bluuebunny/crossref_metadata_2025 huggingface dataset:
Lack of DNA homology in a pair of divergent chromosomes greatly sensitizes them to loss by DNA damage.
Authors: Resnick M A , Skaanild M , Nilsson-Tillgren T
Published: May 2006

Chromosomal DNA is considered a priori to be a target for the induction of numerical (whole chromosome) aneuploidy in mitotic cells. If true, DNA repair would be expected to contribute to genome stability. One type of repair that appears to play an important role in the response of many organisms to DNA-damaging agents involves recombination. Using the yeast Saccharomyces cerevisiae containing a pair of DNA divergent (homoeologous) chromosomes, we have been able to determine the importance of recombinational repair of DNA damage in the maintenance of chromosome number. Specifically, the induction of aneuploidy by ionizing radiation has been examined in diploids that had one chromosome III replaced by a divergent chromosome from Saccharomyces carlsbergensis. The chromosomes are functionally equivalent but lack precise DNA homology over one-half their length. The absence of homology, and thus the opportunity for recombinational repair (presumably of DNA double-strand breaks) in the divergent chromosomes, results in high levels (5-10%) of aneuploidy for chromosome III at doses of radiation resulting in almost no killing. For homologous chromosomes, the frequency of loss is 20-50 times lower.
Molecular basis of adult-onset and chronic GM2 gangliosidoses in patients of Ashkenazi Jewish origin: substitution of serine for glycine at position 269 of the alpha-subunit of beta-hexosaminidase.
Authors: Paw B H , Kaback M M , Neufeld E F
Published: May 2006

Chronic and adult-onset GM2 gangliosidoses are neurological disorders caused by marked deficiency of the A isoenzyme of beta-hexosaminidase; they occur in the Ashkenazi Jewish population, though less frequently than classic (infantile) Tay-Sachs disease. Earlier biosynthetic studies had identified a defective alpha-subunit that failed to associate with the beta-subunit. We have now found a guanosine to adenosine transition at the 3' end of exon 7, which causes substitution of serine for glycine at position 269 of the alpha-subunit [designated 269 (Gly----Ser) substitution]. An RNase protection assay was used to localize the mutation to a segment of mRNA from fibroblasts of a patient with the adult-onset disorder. That segment of mRNA (after reverse transcription) and a corresponding segment of genomic DNA were amplified by the polymerase chain reaction and sequenced by the dideoxy method. The sequence analysis, together with an assay based on the loss of a ScrFI restriction site, showed that the patient was a compound heterozygote who had inherited the 269 (Gly----Ser) mutation from his father and an allelic null mutation from his mother. The 269 (Gly----Ser) mutation, in compound heterozygosity with a presumed null allele, was also found in fetal fibroblasts with an association-defective phenotype and in cells from five patients with chronic GM2 gangliosidosis. It was not found in beta-hexosaminidase A-deficient cells obtained from patients with infantile Tay-Sachs disease nor in cells from individuals who do not have beta-hexosaminidase A deficiency. However, there must be additional mutations with similar consequences, since the 269 (Gly----Ser) substitution was not present in fibroblasts from two patients with juvenile GM2 gangliosidosis even though these had an association-defective alpha-subunit.
Activation of single-channel currents in mouse fibroblasts by platelet-derived growth factor.
Authors: Frace A M , Gargus J J
Published: May 2006

A nonselective cation channel that we characterized in the mouse L-cell membrane becomes quiescent with serum deprivation (arrested cell growth) and rapidly active upon readdition of serum or, specifically, platelet-derived growth factor (PDGF). Using the patch-clamp technique, we find that the predominant channel in the LMTK- cell line is a bursting nonselective cation channel (the NS channel). In cell-attached and inside-out patches, the channel has a conductance of 28 pS; equal selectivity for Na+, K+, and Cs+; and no anion or divalent cation permeability. The channel open probability is voltage insensitive and in inside-out patches does not correlate with intracellular calcium (0.5 nM to 50 microM). When cultures are rendered quiescent by incubation in serum-free medium, channel open probability is virtually 0 as compared to 0.26 (+/- 0.17) in exponentially growing cultures. If mitogenesis is initiated by readdition of serum to quiescent cells while maintaining cell-attached recording, there is a rapid (15-30 s) activation of the channel (n = 12). The open probability of the patch increases (greater than 0.75) for 2-3 min and then decreases. We have attempted applications of several growth factors (fibroblast-derived growth factor, epidermal growth factor, insulin, bombesin, alpha-thrombin, and vasopressin, individually or in combination) but find that only PDGF (5-100 ng/ml; n = 9) produces channel activation. This activation should provide a Na+ entry pathway parallel to that of the Na/H exchanger.
Identification and purification of a single-stranded-DNA-specific exonuclease encoded by the recJ gene of Escherichia coli.
Authors: Lovett S T , Kolodner R D
Published: May 2006

