bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth requirements in vitro of oligodendrocyte cell lines and neonatal rat brain oligodendrocytes.
Authors: Bottenstein J E
Published: May 2006

I have defined the basic requirements for the proliferation of cell lines expressing oligodendrocyte properties and for the survival of galactocerebroside-positive oligodendrocytes derived from neonatal rat brains. Conventional serum-containing medium can be replaced by 01 medium, a chemically defined medium supplemented with insulin, transferrin, sodium selenite, and biotin. Thyroid hormone is not required. When cells are plated directly into O1 medium, the substratum has to be modified by precoating with polylysine and adding fibronectin to the medium prior to the cells. Both cell lines and brain cells can be subcultured numerous times in O1 medium without initial culture in serum-containing medium. Brain cultures can be maintained in O1 medium for several months and contain a significantly higher percentage of mature oligodendrocytes, a lower number of astrocytes, and no fibroblasts as compared to cells maintained in serum-containing medium.
Similarity of the conformation of diphtheria toxin at high temperature to that in the membrane-penetrating low-pH state.
Authors: Zhao J M , London E
Published: May 2006

At high temperature, nicked free monomers of diphtheria toxin undergo a transition to a thermally denatured state, with a midpoint of 45-50 degrees C. In this report, the high-temperature (60 degrees C) conformation has been compared to the native (neutral pH) and low-pH (pH less than 5) conformations. The low-pH and high-temperature conformations are similar although not identical. As in the conformation at low pH, and unlike the toxin in its native conformation, the protein in its conformation at high temperature is hydrophobic, has low fluorescence intensity, and has increased exposure of tryptophan to aqueous solution. As at low pH, at high temperature the circular dichroism spectrum shows at most only partial unfolding of secondary structure. In contrast, the conformation of the toxin in guanidinium chloride is much closer to a random coil. The effects of high temperature and low pH interact in the sense that sensitivity of the native conformation to one is increased by the other. That is, the transition temperature between native and thermally denatured states is decreased as pH is decreased, and the transition pH between neutral-pH and low-pH states is increased as temperature is increased. This implies that there is some region on the protein where high temperature and low pH can disrupt conformation in a similar manner. Taken together, these results indicate that the low-pH and high-temperature conformations can both be defined as partially denatured states, even though unfolding may not be extensive at low pH. Similar behavior may occur in other proteins that undergo functionally important conformational disruption at low pH.
A mutant of Escherichia coli fumarate reductase decoupled from electron transport.
Authors: Weiner J H , Cammack R , Cole S T , Condon C , Honoré N , Lemire B D , Shaw G
Published: May 2006

The terminal electron-transfer enzyme fumarate reductase of Escherichia coli is a complex iron-sulfur flavoenzyme composed of four nonidentical subunits organized into two domains: FrdA and -B (a membrane-extrinsic catalytic domain) and FrdC and -D (a transmembrane anchor domain). We have identified a mutation within the membrane-intrinsic domain that alters the electron transfer properties of the iron-sulfur and flavin redox centers of the catalytic domain. Functional electron flow from the quinone analog 2,3-dimethyl-1,4-naphthoquinone or from the electron transport chain is impaired. However, the mutant enzyme can be reduced normally by single-electron donors such as the dye benzyl viologen. The mutant phenotype results from a single A----G transition changing His-82, within the second transmembrane alpha-helix of the FrdC anchor sequence, to an arginine. The mutation, physically located within the anchor domain, is manifested by altered catalytic properties, indicating that the intrinsic and extrinsic domains are conformationally connected. These results confirm the important role of the anchor subunits in functional electron transport and have implications for communication between intrinsic and extrinsic domains of membrane proteins.
Isolation and characterization of Caenorhabditis elegans DNA sequences homologous to the v-abl oncogene.
Authors: Goddard J M , Weiland J J , Capecchi M R
Published: May 2006

