bluuebunny/crossref_metadata_2025 huggingface dataset:
From elongator tRNA to initiator tRNA.
Authors: Varshney U , Lee C P , RajBhandary U L
Published: May 2006

We show that the two most important properties needed for a tRNA to function in initiation in Escherichia coli are its ability to be formylated and its ability to bind to the ribosomal P site. This conclusion is based on conversion of two different elongator tRNAs to ones that can act as initiators in E. coli. We transplanted the features unique to E. coli and eubacterial initiator tRNAs to E. coli elongator methionine tRNA (tRNA(Met)) along with an anticodon sequence change and analyzed their activities in initiation in E. coli. Introduction of a C1.A72 mismatch at the end of the acceptor stem of tRNA(Met), which generates the minimal features necessary for formylation, produces a tRNA with very low activity in initiation. Subsequent introduction of three consecutive G.C base pairs at the bottom of the anticodon stem, which is necessary for ribosomal P site binding, produces a tRNA with significant activity in initiation. Furthermore, introduction of the features necessary for formylation and for ribosomal P site binding into E. coli elongator glutamine tRNA produces a tRNA that initiates protein synthesis in E. coli.
Identification and analysis of all components of a gel retardation assay by combination with immunoblotting.
Authors: Demczuk S , Harbers M , Vennström B
Published: May 2006

A better method was developed for analysis and identification of protein and DNA components of gel-shift assays. The protein-DNA complexes, separated in polyacrylamide gels, were transferred onto stacked nitrocellulose and anion-exchange membranes. The proteins, bound to nitrocellulose, were identified by immunoblotting, while the DNA, which bound only to the anion-exchange membrane, was detected by autoradiography. The technique readily identified thyroid hormone receptors interacting with response elements representing inverted or direct repeats of the consensus half-site AGGTCA. In addition, specific antisera identified both the thyroid hormone and the retinoic acid receptors in heterodimeric complexes. Adding a third membrane and digoxigenin-labeled DNA probes allowed separate detection of [125I]T3 (labeled 3,5,3'-L-triiodothyronine), DNA, and protein from a single gel-shift reaction. The usefulness of this technique was also demonstrated by detecting the transcription factors P75gag-v-erbA and Jun in shifted complexes. Finally, proteins and DNA transferred to anion-exchange membranes can be eluted and subjected to further study. The combination of the gel shift and the immunoblot approaches (called "Shift-Western blotting") allows identification of the individual components of protein-DNA complexes containing multiple transcription factors, their cognate DNA elements and, when applicable, also the ligand.
Blockade of human immunodeficiency virus type 1 production in CD4+ T cells by an intracellular CD4 expressed under control of the viral long terminal repeat.
Authors: Buonocore L , Rose J K
Published: May 2006

A retroviral vector was constructed in which a gene encoding a mutated soluble CD4 protein that is retained in the endoplasmic reticulum (sCD4-KDEL) is expressed under control of human immunodeficiency virus type 1 (HIV-1) regulatory elements. HIV-1 infection of a human T-cell line transduced with this vector led to induction of sCD4-KDEL synthesis and a block in transport of the HIV envelope protein to the cell surface. There was a complete block to maturation of infectious HIV-1 in the transduced cells, no viral spread, and little or no syncytium formation. Infected cells gradually disappeared from the culture over a period of 2 months. This intracellular trap for HIV has potential application in gene therapy for AIDS.
Progression toward tumor cell phenotype is enhanced by overexpression of a mutant p53 tumor-suppressor gene isolated from nasopharyngeal carcinoma.
Authors: Sun Y , Nakamura K , Wendel E , Colburn N
Published: May 2006

We recently reported the detection of a heterozygous G-->C point mutation at codon 280 of p53 in nasopharyngeal carcinoma, which causes an Arg-->Thr substitution. To test whether this mutant p53 has gained function as an oncogene, we overexpressed the mutant p53 in nontumorigenic cells of two model systems: (i) human Saos-2 cells lacking endogenous p53 and (ii) mouse JB6 variants that bear endogenous wild-type p53. Although they have no growth advantage over the neomycin controls in monolayer culture, human Saos-2 transfectants overexpressing mutant p53 do show enhanced progression to tumor cell phenotype, as assayed by anchorage-independent growth and in vivo tumorigenicity. The enhancement is seen only in transfectants expressing higher levels of p53 protein. In the mouse JB6 system, the mutant p53 functions dominantly in the presence of endogenous wild-type p53 to enhance progression of preneoplastic promotion-sensitive cells toward anchorage-independent phenotype. Mouse JB6 transfectants of mutant p53 are, however, not tumorigenic in nude mice. We conclude from these studies that the G-->C point mutation of p53 at codon 280 is a gain-of-function mutation that appears to operate dominantly and that the mutant p53-thr280 has only moderate oncogenic activity. This mutation may cooperate with other yet-to-be isolated genes in the genesis of nasopharyngeal carcinoma.
Highly lytic CD8+, alpha beta T-cell receptor cytotoxic T cells with major histocompatibility complex (MHC) class I antigen-directed cytotoxicity in beta 2-microglobulin, MHC class I-deficient mice.
Authors: Apasov S , Sitkovsky M
Published: May 2006