The Escherichia coli recJ gene product was overproduced using a plasmid that carries the recJ gene downstream of a strong regulatable promoter and a strong ribosome-binding site. Overexpression of recJ produced a concomitant increase in the levels of single-stranded-DNA-specific nuclease activity present in crude cell extracts. This nuclease activity was purified to homogeneity and found to reside in a 60-kDa polypeptide. This polypeptide was induced with recJ overexpression and had the size and N-terminal amino acid sequence identical to the predicted RecJ protein sequence. The RecJ nuclease degraded linear single-stranded DNA but did not have exonuclease activity on linear double-stranded substrates or endonuclease activity on either single-stranded or double-stranded substrates. The RecJ exonuclease had greater activity on duplex DNA molecules with 5'-rather than 3'-single-stranded tails.
Yeast GCN4 transcriptional activator protein interacts with RNA polymerase II in vitro.
Authors: Brandl C J , Struhl K
Published: May 2006

Regulated transcription by eukaryotic RNA polymerase II (Pol II) requires the functional interaction of multiple protein factors, some of which presumably interact directly with the polymerase. One such factor, the yeast GCN4 activator protein, binds to the upstream promoter elements of many amino acid biosynthetic genes and induces their transcription. Through the use of affinity chromatography involving GCN4- or Pol II-Sepharose columns, we show that GCN4 interacts specifically with Pol II in vitro. Purified Pol II is retained on the GCN4-Sepharose column under conditions in which the vast majority of proteins flow through. Moreover, Pol II can be selectively isolated from more complex mixtures of proteins. Conversely, GCN4 protein, synthesized in vitro or in Escherichia coli, specifically binds to the Pol II-Sepharose column under equivalent conditions. Using deletion mutants, we also show that the DNA-binding domain of GCN4 is both necessary and sufficient for this interaction. We suggest the possibility that this GCN4-Pol II interaction may be important for transcription in vivo.
Monte Carlo simulation of equilibrium globular protein folding: alpha-helical bundles with long loops.
Authors: Sikorski A , Skolnick J
Published: May 2006

To help elucidate the general rules of globular protein folding, computer simulations of the conformational transition in model proteins having the left-handed, four-helix bundle motif in which the helices are joined by one or two long loops, as in apoferritin and somatotropin, respectively, have been undertaken. In the context of simple tetrahedral lattice protein models, these unique native helix bundle motifs can be obtained by a set of interactions similar to those found in previous simulations of the folding of four-member alpha-helical bundles with tight bends and beta-barrel proteins including the Greek key motif. The essential features sufficient to produce the four-helix bundle motif with long loops are as follows: (i) a general pattern of hydrophobic and hydrophilic type residues which differentiate the interior from the exterior of the molecule; (ii) the existence of hydrophilic regions in the amino acid sequence that, on the basis of short-range interactions, are indifferent to loop formation but that interact favorably with all the exterior residues of the helix bundle. Thus, these simulations indicate that, to reproduce all varieties of the left-handed four-helix bundle motif, site-specific interactions are not required.
Acidic fibroblast growth factor mRNA is expressed by cardiac myocytes in culture and the protein is localized to the extracellular matrix.
Authors: Weiner H L , Swain J L
Published: May 2006

Acidic and basic fibroblast growth factors are heparin-binding proteins that induce cellular proliferation, mesodermal development, and vascular growth. As such, they may be important in cardiac development and disease. To determine whether cardiac myocytes contain fibroblast growth factors, neonatal rat cardiac myocytes were studied in primary culture and compared to primary cultures of nonmyocyte cardiac cells. Northern blot analysis revealed a 4.0-kilobase mRNA in myocytes that hybridized to acidic fibroblast growth factor cDNA and was not detectable in nonmyocyte cultures. Western blot analysis demonstrated the accumulation of a 15-kDa peptide with immunological identity to acidic fibroblast growth factor in extracts of extracellular matrix from myocyte cultures that was not detectable in similar extracts of nonmyocyte extracellular matrix. No acidic fibroblast growth factor-like protein was detectable in cellular lysates from either myocyte or nonmyocyte cultures. These results demonstrate that neonatal cardiac myocytes express acidic fibroblast growth factor mRNA and deposit a protein with immunological identity to acidic fibroblast growth factor into the extracellular matrix. The results suggest that acidic fibroblast growth factor produced by cardiac myocytes may mediate, through both paracrine and autocrine mechanisms, such diverse processes as myocyte differentiation, cellular proliferation, and vascular growth in the heart.
Modulation of mitogenic stimuli by angiogenin correlates with in vitro phosphatidylinositol bisphosphate synthesis.
Authors: Heath W F , Moore F , Bicknell R , Vallee B L
Published: May 2006

