atomic emission spectroscopy. Mean tumor boron concentration (mean +/- standard error) was 35.9 +/- 4.6 (n = 15), 22.5 +/- 6.0 (n = 9), and 7.0 +/- 1.1 micrograms of boron per g (n = 6) at 2, 6, and 12 hr, respectively, after borocaptate sodium infusion. Peritumor boron concentrations were elevated above that of normal brain in half of the dogs. Normal brain boron concentration (mean +/- standard error) was 4.0 +/- 0.5, 2.0 +/- 0.4, and 2.0 +/- 0.3 micrograms of boron per g at 2, 6, and 12 hr after infusion, respectively. Some cranial and systemic tissues, and blood, had high boron concentration relative to tumor tissue. Geometric dose sparing should partly offset these relatively high normal tissue and blood concentrations. Borocaptate sodium biodistribution is favorable because tumor boron concentrations of recommended magnitude for boron neutron capture therapy were obtained and there was a high tumor-to-normal brain boron concentration ratio.
Recycling and phosphorylation of eukaryotic initiation factor 2 on 60S subunits of 80S initiation complexes and polysomes.
Authors: Ramaiah K V , Dhindsa R S , Chen J J , London I M , Levin D
Published: May 2006

Phosphorylation of the alpha-subunit (38 kDa) of eukaryotic initiation factor 2 (eIF-2 alpha) regulates initiation of protein synthesis in eukaryotic cells. This phosphorylation is enhanced in cycloheximide-treated heme-deficient reticulocyte lysates in which polysomes are maintained. In early heme deficiency prior to polysome disaggregation, eIF-2(alpha P) accumulates primarily on the 60S subunits of polysomes. Further, isolated polysomes contain eIF-2 alpha that is efficiently phosphorylated in vitro by heme-regulated inhibitor (HRI). Immunoblot analysis of eIF-2 distribution in sucrose gradients of actively protein-synthesizing lysates indicates that eIF-2 is distributed at low levels throughout the polysome profiles. These findings suggest that polysome-bound eIF-2 alpha is a target of HRI under physiological conditions. The presence of eIF-2 on the 60S subunits of polysomes is incompatible with the conventional model in which eIF-2 is recycled during the joining of the 48S preinitiation complex and the 60S subunit to form the 80S initiation complex. A modified model is presented with emphasis on the translocation of eIF-2 from the 40S ribosomal subunit of the 48S preinitiation complex (eIF-2.GTP.Met-tRNA(f).40S.mRNA) to the 60S subunit of the 80S initiation complex.
Chi sequence protects against RecBCD degradation of DNA in vivo.
Authors: Dabert P , Ehrlich S D , Gruss A
Published: May 2006

RecBCD is a multifunctional enzyme involved in DNA degradation and homologous recombination. It also produces an endonucleolytic cleavage near properly oriented chi sites (5'-GCTGGTGG-3'). Plasmids are not known to be affected by either RecBCD enzyme or the presence of a chi site. We report here that plasmids that replicate by a rolling circle mechanism accumulate large amounts of high molecular weight linear multimers (HMW), either if they contain a chi site or if RecBCD is absent. An in vivo inducible system for rolling circle replication was constructed to study RecBCD and its interactions with chi. Results show that (i) HMW accumulation is chi orientation dependent, and (ii) a succession of chi sites prevents degradation of HMW by RecBCD enzyme. These results demonstrate chi activity in plasmids. The rolling circle mechanism produces a sigma structure during plasmid replication; we propose that the double-stranded DNA tail of this sigma form allows RecBCD entry; the tail is degraded unless it is protected by a chi site. By analogy, a principal role of chi in the survival of lambda red-gam- mutants in wild-type strains may be to protect rolling circle concatemers (in late replication) from degradation by RecBCD.
Activity of the Hsp70 chaperone complex--DnaK, DnaJ, and GrpE--in initiating phage lambda DNA replication by sequestering and releasing lambda P protein.
Authors: Hoffmann H J , Lyman S K , Lu C , Petit M A , Echols H
Published: May 2006