DNA sequences homologous to the v-abl oncogene were isolated from a Caenorhabditis elegans genomic library by their ability to hybridize with a v-src probe. The DNA sequence of 2465 nucleotides of one clone was determined. This region corresponds to the 5' protein kinase domain of v-abl plus approximately equal to 375 base pairs toward the 3' end. Four potential introns were identified. The homology between the deduced amino acid sequence of the C. elegans clone and that of the 1.2-kilobase-pair protein kinase region of v-abl is 62%. The tyrosine residue corresponding to the tyrosine that is phosphorylated in the v-src protein is conserved in the C. elegans sequence. When 95 amino acids around this tyrosine were compared with the corresponding sequences of Drosophila c-abl, v-abl, and v-src, the identities were 83%, 79%, and 56%, respectively. Hybridization of the cloned DNA with C. elegans poly(A)+ RNA revealed a major transcript of 4.4 kilobases.
Complete reactivation of X chromosomes from human chorionic villi with a switch to early DNA replication.
Authors: Migeon B R , Schmidt M , Axelman J , Cullen C R
Published: May 2006

Mammalian sex-dosage compensation is mediated by maintaining activity of only one X chromosome. The asynchronous DNA synthesis characterizing the silent human X chromosome is thought to be reversible only during ontogeny of oocytes. We have previously shown that the glucose-6-phosphate dehydrogenase (G6PD) locus (G6PD) on the allocyclic X chromosome in chorionic villi is partially expressed. We now show that in hybrids derived from a clone of chorionic villi cells (heterozygous for G6PD A) and mouse A9 cells, the loci for G6PD, hypoxanthine phosphoribosyltransferase (HPRT) and phosphoglycerate kinase are expressed on both human X chromosomes; the human X chromosomes carrying either G6PD A or B replicate synchronously with each other and with murine chromosomes. The X chromosome with G6PD A was identified as the original late-replicating X, because methylation in the body of the HPRT gene on this chromosome remained characteristic of the inactive X chromosome. These results indicate that X-chromosome inactivation is completely reversible in cells of trophoblast origin; induction of full transcriptional activity is accompanied by acquisition of isocyclic replication, showing an intimate relationship between these processes. The molecular events responsible for this reversal may be similar to those occurring during maturation of oocytes. Chorionic villi and derivative hybrids provide in vitro models for exploring early events that program the single active X chromosome.
Detection of hepatitis B virus X product using an open reading frame Escherichia coli expression vector.
Authors: Elfassi E , Haseltine W A , Dienstag J L
Published: May 2006

The genome of the hepatitis B virus (HBV) contains a sequence, designated X, capable of encoding a protein of 154 amino acids. To determine whether the putative protein synthesized from this region is antigenic, we examined the sera of HBV-infected patients for the ability to react with a hybrid protein that contained 133 amino acids encoded by the X region and portions of the bacterial ompF and beta-galactosidase genes. Some HBV-positive sera tested contained antibodies that specifically recognized the hybrid protein. All sera were from patients diagnosed as suffering from chronic active hepatitis. We conclude that the X region of HBV encodes a protein and that this protein is antigenic in some patients.
Use of hydrophobicity profiles to predict receptor binding domains on apolipoprotein E and the low density lipoprotein apolipoprotein B-E receptor.
Authors: De Loof H , Rosseneu M , Brasseur R , Ruysschaert J M
Published: May 2006