125I-labeled angiogenin binds rapidly to the plasma membrane of several cell lines at 37 degrees C (t1/2 less than 1 min) but in comparatively small amounts. Competition with unlabeled angiogenin varies markedly with different cell lines, being most effective in vascular smooth muscle and fibroblast cells. Angiogenin modulates mitogenic stimuli in bovine adrenal capillary endothelial (BACE), rat aortic smooth muscle (RASM), and fibroblast (3T3) cells. Thus, it enhances the mitogenic effect of certain conditioned media on RASM and 3T3 cells, but it inhibits the mitogenic effect on BACE cells. In RASM and 3T3 cells, mitogenesis is increased at low (less than 5 ng/ml) and high (greater than 100 ng/ml) but not at intermediate concentrations of angiogenin. Plasma membranes from RASM and 3T3 cells that have been treated with angiogenin show an enhanced in vitro synthesis of phosphatidylinositol bisphosphate (PtdInsP2) with an angiogenin concentration dependence similar to that of enhanced mitogenesis. PtdInsP2 synthesis arises by activation of a fatty acid (arachidonyl) coenzyme A synthetase and either a plasma membrane fatty acid acyltransferase or phosphatidylinositol kinase(s), or both. Increased PtdInsP2 or the derived second messengers (e.g., diacylglycerol) may mediate modulation of the mitogenic stimulus. The differential mitogenic interaction of angiogenin with several cell types, either stimulation or inhibition, probably reflects the multistep nature of angiogenesis.
Ammonia and thermotaxis: Further evidence for a central role of ammonia in the directed cell mass movements of
Dictyostelium discoideum
Authors: Bonner J. T. , Har D. , Suthers H. B.
Published: May 2006

Evidence is presented to support the hypothesis that, in addition to its possible role in mediating chemo- and phototaxis, ammonia (NH
3
) is also the key substance responsible for directing thermotaxis of the migrating slugs of
Dictyostelium discoideum
. NH
3
is produced by the cells of the slug and we show that high and low concentrations of NH
3
decrease the speed of the amoebae while intermediate concentrations increase their speed. NH
3
production by amoebae is affected by temperature: the greater the temperature, the more NH
3
is produced. From these facts we speculate that both the positive and the negative thermotaxis found in slugs can be explained by temperature gradients stimulating regional differences in NH
3
production, and depending upon the temperature, the amount of NH
3
will either be in the range that stimulates or inhibits the rate of movement. If this explanation is correct, then minute localized differences in the production of NH
3
and their differential effect on cell speed could account for all the directed movements of the cell masses of these slime molds.
3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis thaliana is structurally distinct from the yeast and animal enzymes.
Authors: Learned R M , Fink G R
Published: May 2006

We have isolated the Arabidopsis thaliana gene (HMG1) encoding 3-hydroxy-3-methylglutaryl-CoA reductase [HMG-CoA reductase; (S)-mevalonate:NAD+ oxido-reductase (CoA-acylating), EC 1.1.1.88], the catalyst of the first committed step in isoprenoid biosynthesis. cDNA copies of the plant gene were identified by hybridization with a short, highly conserved segment of yeast HMG-CoA reductase as probe. DNA sequence analysis reveals that the COOH-terminal domain of the Arabidopsis HMG-CoA reductase (containing the catalytic site of the enzyme) is highly conserved with respect to the yeast, mammalian, and Drosophila enzymes, whereas the membrane-bound amino terminus of the Arabidopsis protein is truncated and lacks the complex membrane-spanning architecture of the yeast and animal reductases. Expression of the Arabidopsis gene from the yeast GAL1 promoter in a yeast mutant lacking HMG-CoA reductase activity suppresses the growth defect of the yeast mutant. Taken together, the sequence similarity to other cloned HMG-CoA reductase genes and the suppression of the yeast hmg- mutant provide strong evidence that the novel Arabidopsis gene we have cloned encodes a functional HMG-CoA reductase enzyme.
Gene-controlled cytosine demethylation in the promoter region of the
Ac
transposable element in maize
Authors: Schwartz Drew
Published: May 2006

The transposase product of the
Ac
transposable element causes demethylation of the
Bam
HI site in the promoter region of
Ac
. This site remains methylated in
Ds9
, the deletional derivative of
Ac
that fails to make an active transposase. In the presence of an active
Ac
element the
Bam
HI site of
Ds9
is demethylated. Transcription of the element is correlated with the demethylation of the promoter region. Evidence is presented that supports the model that the trans-acting protein causes site-specific demethylation by inducing a configuration change in chromatin structure making the site inaccessible to the methylase. A chromosome alteration that involves the insertion of the 4.5-kilobase
Ac
element results in the methylation of
Pvu
I target sites about 1 kilobase removed from the point of insertion.
Structural homology of complement protein C6 with other channel-forming proteins of complement.
Authors: Chakravarti D N , Chakravarti B , Parra C A , Muller-Eberhard H J
Published: May 2006

The amino acid sequence of the amino-terminal half of the complement protein C6 has been found to show overall structural homology with the homologous regions of the channel-forming proteins C7, C8 alpha, C8 beta, and C9. In addition, two specific cysteine-rich segments common to the amino-terminal regions of C7, C8 alpha, C8 beta, and C9 also occur in their expected positions in C6, suggesting functional significance. Two cDNA clones encoding C6 were isolated from a human liver library in the bacteriophage vector lambda gt11. The predicted protein sequence contains an apparent initiation methionine and a putative signal peptide of 21 residues, as well as a site for N-glycosylation at residue 303. The sequence of the C6 protein reported here has 47-52% similarity with C7, C8 alpha, C8 beta, and C9, as well as 31-38% similarity with thrombospondin, thrombomodulin, and low density lipoprotein receptor. The sequence data have been interpreted by using computer algorithms for estimation of average hydrophobicity and secondary structure.
Cytolytic T-cell clones against an autologous human melanoma: specificity study and definition of three antigens by immunoselection.
Authors: Knuth A , Wölfel T , Klehmann E , Boon T , Meyer zum Büschenfelde K H
Published: May 2006