Initiation of DNA replication by phage lambda requires the ordered assembly and disassembly of a specialized nucleoprotein structure at the origin of replication. In the disassembly pathway, a set of Escherichia coli heat shock proteins termed the Hsp70 complex--DnaK, DnaJ, and GrpE--act with ATP to release lambda P protein from the nucleo-protein complex, freeing the DnaB helicase for its DNA-unwinding reaction. To investigate the mechanism of the release reaction, we have examined the interaction between P and the three heat shock proteins by glycerol gradient sedimentation and gel electrophoresis. We have discovered an ATP-dependent ternary interaction between P, DnaK, and DnaJ; this P.DnaK.DnaJ complex is dissociated by GrpE. We have concluded that the function of the Hsp70 complex in sequestering and releasing P protein provides for the critical step in the disassembly pathway. Based on our data and other work on protein folding, the formation of the P.DnaK.DnaJ complex might involve a conformational shift to a folding intermediate of P.
Genomic structure of the human caldesmon gene.
Authors: Hayashi K , Yano H , Hashida T , Takeuchi R , Takeda O , Asada K , Takahashi E , Kato I , Sobue K
Published: May 2006

The high molecular weight caldesmon (h-CaD) is predominantly expressed in smooth muscles, whereas the low molecular weight caldesmon (l-CaD) is widely distributed in nonmuscle tissues and cells. The changes in CaD isoform expression are closely correlated with the phenotypic modulation of smooth muscle cells. During a search for isoform diversity of human CaDs, l-CaD cDNAs were cloned from HeLa S3 cells. HeLa l-CaD I is composed of 558 amino acids, whereas 26 amino acids (residues 202-227 for HeLa l-CaD I) are deleted in HeLa l-CaD II. The short amino-terminal sequence of HeLa l-CaDs is different from that of fibroblast (WI-38) l-CaD II and human aorta h-CaD. We have also identified WI-38 l-CaD I, which contains a 26-amino acid insertion relative to WI-38 l-CaD II. To reveal the molecular events of the expressional regulation of the CaD isoforms, the genomic structure of the human CaD gene was determined. The human CaD gene is composed of 14 exons and was mapped to a single locus, 7q33-q34. The 26-amino acid insertion is encoded in exon 4 and is specifically spliced in the mRNAs for both h-CaD and l-CaDs I. Exon 3 is the exon that encodes the central repeating domain specific to h-CaD (residues 208-436) together with the common domain in all CaD (residues 73-207 for h-CaD and WI-38 l-CaDs, and residues 68-201 for HeLa l-CaDs). The regulation of h- and l-CaD expression is thought to depend on selection of the two 5' splice sites within exon 3. Thus, the change in expression between l-CaD and h-CaD might be caused by this splicing pathway.
Regulation of intrasteric inhibition of the multifunctional calcium/calmodulin-dependent protein kinase.
Authors: Cruzalegui F H , Kapiloff M S , Morfin J P , Kemp B E , Rosenfeld M G , Means A R
Published: May 2006

A regulatory region involved in both autoinhibition and calmodulin (CaM) binding has previously been identified in the multifunctional Ca2+/CaM-dependent protein kinase (CaM kinase II). We have tested the role of various segments of the regulatory region in autoinhibition by the analysis of a series of truncation, substitution, and deletion mutants of the CaM kinase II alpha subunit (CaM kinase II alpha). Unexpectedly, the sequence Lys-Lys-Phe-Asn at positions 291-294, adjacent to the CaM binding domain, was found to be sufficient to maintain an inhibited state in a truncated form of the kinase. However, these residues are not essential in the context of the full-length protein, indicating the importance of additional residues from the overlapping CaM binding domain. We propose here a molecular model for CaM kinase II alpha based on the three-dimensional structure of the cAPK-PKI-(5-24) (protein kinase inhibitor fragment) complex. It is predicted from this model that autoinhibition is of the pseudosubstrate variety and that autophosphorylation of Thr-286 could occur by an intersubunit reaction in the holoenzyme complex.
c-Jun represses the human insulin promoter activity that depends on multiple cAMP response elements.
Authors: Inagaki N , Maekawa T , Sudo T , Ishii S , Seino Y , Imura H
Published: May 2006