Targeted disruption of the beta 2-microglobulin (beta 2m) gene results in major histocompatibility complex (MHC) class I deficiency and virtual disappearance of functional CD8+ cytotoxic T lymphocytes (CTLs) in beta 2m-deficient (beta 2m-/-) mice. We asked whether the beta 2m-/- mice are able to reject tumor cells injected i.p. and what is the cellular composition of peritoneal exudate leukocytes (PELs) from such mice. We found that beta 2m-/- mice do reject MHC class I-bearing tumor cells injected i.p. Surprisingly, analysis of PEL CTLs obtained from i.p. tumor-injected beta 2m -/- mice revealed the presence of a large proportion of functional, tumor-destroying CD8+, CD4-, alpha beta T-cell receptor-positive, CD3+, Thy-1+, MHC class I-negative CTLs with strong MHC class I-directed cytotoxic activity. These results call for careful studies of local accumulation of CD8+ CTLs in beta 2m -/- mouse models and suggest that the dramatic decrease in MHC class I expression caused by beta 2m gene disruption does not prevent CD8+/CD4- cell selection and expansion.
Transcription initiation factor activity of vaccinia virus capping enzyme is independent of mRNA guanylylation.
Authors: Harris N , Rosales R , Moss B
Published: May 2006

Cytoplasmic extracts of vaccinia virus-infected HeLa cells blocked in DNA replication were capable of transcribing templates containing the minimal promoter sequences derived from three viral intermediate-stage genes (A1L, A2L, and G8R) but not promoters from early or late genes. One of three isolated components required for transcription copurified with the viral capping enzyme, a heterodimeric protein responsible for forming the 7-methyl-guanosine(5')triphospho(5')nucleoside [m7G(5')ppp(5')N-] structure at the 5' end of mRNAs, as had been reported using a template with another intermediate promoter [Vos, J. C., Sasker, M. & Stunnenberg, H. G. (1991) EMBO J. 10, 2553-2558]. Transcription factor activity was associated with partially, purified capping enzyme from infected cell extracts, homogeneous enzyme from purified virions, and recombinant viral enzyme from Escherichia coli. By transcribing truncated templates of different sizes, we determined that RNA chains of 35 nt were capped whereas those of 15 nt were not. Nevertheless, the capping enzyme was required for formation of short uncapped transcripts, indicating that capping and transcription initiation factor activities are independent functions.
Expression of the major protein kinase C substrate, the acidic 80-kilodalton myristoylated alanine-rich C kinase substrate, increases sharply when Swiss 3T3 cells move out of cycle and enter G0.
Authors: Herget T , Brooks S F , Broad S , Rozengurt E
Published: May 2006

The expression of the major protein kinase C (PKC) substrate, originally called "80K" for acidic SDS/PAGE-observed 80-kDa PKC substrate and now called "MARCKS" for myristoylated alanine-rich C kinase substrate, in Swiss 3T3 fibroblasts changes strikingly (15- to 22-fold) during transitions of cell growth. Quiescent cells in G0 express high levels of MARCKS mRNA and protein. However, plating these cells in fresh medium at low density to stimulate multiple rounds of cell division caused a striking down-regulation of MARCKS expression. The mRNA level declined to a minimum of 4.5% compared with quiescent control cells 6 hr after plating, and protein levels declined during the same period to 6.5% of the control value. This rapid down-regulation was independent of PKC activation and length of exposure to trypsin (1-10 min) but required plating in medium containing fresh serum. MARCKS mRNA and protein levels remained down-regulated for 3 days, during which time the cells were actively progressing through the cell cycle as judged by fluorescence-activated cell sorting analysis. However, on reaching quiescence, the expression of MARCKS mRNA and protein increased markedly. Furthermore, the rate of recovery of MARCKS mRNA and protein levels was shown to be dependent on the supply of serum-derived growth factors in the medium. Addition of hydroxyurea to arrest the cells in S phase or at the G1/S boundary rather than G0 completely prevented the recovery of MARCKS protein. The down-regulation of MARCKS following plating and its serum-dependent recovery was also demonstrated in tertiary cultures of mouse embryo fibroblasts. The results suggest that MARCKS may play a role in the regulation of entry and exit of cells from G0.
Markedly prolonged incubation period of hepatitis B in a chimpanzee passively immunized with a human monoclonal antibody to the a determinant of hepatitis B surface antigen.
Authors: Ogata N , Ostberg L , Ehrlich P H , Wong D C , Miller R H , Purcell R H
Published: May 2006

The protective efficacy of a human monoclonal antibody directed against the a determinant of hepatitis B virus (HBV) surface antigen was studied in a chimpanzee. A single high dose of 5 mg/kg (body weight) of monoclonal antibody SDZ OST 577 was intravenously administered to a chimpanzee, followed by intravenous challenge with 10(3.5) chimpanzee infectious doses of a wild-type HBV, the MS-2 strain (ayw subtype). The passively acquired antibody to HBV surface antigen could be detected for 40 weeks. Serum HBV DNA tested by a "nested" polymerase chain reaction assay was negative through the 36th week after virus challenge but became positive by the 38th week. The chimpanzee subsequently developed acute hepatitis B approximately 1 year after challenge. The nucleotide sequence of the a determinant of the surface gene of the replicated virus was identical with that of the inoculated wild-type virus. Thus, a human monoclonal antibody directed against the a determinant of HBV surface antigen delayed but did not prevent experimental infection of HBV and hepatitis in the chimpanzee. Our results indicate an incomplete ability of this antibody to protect against HBV infection in vivo after a single infusion.
Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin.
Authors: Savarino S J , Fasano A , Watson J , Martin B M , Levine M M , Guandalini S , Guerry P
Published: May 2006