Cytolytic T-lymphocyte (CTL) clones against an autologous melanoma (SK-MEL-29) were generated by mixed lymphocyte tumor culture and subsequent cloning of responder lymphocytes at limiting dilutions. These CTL clones lysed autologous melanoma but not autologous Epstein-Barr virus-transformed B cells and none of the allogeneic tumor targets included in the specificity analysis. The lysis of autologous melanoma targets could be inhibited by monoclonal antibodies against monomorphic HLA class I determinants. For proliferation of CTLs, the stimulation with the relevant target antigen on autologous tumor cells was essential. Immunoselection experiments carried out with two CTL clones revealed the existence of melanoma subclones that were resistant to lysis by the CTL clones used for immunoselection but were still lysed by other autologous CTL clones. This analysis allowed us to identify three stable simultaneously expressed antigens on the melanoma cells defined by autologous CTLs.
Fluorinated 4-quinolones induce hyperproduction of interleukin 2.
Authors: Riesbeck K , Andersson J , Gullberg M , Forsgren A
Published: May 2006

The fluorinated 4-quinolones are a "new" group of antibiotics with a broad antibacterial spectrum. They are already widely used in clinical practice. Previous studies have shown that these drugs increase the uptake of [3H]thymidine into DNA of mitogen-stimulated lymphocytes but inhibit cell growth and immunoglobulin secretion. This study shows that the 4-quinolones strongly (up to 100 times) increase the recovery of interleukin 2 (IL-2) in culture supernatants of phytohemagglutinin (PHA)-stimulated normal human lymphocytes and also prolong the kinetics of IL-2 production. The effect was significant at clinically achievable concentrations (5 micrograms/ml). In addition to hyperproduction of IL-2, the level of RNA hybridizing with a human IL-2 cDNA probe was also intensely elevated (16-32 times) in PHA-stimulated lymphocytes cultured with ciprofloxacin (80 micrograms/ml). The mechanism responsible for 4-quinolone-mediated effects on T cells is at present unclear, but evidence is presented that suggests the effect is not exerted at the level of protein kinase C activation. Ciprofloxacin at 80 micrograms/ml also decreased the expression of IL-2 receptors measured by immunofluorescence with CD 25 antibodies and a radiolabeled IL-2 binding assay. At the same concentration of ciprofloxacin, there was a very low expression of the transferrin receptor and the cell size increased very little in human lymphocytes after PHA stimulation. The enhanced IL-2 production by 4-quinolones may contribute to side effects reported when these drugs are used for treatment of patients.
Purification and sequencing of neuropeptides contained in neuron R15 of Aplysia californica.
Authors: Weiss K R , Bayley H , Lloyd P E , Tenenbaum R , Kolks M A , Buck L , Cropper E C , Rosen S C , Kupfermann I
Published: May 2006

R15 is a large identified neuron present in the abdominal ganglion of the mollusc Aplysia. Previous studies have indicated that this neuron may play a role in water balance and possibly renovascular functions. A peptidic factor contained in the neuron R15 has been shown to increase the water content of Aplysia. To determine the structure of the peptides contained in R15, we purified the extracts of 820 R15 cells by means of two steps of reverse-phase HPLC. The purification yielded a number of peptides, only one of which, R15 alpha 1, resulted in water uptake when injected into animals. Determination of the amino acid content and sequence analysis of the R15 alpha 1 peptide demonstrated that this peptide contains 38 residues, including two cysteines. The peptide failed to react with iodoacetate, indicating that the two cysteines are connected by a disulfide bridge. To confirm the assigned structure, the peptide was synthesized with a disulfide bridge. The chromatographic properties and bioactivity of the synthetic material were identical to those of the native peptide. Several other R15 peptides were inactive in the bioassay for water uptake. The sequence of one of these peptides (R15 beta) was determined, and it was established that the peptide contains 28 residues. Amino acid analysis of three other peaks was performed. One of these peaks contained a peptide (R15 beta f) whose amino acid composition suggests that it is a fragment of the R15 beta peptide. The other two peaks contained peptides with identical amino acid compositions, suggesting that they are variants of a single peptide (R15 gamma). The amino acid sequences of all the peptides identified in neuron R15 correspond to stretches of a polyprotein encoded by a recently sequenced R15 cDNA.
Voltage dependence of adaptation and active bundle movement in bullfrog saccular hair cells.
Authors: Assad J A , Hacohen N , Corey D P
Published: May 2006