Glucose is known to increase the cAMP concentration in pancreatic beta cells. To determine the mechanism by which cAMP augments insulin gene expression, we first identified the cAMP response elements (CREs) of the human insulin gene. In DNase I footprint analysis, the bacterially synthesized CRE-binding protein, CRE-BP1, protected four sites: two sites in the region upstream from the insulin core promoter, one site in the first exon, and one site in the first intron. To examine the roles of those four sites, we constructed a series of DNA plasmids in which the wild-type and mutant insulin promoters were linked to the chloramphenicol acetyl-transferase gene. Studies of the transcriptional activity of these plasmids after transfection into hamster insulinoma (HIT) cells showed that these four sites contributed additively to the cAMP inducibility of the insulin promoter. Surprisingly, the c-jun protooncogene product (c-Jun) repressed the cAMP-induced activity of the insulin promoter in a cotransfection assay with the c-Jun expression plasmid. Northern blot analysis demonstrated that the level of c-jun mRNA was dramatically increased by glucose deprivation in HIT cells. These results suggest that glucose may regulate expression of the human insulin gene through multiple CREs and c-Jun.
Tamoxifen prevents induction of hepatic neoplasia by zeranol, an estrogenic food contaminant.
Authors: Coe J E , Ishak K G , Ward J M , Ross M J
Published: May 2006

Zeranol (alpha-zearalanol) is a beta-resorcylic acid lactone (RAL) that has estrogen activity. It is synthesized by molds and is difficult to avoid in human food products. We tested the ability of this mycoestrogen to damage the liver of the Armenian hamster, a rodent that is especially sensitive to hepatotoxic effects of exogenous estrogens. Zeranol induced acute hepatotoxicity and, subsequently, hepatic carcinogenesis; both effects were blocked by tamoxifen, suggesting estrogen receptor mediation. Because zeranol is acting alone as a primary initiator of hepatic neoplasms, this model provides an unusual opportunity to study the pathogenesis of estrogen-initiated tumorigenesis.
Activation of Src-like protein-tyrosine kinase Lyn and its association with phosphatidylinositol 3-kinase upon B-cell antigen receptor-mediated signaling.
Authors: Yamanashi Y , Fukui Y , Wongsasant B , Kinoshita Y , Ichimori Y , Toyoshima K , Yamamoto T
Published: May 2006

Crosslinking of membrane-bound immunoglobulins, which are B-cell antigen receptors, causes proliferation and differentiation of B cells or the inhibition of their growth. The receptor-mediated signaling involves tyrosine phosphorylation of cellular proteins. The Src-like protein-tyrosine kinase Lyn is expressed preferentially in B cells and is an intracytoplasmic constituent of the B-cell antigen receptor complex. Crosslinking of membrane-bound immunoglobulin M with antibody induced rapid increases in the kinase activities of Lyn and Lyn-associated phosphatidylinositol 3-kinase. Crosslinking of B-cell antigen receptor also induced association of Lyn with an 85-kDa noncatalytic subunit of phosphatidylinositol 3-kinase. Thus, Lyn is functionally associated with membrane-bound immunoglobulin M and seems likely to participate in B-cell antigen receptor-mediated signaling.
Onset of cell-specific gene expression in the developing mouse pancreas.
Authors: Gittes G K , Rutter W J
Published: May 2006