Enteroaggregative Escherichia coli (EAggEC) are associated with persistent diarrhea in young children. Some of these organisms produce a low-molecular-weight, heat-stable, plasmid-encoded enterotoxin that has been named EAggEC heat-stable enterotoxin 1 (EAST1). We have cloned a 4.4-kb DNA fragment from the virulence plasmid of prototype EAggEC strain 17-2, which expresses enterotoxic activity as measured by electrogenic response in Ussing chambers mounted with rabbit ileal tissue. DNA-sequence analysis of this fragment identified an open reading frame (ORF) encoding a cysteine-rich polypeptide of 38 amino acids (M(r), 4100). Insertional and deletional mutations in this ORF resulted in loss of enterotoxic activity. The ORF was cloned into a T7 expression vector, and postinduction culture filtrates exhibited enterotoxic activity and increased ileal tissue cGMP levels. A synthetic peptide consisting of predicted amino acid residues 8-29 also showed enterotoxic activity. These data indicate that this ORF, named astA (EAggEC heat-stable enterotoxin), represents the EAST1 structural gene. EAST1 shows significant homology with the enterotoxic domain of heat-stable enterotoxin a (STa) of enterotoxigenic E. coli and with guanylin, a mammalian analog of STa. Unlike STa, which requires six cysteines and three disulfide linkages for full biological activity, both EAST1 and guanylin contain four cysteine residues. Based on the cGMP data and the sequence homology to STa and guanylin, it is predicted that EAST1 stimulates the particulate form of guanylate cyclase through the same receptor-binding region as STa and guanylin.
Strategies to protect crop plants against viruses: pathogen-derived resistance blossoms.
Authors: Wilson T M
Published: May 2006

Since 1986, the ability to confer resistance against an otherwise devastating virus by introducing a single pathogen-derived or virus-targeted sequence into the DNA of a potential host plant has had a marked influence on much of the research effort, focus, and short-term objectives of plant virologists throughout the world. The vast literature on coat protein-mediated protection, for example, attests to our fascination for unraveling fundamental molecular mechanism(s), our (vain) search for a unifying hypothesis, our pragmatic interest in commercially exploitable opportunities for crop protection, and our ingenuity in manipulating transgene constructions to broaden their utility and reduce real or perceived environmental risk issues. Other single dominant, pathogen-derived plant resistance genes have recently been discovered from a wide variety of viruses and are operative in an ever-increasing range of plant species. Additional candidates seem limited only by the effort invested in experimentation and by our ingenuity and imagination. This review attempts to consider, in a critical way, the current state of the art, some exceptions, and some proposed rules. The final impression, from all the case evidence considered, is that normal virus replication requires a subtle blend of host- and virus-coded proteins, present in critical relative concentrations and at specific times and places. Any unregulated superimposition of interfering protein or nucleic acid species can, therefore, result in an apparently virus-resistant plant phenotype.
CD22 associates with the human surface IgM-B-cell antigen receptor complex.
Authors: Leprince C , Draves K E , Geahlen R L , Ledbetter J A , Clark E A
Published: May 2006

The B-cell surface molecule CD22, when cross-linked, modulates signaling through the surface IgM (sIgM)-B-cell receptor (BCR) complex. Here we analyzed the basis of this interaction between CD22 and the human sIgM complex. After lysis of B cells or B-cell lines in digitonin, CD22 coimmunoprecipitated a kinase activity that in vitro-phosphorylated two polypeptides of 150 and 130 kDa on tyrosine residues. By immunoblot analysis with a rabbit anti-serum specific for a synthetic peptide of CD22, we found these proteins to be CD22 itself. Furthermore, the phosphorylated 150-kDa CD22 was found in the sIgM-BCR complex maintained by digitonin, along with Ig alpha/mb-1, Ig beta/B29, and a 75-kDa polypeptide precipitated by an antiserum specific to protein-tyrosine kinase PTK72. CD22 is likely to be an important signaling partner in the sIgM-BCR complex since it is very rapidly and strikingly phosphorylated after sIgM is cross-linked and since it contains the antigen recognition homology I (ARHI) motif, present in other antigen receptor molecules.
Pattern formation and handedness in the cytoskeleton of human platelets.
Authors: Hagmann J
Published: May 2006

The cytoskeletal patterns of human platelets spread on a glass surface are analyzed. F-actin is arranged in patterns of parallel microfilaments, microfilaments forming triangles, or microfilaments radiating tangentially from a central ellipse or circle. Vinculin, a cytoskeletal protein, is located at both ends of the filaments. In platelets with tangentially radiating microfilaments, vinculin patches are aligned on the branches of a two-armed spiral. The spirals are always left-handed. Talin and two integrins (gpIIb-IIIa, vitronectin receptor), proteins usually associated with focal contacts in tissue culture cells, are not concentrated at the ends of microfilaments in human platelets. It is suggested that the distribution of vinculin is due to competitive aggregation of vinculin close to the inner leaflet of the ventral plasma membrane and that sites of cytoskeleton-membrane linkage are important for generating supramolecular asymmetries of biological systems.
Persistent induction of c-fos and c-jun expression by asbestos.
Authors: Heintz N H , Janssen Y M , Mossman B T
Published: May 2006