Hair cells of the bullfrog sacculus adapt to maintained displacement stimuli in a manner that suggests an active regulation of the tension stimulus reaching transduction channels. We have examined adaptation in dissociated hair cells by whole-cell patch-clamp recording and video microscopy. Adaptation was present in these cells, and it depended on extracellular calcium. The adaptation rate--as well as the position of the resting current-displacement curve--also depended on membrane potential, suggesting that calcium passes into the cytoplasm to reach its site of action. After abrupt hyperpolarization, the adaptation rate increased within milliseconds, suggesting that the calcium site is within a few micrometers of the ion channels through which calcium enters. The voltage dependence of the resting current-displacement curve, together with the "gating springs" hypothesis for transduction, predicts movement of the bundle away from the kinocilium when the cell is depolarized. This was observed.
Repair of N-methylpurines in specific DNA sequences in Chinese hamster ovary cells: absence of strand specificity in the dihydrofolate reductase gene.
Authors: Scicchitano D A , Hanawalt P C
Published: May 2006

We have developed a quantitative method for examining the removal of N-methylpurines from specific genes to investigate their possible differential repair throughout the genome. Chinese hamster ovary cells were exposed to dimethyl sulfate, and the isolated DNA was treated with an appropriate restriction endonuclease. The DNA was heated to convert remaining N-methylpurines to apurinic sites to render them alkaline-labile. Duplicate samples heated in the presence of methoxyamine to protect the apurinic sites from alkaline hydrolysis provided controls to assess total DNA. After alkaline hydrolysis, agarose gel electrophoresis, Southern transfer, and probing for the fragment of interest, the ratios of band intensities of the test DNA sample to its methoxyamine-treated control counterpart were calculated to yield the percentage of fragments containing no alkaline-labile sites. The frequency of N-methylpurines was measured at different times after dimethyl sulfate treatment to study repair. We found no differences between the rates of repair of N-methylpurines in the active dihydrofolate reductase gene and a nontranscribed region located downstream from it in treated cells. Also, similar rates of repair were observed in the transcribed and nontranscribed strands of the gene, in contrast to previous results for the removal of cyclobutane pyrimidine dimers. Thus, there does not appear to be a coupling of N-methylpurine repair to transcription in Chinese hamster ovary cells. However, the repair in the dihydrofolate reductase domain appears to be somewhat more efficient than that in the genome overall. Our method permits the quantifying at the defined gene level of abasic sites or of any DNA adduct that can be converted to them.
Differential use of termination codons in ciliated protozoa.
Authors: Harper D S , Jahn C L
Published: May 2006

Sequence analysis of genes in four species of ciliated protozoa and analysis of tRNAs in Tetrahymena has demonstrated that TAG and TAA encode glutamine or glutamic acid in these organisms and TGA is the only stop codon. Thus, it has generally been assumed that all ciliates use a nonuniversal genetic code in which TGA acts as the sole termination codon. We have sequenced the linear DNA molecules that carry an actin gene and a beta-tubulin gene from the ciliate Euplotes crassus. These genes are shown to use TAA as a termination codon based on homology to known actin and beta-tubulin gene sequences. In addition, we have sequenced a portion of the 3' terminus of the E. crassus H4 histone gene and show that it also uses TAA as a termination codon. These data indicate that the timing of genetic code changes in the ciliates must be reconsidered.
Physical association of CD4 and the T-cell receptor can be induced by anti-T-cell receptor antibodies.
Authors: Rojo J M , Saizawa K , Janeway C A
Published: May 2006

CD4 can physically associate with the CD3-T-cell receptor complex as visualized in cocapping experiments. This association occurs when the T-cell receptor is cross-linked by certain anti-variable region antibodies that appear to induce a conformational change in the receptor such that it associate with CD4. Similar association has been observed in earlier studies with the same cloned helper T cell when the physiological ligand, antigen-class II major histocompatibility complex molecule, is bound by the T-cell receptor. The ability of anti-T-cell receptor antibodies to induce the T-cell receptor-CD4 association correlates with a 100-fold increase in the ability of the antibody to activate the T cell. This suggests that the complex of CD4 and the T-cell receptor act synergistically in T-cell activation, thus readily explaining the commonly observed association of CD4 expression with class II major histocompatibility complex-restricted antigen recognition. This association could also play a role in infection by human immunodeficiency virus.
Nerve growth factor receptor immunoreactivity is transiently associated with the subplate neurons of the mammalian cerebral cortex.
Authors: Allendoerfer K L , Shelton D L , Shooter E M , Shatz C J
Published: May 2006

Nerve growth factor and its receptor (NGFR) are known to be present in diverse embryonic and neonatal central nervous system tissues, including the cerebral cortex. However, the identity of the cortical cells expressing NGFR immunoreactivity has not been established. We have used immunolabeling coupled with [3H]thymidine autoradiography to identify such cells in ferret and cat brain. Polyclonal antibodies raised against a synthetic peptide corresponding to a conserved amino acid sequence of the NGFR were used for this purpose. Western (immunologic) blot analyses show that these antibodies specifically recognize NGFR and precursor proteins. In both species, NGFR immunoreactivity is primarily associated with the early generated and transient subplate neuron population of the developing neocortex, as indicated by the following evidence: the immunoreactive cells (i) are located directly beneath the developing cortical plate, (ii) frequently have the inverted pyramid shape characteristic of subplate neurons, and (iii) can be labeled by an injection of [3H]thymidine on embryonic day (E) 28, a time when only subplate neurons are being generated. Intense NGFR immunostaining is seen on the cell bodies of these neurons as early as E30, several days after their last round of cell division, and this immunostaining remains strong for approximately 3 weeks. The NGFR immunoreactivity begins to decline around E52 and has disappeared from the region altogether by E60, at which time subplate neurons begin to die. The cellular localization and timing of expression suggest that the NGFR may play a role in the maintenance of subplate neurons and in the maturation of the cerebral cortex.
Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.
Authors: Daniell H , Vivekananda J , Nielsen B L , Ye G N , Tewari K K , Sanford J C
Published: May 2006