A central question in developmental biology has been the initiation of cell-specific gene expression and its temporal relationship to morphogenesis. We have coupled embryo microdissection with the exquisite sensitivity of the polymerase chain reaction to define the onset of cell-specific gene expression during pancreatic organogenesis. Using the precise assignment of gestational age by the number of somites in each embryo, we determined the onset of transcription of major genes of the endocrine and exocrine pancreas during mouse development to within 2-3 hr. Somatostatin mRNA was detected at the 10-somite stage throughout the foregut, consistent with the presence of somatostatin-producing cells throughout the adult gut. Mature mRNA for insulin and glucagon first appears surprisingly early, at the 20-somite stage in the wall of the embryonic foregut and is restricted to only the area of the duodenum from which the pancreas will arise 10-12 hr later. In contrast, exocrine gene transcription begins 24 hr after formation of the pancreatic diverticulum. Thus cell-specific gene expression in the endocrine pancreas begins in a "pre-morphogenetic phase." This early expression of insulin and glucagon could reflect the initiation of an endocrine cell lineage.
Environmental education.
Authors: Lynch D R , Hutchinson C E
Published: May 2006

The need for a new profession devoted to environmental matters is asserted. The qualities of such a profession are sketched, and it is argued that new initiatives in environmental education are needed in the form of graduate, professional programs with primary emphasis on practice. An example 2-year program is presented. A fundamental requirement is scientific competence; undergraduate preparation in the sciences or engineering is mandatory. The graduate curriculum itself is built on three primary cores: environmental science and engineering, business and management, and public policy. Additionally, an environmental round table is proposed as a focal point for academic, industrial, governmental, and public discussion on environmental matters. The round table would provide oversight for the professional educational program and an affiliated research institute.
Structural and enzymatic characterization of a purified prohormone-processing enzyme: secreted, soluble Kex2 protease.
Authors: Brenner C , Fuller R S
Published: May 2006

The prohormone-processing Kex2 protease of the budding yeast Saccharomyces cerevisiae can be converted from an intracellular membrane protein to a soluble, secreted, and active form by deletion of the transmembrane domain and C-terminal tail. One such molecule was purified to near homogeneity from the culture medium of an overexpressing yeast strain. Amino acid sequence analysis revealed that the N terminus of mature Kex2 protease is created by a potentially autoproteolytic cleavage at Lys108-Arg109, prior to the domain homologous to subtilisin, followed by trimming of Leu-Pro and Val-Pro dipeptides by the Ste13 dipeptidyl aminopeptidase. Kinetic parameters were examined using fluorogenic peptidyl-methylcoumarin amide substrates. Initial burst titration indicated that the preparation was entirely active. Measurements of dependence of activity on pH yielded a simple curve suggesting titration of a single ionizable group. Activity was half-maximal at pH 5.7 and nearly constant from pH 6.5 to 9.5. Discrimination between substrates was as great as 360-fold in Km and 130-fold in kcat. Substrates with a Lys-Arg dipeptide preceding the cleaved bond were preferred, having kcat/Km values up to 1.1 x 10(7) sec-1.M-1. The enzyme cleaved substrates having Arg-Arg, Pro-Arg, Ala-Arg, and Thr-Arg with increased Km but with unchanged kcat. In contrast, the enzyme displayed a dramatically lower kcat for a Lys-Lys substrate with a smaller increase in Km. Thus the two residues preceding the cleaved bond may play distinct roles in the selectivity of binding and cleavage of prohormone substrates.
Retinoic acid-dependent transactivation of major histocompatibility complex class I promoters by the nuclear hormone receptor H-2RIIBP in undifferentiated embryonal carcinoma cells.
Authors: Nagata T , Segars J H , Levi B Z , Ozato K
Published: May 2006