To investigate the mechanisms of asbestos-induced carcinogenesis, expression of c-fos and c-jun protooncogenes was examined in rat pleural mesothelial cells and hamster tracheal epithelial cells after exposure to crocidolite or chrysotile asbestos. In contrast to phorbol 12-myristate 13-acetate, which induces rapid and transient increases in c-fos and c-jun mRNA, asbestos causes 2- to 5-fold increases in c-fos and c-jun mRNA that persist for at least 24 hr in mesothelial cells. The induction of c-fos and c-jun mRNA by asbestos in mesothelial cells is dose-dependent and is most pronounced with crocidolite, the type of asbestos most pathogenic in the causation of pleural mesothelioma. Induction of c-jun gene expression by asbestos occurs in tracheal epithelial cells but is not accompanied by a corresponding induction of c-fos gene expression. In both cell types, asbestos induces increases in protein factors that bind specifically to the DNA sites that mediate gene expression by the AP-1 family of transcription factors. The persistent induction of AP-1 transcription factors by asbestos suggests a model of asbestos-induced carcinogenesis involving chronic stimulation of cell proliferation through activation of the early response gene pathway that includes c-jun and/or c-fos.
The two motility systems of Myxococcus xanthus show different selective advantages on various surfaces.
Authors: Shi W , Zusman D R
Published: May 2006

Myxococcus xanthus, a bacterium that forms fruiting bodies, moves by gliding motility utilizing dual motility systems that differ both genetically and morphologically [system A, having at least 21 genetic loci and moving mainly single cells, and system S, having at least 10 genetic loci and moving groups (rafts) of cells] [Hodgkin, J. & Kaiser, D. (1979) Mol. Gen. Genet. 172, 177-191]. In this study, we found that A- and S-gliding-motility systems have different selective advantages on surfaces containing different concentrations of agar. We observed that colonies of A+S- cells (A-motile cells) swarmed better than A-S+ cells (S-motile cells) on relatively firm and dry surfaces (e.g., 1.5% agar). In contrast, colonies of A-S+ cells swarmed much better than A+S- cells on soft and wet surfaces (e.g., 0.3% agar). Individual A-motile cells moved at a rate of 2-4 microns/min on 1.5% agar but they barely moved on 0.3% agar (< 0.5 microns/min); in contrast S-motile cells moved 3-5 times faster on 0.3% agar than on 1.5% agar. Wild-type cells with both A- and S-motility systems were able to move well over a wide range of surfaces. These results suggest that dual motility systems enable the myxobacteria to adapt to a variety of physiological and ecological environments and show similarities in function to the dual motility systems of flagellated bacteria such as Vibrio spp.
Baculovirus expression of the maize mitochondrial protein URF13 confers insecticidal activity in cell cultures and larvae.
Authors: Korth K L , Levings C S
Published: May 2006

The URF13 protein, which is encoded by the mitochondrial gene T-urf13, is responsible for cytoplasmic male sterility and pathotoxin sensitivity in the Texas male-sterile cytoplasm (cms-T) of maize. Mitochondrial sensitivity to two host-specific fungal toxins (T toxins) is mediated by the interaction of URF13 and T toxins to form pores in the inner mitochondrial membrane. A carbamate insecticide, methomyl, mimics the effects of T toxins on isolated cms-T mitochondria. URF13 was expressed in Spodoptera frugiperda (fall army-worm) cells (Sf9) in culture and in Trichoplusia ni (cabbage looper) larvae with a baculovirus vector. In insect cells, URF13 forms oligomeric structures in the membrane and confers T toxin or methomyl sensitivity. Adding T toxin or methomyl to Sf9 cells producing URF13 causes permeabilization of plasma membranes. In addition, URF13 is toxic to insect cells grown in culture without T toxins or methomyl; even a T-toxin-insensitive mutant form of URF13 is lethal to cell cultures. Baculoviruses expressing URF13 are lethal to T. ni larvae, at times postinjection comparable to those obtained by injecting a baculovirus expressing an insect neurotoxin. This result suggests that URF13 could be useful as a biological control agent for insect pests. Our data indicate that URF13 has two independent mechanisms for toxicity, one that is mediated by T toxin and methomyl and one that is independent of these toxins. Similarly, male sterility and toxin sensitivity in cms-T maize may be due to independent mechanisms.
Visualization of the interaction of a regulatory protein with RNA in vivo.
Authors: Bertrand E , Fromont-Racine M , Pictet R , Grange T
Published: May 2006

We have adapted to RNA molecules the ligation-mediated polymerase chain reaction (LMPCR) procedure of genomic sequencing [Mueller, P. R. & Wold, B. (1989) Science 246, 780-786]. This new procedure, the reverse ligation-mediated PCR (RLPCR), is sufficiently sensitive to allow "in vivo" footprinting of minor RNA species. It is based on the ligation of an RNA linker of known sequence to every 5' end resulting from the cleavage of total cellular RNA. Target RNA molecules are specifically reverse-transcribed and the resulting products are amplified by PCR. The localization of the initial 5' ends is ultimately determined on a sequencing gel. To demonstrate the validity of this strategy, we have used RNase T1 treatment of permeabilized cells and RLPCR and have detected in vivo iron-depletion-dependent footprints on two iron-responsive elements of the transferrin receptor mRNA.
MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2.
Authors: Kozopas K M , Yang T , Buchan H L , Zhou P , Craig R W
Published: May 2006