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.
Identification of the proteolytically activated form of protein kinase C in stimulated human neutrophils.
Authors: Pontremoli S , Michetti M , Melloni E , Sparatore B , Salamino F , Horecker B L
Published: May 2006

The proteolytically activated form of protein kinase C has been identified in human neutrophils by using a monoclonal antibody that recognizes both the native kinase and the catalytically active proteolytic fragment (protein kinase M). Stimulation with fMet-Leu-Phe results in the conversion of approximately 30% of native protein kinase C to protein kinase M, with little evidence of further degradation. Stimulation with phorbol 12-myristate 13-acetate, on the other hand, causes only a transient formation of protein kinase M, with complete loss of total kinase activity. These differences are related to the differences in biochemical responses, reported earlier, in neutrophils exposed to these two activators.
Trans-dominant negative mutants of Fos and Jun.
Authors: Ransone L J , Visvader J , Wamsley P , Verma I M
Published: May 2006

Jun and Fos nuclear oncoproteins form a complex that regulates transcription from promoters containing activator protein AP-1 binding sites. The leucine-zipper and basic-region domains of both Fos and Jun are necessary for formation of the heterodimer that binds to DNA. Reciprocal mutations in the basic region of Fos or Jun can influence the binding of the heterodimer to DNA, implying a symmetrical binding site. DNA-binding mutants of Jun exhibit increased affinity for Fos and are capable of suppressing wild-type Fos-Jun DNA-binding activity. In contrast, mutations in the basic domain of Fos, which prevent binding to DNA in association with Jun, do not significantly diminish the ability of the wild-type heterodimer to bind to DNA. These dominant negative mutants are functional in vivo and can be exploited to study the role of Fos and Jun in normal and transformed cells.
Enhanced resolution of DNA restriction fragments: a procedure by two-dimensional electrophoresis and double-labeling.
Authors: Yi M , Au L C , Ichikawa N , Ts'o P O
Published: May 2006

A probe-free method was developed to detect DNA rearrangement in bacteria based on the electrophoretic separation of twice-digested restriction fragments of genomic DNA into a two-dimensional (2-D) pattern. The first restriction enzyme digestion was done in solution, followed by electrophoresis of the restriction fragments in one dimension. A second restriction enzyme digestion was carried out in situ in the gel, followed by electrophoresis in a second dimension perpendicular to the first electrophoresis. The 2-D pattern provides for the resolution of 300-400 spots, which are defined and indexed by an "x,y" coordinate system with size markers. This approach has greatly increased the resolution power over conventional one-dimensional (1-D) electrophoresis. To study DNA rearrangement, a 2-D pattern from a test strain was compared with the 2-D pattern from a reference strain. After the first digestion, genomic DNA fragments from the test strain were labeled with 35S, while those from the reference strain were labeled with 35P. This was done to utilize the difference in the energy emission of 35S and 32P isotopes for autoradiography when two x-ray films were exposed simultaneously on top of the gel after the 2-D electrophoresis. The irradiation from the decay of 35S exposed only the lower film, whereas the irradiation from the decay of 32P exposed both the lower and upper films. Different DNA fragments existed in the test DNA compared with the reference DNA can be identified unambiguously by the differential two 2-D patterns produced on two films upon exposure to the 35S and 32P fragments in the same gel. An appropriate photographic procedure further simplified the process, allowing only the difference in DNA fragments between these two patterns to be shown in the map. We have utilized the difference map obtained from Escherichia coli strains HB101 and HB101 (lambda) genomic DNA to show the incorporation of one copy of phage lambda DNA without the use of a lambda DNA probe. This is the same test system that was used previously.
Glucocorticoid receptor-dependent disruption of a specific nucleosome on the mouse mammary tumor virus promoter is prevented by sodium butyrate.
Authors: Bresnick E H , John S , Berard D S , LeFebvre P , Hager G L
Published: May 2006