H-2RIIBP is a member of the nuclear hormone receptor superfamily that binds to the region II enhancer of major histocompatibility complex (MHC) class I genes. The binding occurs through the GG(T/A)CA motif present also in many other genes. The role of H-2RIIBP in developmental regulation of MHC class I genes has been studied in undifferentiated N-Tera2 embryonal carcinoma cells by transient cotransfection of an expressible H-2RIIBP plasmid and a chloramphenicol acetyltransferase reporter gene linked to the MHC class I promoter. Transfection of the expression plasmid led to production of H-2RIIBP transcripts and enhanced MHC class I promoter activity in cells that were treated with retinoic acid but not yet differentiated. Retinoic acid concentrations required for transactivation overlapped with those capable of inducing morphological differentiation and expression of endogenous MHC class I genes in these cells. This enhancement was mediated by region II, as a heterologous thymidine kinase promoter driven by region II also served as a target for H-2RIIBP transactivation. Deletion of the bulk of the DNA-binding domain or the ligand-binding domain of H-2RIIBP, but not of the N-terminal domain, abolished transactivation, indicating that the former two domains are critical for the enhancement. Moreover, H-2RIIBP transactivation exhibited a strict cell-type restriction. As observed in other cell lines, N-Tera2 cells that had undergone differentiation failed to elicit transactivation, suggesting that H-2RIIBP acts in concert with a cofactor expressed in undifferentiated N-Tera2 cells that requires retinoic acid for its function. These results suggest that H-2RIIBP can function as a developmentally specific transcription factor for MHC class I genes.
A BamHI repeat element is predominantly associated with the degenerating neo-Y chromosome of Drosophila miranda but absent in the Drosophila melanogaster genome.
Authors: Ganguly R , Swanson K D , Ray K , Krishnan R
Published: May 2006

In Drosophila miranda, females have two X1 and two evolving X2 chromosomes, and males have one of each of these two X chromosomes and a Y chromosome. In males, the homologue of the X2 chromosome, the neo-Y chromosome, is attached to the Y chromosome and is under the process of degenerative evolution. We have examined a developmentally regulated X2/neo-Y chromosome-linked gene, 549mr, of D. miranda and found that the neo-Y chromosome-linked copy of this gene (549mr-NY) contains an insertional DNA. We discovered that sequences similar to those in the insertional DNA are present in multiple copies in the genome of both sexes of D. miranda but are more abundant in the males. The insertional DNA also identified a 1.1-kilobase BamHI repeat that is present in at least 6-fold excess in the male genome as compared to the female. This BamHI repeat and similar DNA sequences are predominantly concentrated on the evolving neo-Y chromosome, but very few are found on the homologous X2 and other chromosomes. The BamHI repeat also hybridizes with 2.0- and 1.8-kb RNAs and many other RNA species, which together are also approximately 6-fold greater in males. No sequences similar to the BamHI repeat are found in Drosophila melanogaster. Moreover, the BamHI repeat is not homologous to P, copia, or other D. melanogaster transposable elements. This repeat, named the NY element, may be involved in gene disruption and the process of degenerative evolution of the neo-Y chromosome.
Functional complementation of internal deletion mutants in the lactose permease of Escherichia coli.
Authors: Bibi E , Kaback H R
Published: May 2006

Using the lactose permease of Escherichia coli, a well-characterized membrane protein with 12 transmembrane domains, we demonstrated that certain paired in-frame deletion constructs complement each other functionally. Although cells expressing the deletion mutants individually are unable to catalyze active lactose accumulation, cells simultaneously expressing specific pairs of deletions catalyze transport up to 60% as do cells expressing wild-type permease. Moreover, complementation clearly does not occur at the level of DNA but probably occurs at the protein level. Remarkably, functional complementation is observed only with pairs of permease molecules containing large deletions and is not observed with missense mutations or point deletions. Although the mechanism of functional complementation is obscure, the findings indicate that certain pairs of permease molecules containing specific internal deletions can interact to form a functional complex in the same way phenomenologically as do independently expressed polypeptides corresponding to different N- and C-terminal portions of the permease.