During their lifespan, immature cells normally pass through sequential transitions to a differentiated state and eventually undergo cell death. This progression is aberrant in cancer, although the transition to differentiation can be reestablished in inducible leukemia cell lines. This report describes a gene, MCL1, that we isolated from the ML-1 human myeloid leukemia cell line during phorbol ester-induced differentiation along the monocyte/macrophage pathway. Our results demonstrate that expression of MCL1 increases early in the induction, or "programming," of differentiation in ML-1 (at 1-3 hr), before the appearance of differentiation markers and mature morphology (at 1-3 days). They further show that MCL1 has sequence similarity to BCL2, a gene involved in normal lymphoid development and in lymphomas with the t(14;18) chromosome translocation. MCL1 and BCL2 do not fall into previously known gene families. BCL2 differs from many oncogenes in that it inhibits programmed cell death, promoting viability rather than proliferation; this parallels the association of MCL1 with the programming of differentiation and concomitant maintenance of viability but not proliferation. Thus, in contrast to proliferation-associated genes, expression of MCL1 and BCL2 relates to the programming of differentiation and cell viability/death. The discovery of MCL1 broadens our perspective on an emerging MCL1/BCL2 gene family and will allow further comparison with oncogene families.
Biochemical interaction of the Escherichia coli RecF, RecO, and RecR proteins with RecA protein and single-stranded DNA binding protein.
Authors: Umezu K , Chi N W , Kolodner R D
Published: May 2006

The Escherichia coli RecF, RecO, and RecR proteins were analyzed for their effect on RecA-mediated pairing of single-stranded circular DNA and homologous linear duplex DNA substrates. As shown by other workers, joint molecule formation by RecA was inhibited by E. coli single-stranded DNA binding protein (SSB) when it was added to single-stranded DNA before RecA. This inhibitory effect was overcome by the addition of RecO and RecR or RecF, RecO, and RecR. Both the rate and extent of joint molecule formation were restored to the maximal level observed when SSB was added after RecA. RecF, RecO, and RecR proteins had no effect on the conversion of joint molecules to final products and only appeared to stimulate an early step in the pairing reaction. The stimulatory effect of RecF, RecO, and RecR was not seen without SSB or when SSB was added after RecA. RecF protein by itself inhibited reactions in mixtures containing RecA and SSB, and this inhibition was overcome by the addition of RecO and RecR. These data suggest that RecO and RecR, and possibly RecF, help RecA overcome inhibition by SSB and utilize SSB-single-stranded-DNA complexes as substrates.
Appearance of heparin-binding EGF-like growth factor in wound fluid as a response to injury.
Authors: Marikovsky M , Breuing K , Liu P Y , Eriksson E , Higashiyama S , Farber P , Abraham J , Klagsbrun M
Published: May 2006

Wound fluid was obtained from porcine partial-thickness excisional wounds and analyzed for heparin-binding growth factors. Two heparin-binding growth factor activities were detected, a relatively minor one that was eluted from a heparin affinity column with 0.65 M NaCl and a major one that was eluted with 1.1 M NaCl. These activities were not present in wound fluid 1 hr after injury but appeared 1 day after injury, were maximal 2-3 days after injury, and were not detectable by 8 days after injury. The heparin-binding growth factor eluted with 0.65 M NaCl was identified as a platelet-derived growth factor (PDGF)-like activity by the use of specific anti-PDGF neutralizing antibodies. The heparin-binding growth factor eluted with 1.1 M NaCl was shown to be structurally related to heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) by several criteria, including binding to heparin affinity columns and elution with 1.1 M NaCl, competition with the binding of 125I-EGF to the EGF receptor, triggering phosphorylation of the EGF receptor, immunodetection on a Western blot, and stimulation of fibroblast and keratinocyte growth. It was concluded that HB-EGF is a major growth factor component of wound fluid and, since it is mitogenic for fibroblasts and keratinocytes, that it might play an important role in wound healing.
Increasing the chemical potential of the germ-line antibody repertoire.
Authors: Sarvetnick N , Gurushanthaiah D , Han N , Prudent J , Schultz P , Lerner R
Published: May 2006

To augment the chemical potential of the immunological repertoire, a metal ion-binding light chain has been introduced into the murine genome. Mice containing the transgene were subsequently immunized with a fluorescein conjugate. The transgenic light chain was found at a high frequency in the anti-fluorescein memory B-cell compartment. This general method should be applicable to other cofactors and small molecules and should lead to generation of antibodies with unique catalytic activities.
Isolation and characterization of major histocompatibility complex class IIB genes from the nurse shark.
Authors: Bartl S , Weissman I L
Published: May 2006

The major histocompatibility complex (MHC) contains a set of linked genes which encode cell surface proteins involved in the binding of small peptide antigens for their subsequent recognition by T lymphocytes. MHC proteins share structural features and the presence and location of polymorphic residues which play a role in the binding of antigens. In order to compare the structure of these molecules and gain insights into their evolution, we have isolated two MHC class IIB genes from the nurse shark, Ginglymostoma cirratum. Two clones, most probably alleles, encode proteins which differ by 13 amino acids located in the putative antigen-binding cleft. The protein structure and the location of polymorphic residues are similar to their mammalian counterparts. Although these genes appear to encode a typical MHC protein, no T-cell-mediated responses have been demonstrated in cartilaginous fish. The nurse shark represents the most phylogenetically primitive organism in which both class IIA [Kasahara, M., Vazquez, M., Sato, K., McKinney, E.C. & Flajnik, M.F. (1992) Proc. Natl. Acad. Sci USA 89, 6688-6692] and class IIB genes, presumably encoding the alpha/beta heterodimer, have been isolated.
Variable thermal emission and chlorophyll fluorescence in photosystem II particles.
Authors: Allakhverdiev S I , Klimov V V , Carpentier R
Published: May 2006