Our laboratory has previously developed cell lines derived from mouse NIH 3T3 fibroblasts and C127 mammary tumor cells that stably express mouse mammary tumor virus (MMTV) long terminal repeat fusion genes in bovine papillomavirus-based episomes. Glucocorticoid hormone strongly activates transcription from episomes and induces the disruption of a single nucleosome in an array of phased nucleosomes on the MMTV promoter. Sodium butyrate inhibits the glucocorticoid hormone-dependent development of a nuclease-hypersensitive site that is due to the displacement of this nucleosome, and inhibits induction of RNA transcripts from episomes. Saturation binding studies show that butyrate treatment does not significantly affect the amount or the hormone-binding affinity of the glucocorticoid receptor. In a transient transfection assay, glucocorticoid hormone can activate transcription from a MMTV long terminal repeat-driven luciferase gene construct equivalently in untreated and butyrate-treated cells, indicating that the soluble factors necessary for transactivation of the MMTV promoter are unaffected by butyrate. The differential effect of butyrate on the induction of stable chromatin templates and transiently expressed plasmids suggests that butyrate prevents nucleosome displacement and represses transcription by inducing a modification of chromatin.
Reactivity of primary biliary cirrhosis sera with Escherichia coli dihydrolipoamide acetyltransferase (E2p): characterization of the main immunogenic region.
Authors: Fussey S P , Ali S T , Guest J R , James O F , Bassendine M F , Yeaman S J
Published: May 2006

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial autoantibodies in the serum. The major antigens recognized by the antibodies are the E2 components of the 2-oxo acid dehydrogenase complexes, all of which possess covalently attached lipoic acid cofactors. A bacterial etiology has been proposed for the disease, and patients' antibodies are known to recognize the E2 subunits (E2p) of both mammalian and bacterial pyruvate dehydrogenase complexes. Immunoblotting and ELISA inhibition techniques using extracts of Escherichia coli deletion strains, genetically restructured E2 polypeptides, and isolated lipoyl domains demonstrate that (i) the E2o subunit of the E. coli 2-oxoglutarate dehydrogenase complex is recognized by patients' antibodies; (ii) the main immunogenic region of E2p lies within the lipoly domains; (iii) the presence of a lipoly residue within the domain is crucial for effective recognition by the antibodies; and (iv) octanoylated E2p, octanoylated E2o, and octanoylated lipoyl domain, produced by a mutant deficient in lipoate biosynthesis, are recognized by patients' antibodies but not as effectively as their lipoylated counterparts. These findings indicate that antibodies in PBC patients' sera bind to a unique peptide-cofactor conformation within the lipoyl domains of the E2 polypeptides and that this epitope is partially mimicked by substituting the lipoyl cofactor with an octanoyl group.
Chlorophyll regulates accumulation of the plastid-encoded chlorophyll apoproteins CP43 and D1 by increasing apoprotein stability.
Authors: Mullet J E , Klein P G , Klein R R
Published: May 2006

Chlorophyll apoprotein accumulation in higher plant chloroplasts is controlled by light-dependent chlorophyll formation. Dark-grown plants lack chlorophyll and chlorophyll apoproteins. However, the plastid genes encoding the chlorophyll apoproteins are transcribed; chlorophyll apoprotein mRNA accumulates and associates with polysomes in plastids of dark-grown plants. Pulse-labeling assays revealed a population of short-lived proteins in plastids of dark-grown plants. One of these transiently labeled proteins was CP43, a chlorophyll apoprotein associated with photosystem II. Pulse-chase assays showed that newly synthesized CP43 was rapidly degraded in plastids of dark-grown plants, which lack chlorophyll. In contrast, CP43 synthesized in plastids from illuminated plants was stable. The synthesis of D1, a chlorophyll apoprotein of the photosystem II reaction center, was also analyzed in plastids of dark-grown and illuminated plants. Radiolabel accumulation into full-length D1 was only detected in plastids of illuminated plants. However, D1 translation intermediates of 15-25 kDa were detected in both plastid populations. Pulse-chase assays showed that the 15- to 25-kDa D1 translation products were precursors of mature D1 in plastids of illuminated plants. In contrast, in plastids of dark-grown plants, the 15- to 25-kDa translation intermediates were converted into a 23-kDa polypeptide previously suggested to be a proteolytic product of D1. These results indicate that chlorophyll produced in illuminated plants stabilizes D1 nascent polypeptides, which allows accumulation of mature D1.
Phospholipid in the hexagonal II phase is immunogenic: evidence for immunorecognition of nonbilayer lipid phases in vivo.
Authors: Rauch J , Janoff A S
Published: May 2006

Immunization of mice with phosphatidylethanolamine in the hexagonal II phase but not the bilayer phase resulted in the induction of anti-phospholipid antibodies. These antibodies, which were strongly reactive with phosphatidylethanolamine and crossreactive with cardiolipin, had functional lupus anticoagulant activity and were characteristic of autoantibodies common in patients with autoimmune disease. Recognition of the hexagonal II phase by the afferent limb of the immune system suggests that nonbilayer phospholipids can arise in the course of membrane remodeling and induce the autoantibodies of disease.
An RNA polymerase II promoter containing sequences upstream and downstream from the RNA startpoint that direct initiation of transcription from the same site.
Authors: Nakatani Y , Brenner M , Freese E
Published: May 2006

The gfa gene encodes glial fibrillary acidic protein (GFAP), an intermediate-filament protein expressed primarily in glial cells. We have used in vitro transcription studies to show that the basal level of transcription of the human gene encoding GFAP is controlled by two distinct initiators--i.e., promoter elements that direct transcription from a specific start site. One initiator is located about 25 base pairs upstream from the transcription start site, contains a TATA box, and apparently acts together with a sequence found around the transcription start site. The other initiator is located between +11 and +50 bp downstream from the transcription start site. Most of this second region overlaps with the protein-encoding sequence, which starts at bp +17. The sensitivity of transcription to alpha-amanitin indicates that both initiators are used by RNA polymerase II.
Expression of human alpha-globin and mouse/human hybrid beta-globin genes in murine hemopoietic stem cells transduced by recombinant retroviruses.
Authors: Li C L , Dwarki V J , Verma I M
Published: May 2006

Murine cell lines releasing helper-free recombinant retroviruses containing human alpha-globin and mouse/human hybrid beta-globin genes were generated. The expression of the hybrid beta-globin gene but not the human alpha-globin gene was regulated appropriately in infected mouse erythroid leukemia (MEL) cells. Murine bone marrow cells were infected by coculture with virus-producing cells and transplanted into lethally irradiated syngeneic recipients. Greater than 90% of the spleen colonies (12-15 days), which are derived from hemopoietic multipotential stem cells, showed proviral integration. Various levels of expression of the transduced globin genes were detected in all of the provirus-positive spleen colonies. Proviral sequences and transcripts from the transduced globin genes could also be detected in a few long-term reconstituted recipients in an observation period of 10 months after transplantation.
Refolding of Escherichia coli dihydrofolate reductase: sequential formation of substrate binding sites.
Authors: Frieden C
Published: May 2006

The kinetics of refolding of Escherichia coli dihydrofolate reductase (EC 1.5.1.3) have been examined upon dilution of unfolded enzyme in 4.5 M urea to 1.29 M urea in 0.02 M phosphate buffer (pH 7.2) at 10 degrees C. Changes in the intrinsic protein fluorescence on refolding are characterized by four phases. Based on changes in the amplitudes of these phases, as a consequence of quenching of the intrinsic fluorescence by ligands, it is possible to determine the step at which a ligand binds during the refolding process. The results show that either NADP or NADPH binds to the last species formed in a sequence involving three intermediates between the unfolded and native states. Dihydrofolate, on the other hand, binds during the formation of the second observed intermediate. When refolding is performed in the presence of methotrexate, an analogue of dihydrofolate, and NADPH, NADPH binds, as determined from changes in NADPH fluorescence, to the third observed intermediate rather than the last (fourth) species formed. Measurements of the recovery of enzymatic activity during refolding suggest that dihydrofolate also induces NADPH binding prior to the final observed folding phase. These results define more closely the formation of structural domains during the folding of dihydrofolate reductase.
Internal deletions in the yeast transcriptional activator HAP1 have opposite effects at two sequence elements.
Authors: Kim K S , Pfeifer K , Powell L , Guarente L
Published: May 2006

In this report we study the effects of internal deletions of the yeast transcriptional activator HAP1 (CYP1) on activity at two dissimilar DNA binding sites, upstream activation sequence 1 (UAS1) of CYC1 (iso-1-cytochrome c) and CYC7 (iso-2-cytochrome c). These deletions remove up to 1061 amino acids of the 1483-residue protein and bring the carboxyl-terminal acidic activation domain closer to the amino-terminal DNA-binding domain. Surprisingly, the deletions have opposite effects at the two sites; activity at UAS1 increases with deletion size, while activity at CYC7 decreases. The mutant with the largest deletion, mini-HAP1, has no measurable activity at CYC7 but binds normally to the site in vitro. In contrast, a protein with the DNA-binding domain of HAP1 fused to the acidic activation domain of GAL4 is active at both UAS1 and CYC7. These findings are discussed in the context of two models that suggest how the DNA sequence can alter the activity of the bound HAP1. In a separate experiment, we generate a mutation in the DNA-binding domain of HAP1 that requires the addition of zinc for binding to either UAS1 or CYC7 in vitro. This finding shows that a zinc finger anchors DNA binding to both types of HAP1 sites.
Regulation of the class II-associated invariant chain gene in normal and mutant B lymphocytes.
Authors: Doyle C , Ford P J , Ponath P D , Spies T , Strominger J L
Published: May 2006

The invariant chain protein is intracellularly associated with class II major histocompatibility proteins. In many cases, the expression of these molecules appears to be regulated in a similar manner. Contained within the promoter of the invariant chain gene are sequences (X and I gamma 1) that are similar to the X and Y box elements of class II genes, suggesting that these sequences might be involved in its regulation. DNase I footprinting reveals additional cis-acting elements (I gamma 2 and I gamma 3) that contain sequence similarities to NF-kappa B and/or H2TF1/KBF1 recognition sequences. A series of fusion constructs with the chloramphenicol acetyltransferase reporter gene were used to analyze the role of these sequences (I gamma 1, I gamma 2, I gamma 3, and X and Y elements) in both normal and mutant B lymphocytes. These data suggest the likelihood of multiple X box proteins in B cells, which can act as both negative and positive regulatory factors.