In photosynthetic systems, the absorbed light energy is used to generate electron transport or it is lost in the form of fluorescence and thermal emission. While fluorescence can be readily measured, the detection of thermal deactivation processes can be achieved by the photoacoustic technique. In that case, the pressure wave generated by the thermal deactivations in a sample irradiated with modulated light is detected by a sensitive microphone. The relationships between the yield of fluorescence and thermal emissions measured simultaneously were analyzed by using a spinach photosystem II (PSII)-enriched preparation. It is shown that the quenching of fluorescence due to the photochemical activity of the preparations (photochemical quenching) increases in proportion to the fraction of thermal deactivations that is not immediately released as heat but is stored in photochemical intermediates (energy-storage yield) as the intensity of the photoacoustic modulated measuring beam (35 Hz) is decreased. Maximal levels of fluorescence and thermal emissions were both decreased in similar proportions upon photoreduction of pheophytin (Pheo), the primary acceptor of PSII. The variable components of fluorescence and thermal emissions were strongly decreased upon depletion of Mn from the Mn complex that catalyzes water oxidation and were recovered proportionally during reconstitution with Mn2+ at various Mn2+/reaction center ratios. Finally, depletion of Mn from the Mn complex together with the Fe of the QA-Fe-QB complex that is composed of the secondary quinone acceptors of PSII resulted in an increased initial level of fluorescence Fo and in the loss of the variable components of fluorescence and thermal emissions. The initial Fo and the variable components could be partially recovered by reconstitution of both donor and acceptor sides with Mn2+, Co2+, HCO3- and plastoquinone. It is concluded that the photochemical fluorescence quenching is correlated with a simultaneous "quenching" of a variable component of thermal emission. It is proposed that the measured component of variable thermal emission is related to the decay of the pair [P680+ Pheo-]. The suggestion is also made that a bicarbonate-induced protonation of reduced QA or QB or conformational change in the PSII complex, or both, adds an additional entropic factor to the variable thermal emission component.
Mode of cell proliferation in the developing mouse neocortex.
Authors: Takahashi T , Nowakowski R S , Caviness V S
Published: May 2006

There are two proliferative populations in the developing cerebral wall: the pseudostratified ventricular epithelium (PVE) and the secondary proliferative population (SPP). The present experiments provide on embryonic day 14 (E14) in the mouse direct measures of the values for the proportions of daughter cells that continue to proliferate (proliferative, P fraction, or P) and for those that leave the cell cycle (quiescent, Q fraction, or Q; Q = 1 - P) for both of these populations. The range of values of P for the PVE, 0.62-0.66, would provide for the relatively low rate of neuronal output and for an expansion of the proliferative population appropriate for E14. The even higher values of P for the SPP, 0.73-1.0, would expand this population rapidly in preparation for the high glial cell output to occur later in cerebral histogenesis.
Plant hormone ethylene is a Norrish type II product from enzymically generated triplet 1-butanal.
Authors: Knudsen F D , Campa A , Stefani H A , Cilento G
Published: May 2006

The peroxidase-catalyzed aerobic oxidation of a linear aldehyde, leading to the next lower homologue in the triplet state, is accompanied by Norrish type II product ethylene when the substrate is pentanal. This system appears to provide an example of "photobiochemistry without light" because ethylene is a plant hormone and is formed in lipid peroxidation when the recurrent oxidation of linear aldehydes occurs.
Techniques for measuring sulcal/gyral patterns in the brain as visualized through magnetic resonance scanning: BRAINPLOT and BRAINMAP.
Authors: Andreasen N C , Harris G , Cizadlo T , Arndt S , O'Leary D S , Swayze V , Flaum M
Published: May 2006

A method for measuring sulcal and gyral patterns, using data derived from magnetic resonance (MR) scanning, is described. This method can be applied through two newly developed computer programs, BRAINPLOT and BRAINMAP. These programs provide quantitative measures of brain surface pattern. The method has been validated with postmortem brains, phantoms, and human MR data. The method is robust to detecting differences in brain surface anatomy between atrophic and nonatrophic brains. It appears to offer an efficient, fully automated, and accurate method for analyzing the large amounts of information generated through in vivo neuroimaging techniques.
Molecular cloning, characterization, and elicitation of acetyl-CoA carboxylase from alfalfa.
Authors: Shorrosh B S , Dixon R A , Ohlrogge J B
Published: May 2006

Acetyl-CoA carboxylase [ACCase; acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] catalyzes the ATP-dependent carboxylation of acetyl CoA to produce malonyl CoA. In plants, malonyl CoA is needed for plastid localized fatty acid biosynthesis and for a variety of pathways in the cytoplasm including flavonoid biosynthesis. We have determined the full nucleotide sequence of an ACCase from alfalfa, which appears to represent a cytoplasmic isozyme. Partial cDNAs were isolated from a cDNA library of suspension culture cells that had been elicited for isoflavonoid phytoalexin synthesis. The full-length sequence was obtained by primer extension and amplification of the cDNA with synthetic primers. The sequence codes for a protein of 2257 amino acids with a calculated M(r) of 252,039. The biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase domains, respectively, show approximately 72%, 50%, and 65% sequence similarity to those of animal, diatom, and yeast ACCase sequences. ACCase enzyme activity and transcripts are induced severalfold upon addition of yeast or fungal elicitors to alfalfa cell cultures.
Mass spectrometric molecular-weight determination of highly acidic compounds of biological significance via their complexes with basic polypeptides.
Authors: Juhasz P , Biemann K
Published: May 2006

Highly acidic compounds that are difficult to ionize by matrix-assisted laser desorption ionization give excellent spectra when mixed with a basic peptide or protein to form a noncovalent complex. This phenomenon makes it possible to determine the molecular weights of polysulfated, -sulfonated, and -phosphorylated biomolecules such as cysteic acid-containing peptides, oligonucleotides, heparin-derived oligosaccharides, and suramin (a drug containing two trisulfonated naphthalene moieties). Peptides and small proteins rich in arginine were used as the basic components. The extent of complex formation correlates with the number of phosphate and sulfate groups in the acidic component and with the number of arginines in the basic component. Neither the acidic amino acid residue aspartic and glutamic acid nor the basic lysine and histidine contribute to complex formation. For oligonucleotides, histone H4 was found to be the best complexing agent investigated. The analytical utility of the complex formation is demonstrated by the molecular-mass determination of acidic compounds from 500 to 6000 Da at the picomole or sub-picomole level with an accuracy of +/- 0.1% or better and by the absence of alkali cation adducts.
Development of anti-tumor immunity following thymidine kinase-mediated killing of experimental brain tumors.
Authors: Barba D , Hardin J , Sadelain M , Gage F H
Published: May 2006

Using the 9L experimental brain tumor model, we studied long-term tumor regression and immunologic consequences of tumor killing in a model of in vivo gene transfer of the herpes simplex virus 1 thymidine kinase (HSV-TK) gene and ganciclovir (GCV) treatments. Fibroblasts modified to produce retroviral vectors carrying the HSV-TK gene were implanted into established 9L brain tumors in Fischer 344 rats to carry out gene transfer. Animals were then treated with parenteral GCV. Significant tumor regression was seen following GCV treatments in short-term experiments (17 days) as quantified by measurements of tumor volume. In long-term studies, 7 of 32 (22%) treated animals survived 90 days. Histologic examination of the brains of the successfully treated animals demonstrated residual tumor cells and inflammatory cells consisting predominantly of macrophages/microglia and T cells in the hemisphere with the residual tumor cyst. Rats surviving 90 days rejected repeat tumor injections into the contralateral brain and flank, whereas identical tumor injections in naive animals resulted in both brain and flank tumors. The presence of significant anti-tumor immunity following HSV-TK and GCV treatments suggests that the immune system plays a critical role in the sustained tumor regressions associated with these treatments. These findings show that while HSV-TK and GCV treatments can result in long-term tumor regressions in this model, the success of these treatments could be improved by better understanding the role played by the host's immune systems.
Spatiotemporal expression patterns of chicken ovalbumin upstream promoter-transcription factors in the developing mouse central nervous system: evidence for a role in segmental patterning of the diencephalon.
Authors: Qiu Y , Cooney A J , Kuratani S , DeMayo F J , Tsai S Y , Tsai M J
Published: May 2006

Chicken ovalbumin upstream promoter-transcription factor (COUP-TF) genes encode transcription factors belonging to the orphan subfamily of the steroid/thyroid hormone receptor superfamily. Two COUP-TF counterparts have been cloned from mouse. In an attempt to study the function of these genes in the developing central nervous system (CNS), the spatiotemporal expression patterns of the two mouse genes have been examined by in situ hybridization. Both genes are widely expressed in the developing CNS, with patterns that are overlapping yet distinct from each other. The differential expression of murine COUP-TFI and -II in the diencephalon is striking in that high levels of expression from each gene are confined to specific segmental compartments--the neuromeres. Our results suggest that murine COUP-TFs may play important roles in the development and differentiation of the CNS, including the specification of diencephalic neuromeres.
Costimulatory signals for human T-cell activation induce nuclear translocation of pp19/cofilin.
Authors: Samstag Y , Eckerskorn C , Wesselborg S , Henning S , Wallich R , Meuer S C
Published: May 2006

Resting T lymphocytes that have recognized antigen bound to a major histocompatibility complex molecule with the T-cell receptor require costimulatory signals through accessory receptors, including CD2, CD4, CD8, and CD28, for their clonal growth and expression of their functional repertoires. Absence of costimulation, in contrast, can induce clonal anergy in vitro and selective tolerance in vivo. Here we have defined a potential intracellular messenger for T-cell activation which is strictly regulated by costimulatory signals mediated through accessory receptors: pp19/cofilin, a small actin-binding protein, undergoes dephosphorylation and subsequent translocation from the cytosol into the nucleus. In untransformed T cells this process correlates with functional responses essential for the induction of T-cell proliferation (i.e., production of interleukin 2). Moreover, spontaneous dephosphorylation as well as nuclear translocation of pp19/cofilin occur in the autonomously proliferating T-lymphoma cell line Jurkat.
Biologic and therapeutic significance of MYB expression in human melanoma.
Authors: Hijiya N , Zhang J , Ratajczak M Z , Kant J A , DeRiel K , Herlyn M , Zon G , Gewirtz A M
Published: May 2006

We investigated the therapeutic potential of employing antisense oligodeoxynucleotides to target the disruption of MYB, a gene which has been postulated to play a pathogenetic role in cutaneous melanoma. We found that MYB was expressed at low levels in several human melanoma cell lines. Also, growth of representative lines in vitro was inhibited in a dose- and sequence-dependent manner by targeting the MYB gene with unmodified or phosphorothioate-modified antisense oligodeoxynucleotides. Inhibition of cell growth correlated with specific decrease of MYB mRNA. In SCID mice bearing human melanoma tumors, infusion of MYB antisense transiently suppressed MYB gene expression but effected long-term growth suppression of transplanted tumor cells. Toxicity of the oligodeoxynucleotides was minimal in mice, even when targeted to the murine Myb gene. These results suggest that the MYB gene may play an important, though undefined, role in the growth of at least some human melanomas. Inhibition of MYB expression might be of use in the treatment of this disease.
A cytoplasmic domain is important for the formation of a SecY-SecE translocator complex.
Authors: Baba T , Taura T , Shimoike T , Akiyama Y , Yoshihisa T , Ito K
Published: May 2006

An approach to identifying the interaction site of multicomponent protein assembly has been applied to the membrane-bound SecY-SecE complex, which mediates protein export across the Escherichia coli cytoplasmic membrane. A dominant negative secY allele, secY-d1, inactivates SecY but preserves its ability to interact with SecE. Thus, the mutant protein sequesters SecE in an inactive complex. Second site mutations that disrupt the SecE binding site will suppress the export interference. We introduced insertion/deletion mutations that intragenically suppressed secY-d1. After eliminating knock-out mutations by virtue of the expression of a LacZ alpha sequence that had been attached to the C terminus, we obtained a striking clustering of mutations in cytoplasmic domain 4. On the basis of this result, the secY24 (Ts) substitution mutation in this domain was examined for its effects on interaction with SecE. It indeed suppressed secY-d1. Although the instability associated with excess SecY can be alleviated by overproduction of SecE, the secY24 mutant protein was not stabilized by SecE. The basal-level SecY24 protein was also destabilized at 42 degrees C. SecE was coimmunoprecipitated with SecY+ but not with the SecY24 protein. These results indicate that the secY24 mutation weakens SecY's interaction with SecE. Taken together, we propose that cytoplasmic domain 4 is important for the association between SecY and SecE.
Neurotensin is an autocrine trophic factor stimulated by androgen withdrawal in human prostate cancer.
Authors: Sehgal I , Powers S , Huntley B , Powis G , Pittelkow M , Maihle N J
Published: May 2006

After therapeutic hormone deprivation, prostate cancer cells often develop androgen-insensitive growth through mechanisms thus far undefined. Neuropeptides have been previously implicated as growth factors in some prostate cancers. Here, we demonstrate that androgen-sensitive LNCaP human prostate cancer cells produce and secrete neurotensin following androgen withdrawal. We show that while LNCaP cells express the neurotensin receptor, only androgen-deprived cells exhibit a growth response to exogenous neurotensin. We further demonstrate that androgen-stimulated cells may be refractory to exogenous neurotensin due to androgen induction of a metalloprotease active toward neurotensin. Thus, prostate cancer cells deprived of androgen develop an alternative autocrine growth mechanism involving neurotensin.
The combinatorial synthesis and chemical and biological evaluation of a 1,4-benzodiazepine library.
Authors: Bunin B A , Plunkett M J , Ellman J A
Published: May 2006

A library of 192 structurally diverse 1,4-benzodiazepine derivatives containing a variety of chemical functionalities including amides, carboxylic acids, amines, phenols, and indoles was constructed from three components, 2-aminobenzophenones, amino acids, and alkylating agents, by employing Geysen's pin apparatus [Geysen, H. M., Rodda, S. J., Mason, T. J., Tribbick, G. & Schoofs, P. G. (1987) J. Immunol. Methods 102, 259-274]. Rigorous analytical verification of the chemical integrity and yield of a representative collection of the diverse derivatives was carried out. In addition, the library of derivatives was evaluated for binding to the cholecystokinin A receptor by employing a competitive radio-ligand binding assay. This provided detailed structure versus activity relationships that were confirmed by independent large-scale synthesis and evaluation of several of the 1,4-benzodiazepine derivatives.
Functional unit size of the charybdotoxin receptor in smooth muscle.
Authors: Garcia-Calvo M , Knaus H G , Garcia M L , Kaczorowski G J , Kempner E S
Published: May 2006

Target inactivation analysis was used to determine the functional size of the charybdotoxin (ChTX) receptor in aortic and tracheal sarcolemmal membrane vesicles. This receptor has previously been shown to be an integral component of the high-conductance Ca2+-activated K+ (Maxi-K) channel in these smooth muscles. Exposure of either bovine aortic or bovine tracheal sarcolemma to high-energy irradiation results in disappearance of 125I-labeled ChTX binding activity as a monoexponential function of radiation dose; from these functions molecular masses of 88 +/- 10 kDa and 89 +/- 6 kDa, respectively, can be calculated. Similar results were obtained from radiation inactivation studies with the detergent-solubilized ChTX receptor from aortic sarcolemmal membranes. The effect of radiation on 125I-labeled ChTX binding is to decrease the number of functional ChTX receptors without affecting the affinity of receptors for the toxin, indicating that radiation is destroying, rather than altering, the binding site. The validity of the radiation inactivation technique in these membrane preparations is supported by data obtained in parallel experiments in which target sizes of the alpha 1 subunit of the L-type Ca2+ channel and 5'-nucleotidase were measured. The molecular masses determined for these entities are in excellent agreement with those expected from previous studies. The present data are discussed in terms of the recently determined subunit composition of the smooth muscle Maxi-K channel. In light of the target size, a single alpha beta subunit heterodimer complex could serve as the ChTX receptor.