We have used mean hydrophobicity and hydrophobic moment calculations to predict the receptor binding domains in apolipoprotein E and in the low density lipoprotein apolipoprotein B-E receptor. In apolipoprotein E, two receptor binding domains, residues 136-160 and 214-236, having a high hydrophilicity and a high mean helical hydrophobic moment, were identified. The first domain has been located experimentally and mutations influencing the hydrophobicity parameters of the binding site have been shown to affect the receptor binding. The second domain is probably, either separately or in combination with the first domain, involved in receptor binding or in heparin binding. In the low density lipoprotein apolipoprotein B-E receptor, six protein domains were identified. In the first domain (residues 1-371), eight hydrophilic maxima, organized in pairs through disulfide bonds, form the four experimentally observed receptor binding sites. These sites consist of repeats of 26 amino acids but differ from those reported by others [Yamamoto, T., Davis, C. G., Brown, M. S., Schneider, W. J., Casey, M. L., Goldstein, J. W. & Russell, D. W. (1984) Cell 39, 27-38]. The second, more hydrophobic, domain (residues 372-640) forms the core of the receptor, explaining its homology with the precursor of mouse epidermal growth factor, while the cysteine residues in the third domain (residues 641-699), interacting with those of the first domain, further stabilize the molecule. Beyond the fourth hydrophilic domain (residues 700-767), to which carbohydrates are linked, a very hydrophobic membrane spanning region (residues 768-789) could be detected easily. The last domain (residues 790-839), situated in the cytoplasma, contains hydrophilic maxima, as this region might interact with clathrin-related proteins. These data suggest that hydrophobicity analysis can detect and predict protein domains: hydrophilic receptor sites as well as hydrophobic core-forming and membrane-spanning regions.
Fractionation and characterization of a yeast mRNA splicing extract.
Authors: Cheng S C , Abelson J
Published: May 2006

We have fractionated a yeast whole cell extract that can accurately splice synthetic actin and CYH2 pre-mRNAs. Three fractions, designated I, II, and III, have been separated by use of ammonium sulfate fractionation and chromatography on heparin agarose. Each fraction alone has no splicing activity. Fractions I and II allow the first step of the splicing reaction to proceed, giving rise to the splicing intermediates, free exon 1, and intron-exon 2. Addition of fraction III completes the reaction. Micrococcal nuclease treatment of the whole cell extract or of either fraction I or II abolished splicing activity, indicating that fractions I and II have RNA moieties that are required in the splicing reaction. The nature of the RNAs was examined using antibodies directed against the trimethylated cap structure unique to small nuclear RNAs. Preincubation of the whole cell extract with protein A-Sepharose coupled to trimethylated cap antibody abolished splicing activity. This indicates that at least one essential RNA component contains a trimethyl cap. Thus, in yeast as in mammalian systems, small nuclear RNAs are involved in mRNA splicing.
Cathepsin B: association with plasma membrane in metastatic tumors.
Authors: Sloane B F , Rozhin J , Johnson K , Taylor H , Crissman J D , Honn K V
Published: May 2006

The subcellular localization of cathepsin B activity (EC 3.4.22.1) in three murine melanomas of increasing metastatic potential (Cloudman less than B16-F1 less than B16 amelanotic) was determined. Cathepsin B activity was localized in the heavy mitochondrial fraction of normal murine liver but in the light mitochondrial fraction of the metastatic melanomas; the localization of three other lysosomal hydrolases did not shift. Further purification of the light mitochondrial fraction into L-1 (density = 1.045 g/ml) and L-2 (density = 1.07 g/ml) fractions was achieved on a 30% iso-osmotic Percoll gradient. The L-1 fraction of liver and melanomas contained Na+, K+-ATPase activity; the L-2 fraction of liver contained four lysosomal hydrolase (cathepsins B and H, N-acetyl-beta-glucosaminidase, and beta-glucuronidase) and glucose-6-phosphatase activities. Ultrastructural examination revealed that the L-1 fraction consisted of membrane vesicles and the L-2 fraction of secondary lysosomes. In the B16 melanomas cathepsin B and N-acetyl-beta-glucosaminidase activities were found in both L-1 and L-2 fractions. Specific activities of the two enzymes in the plasma membrane (L-1) fractions increased in correspondence with metastatic potential. Cathepsin H and beta-glucuronidase activities were not localized in the plasma membrane fractions of the B16 melanomas. Localization of hydrolytic enzymes in the plasma membrane of metastatic tumor cells could result in focal dissolution of the extracellular matrix and thereby invasion and metastasis.
Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture.
Authors: Berthois Y , Katzenellenbogen J A , Katzenellenbogen B S
Published: May 2006

Although much attention has been paid to the removal of hormones from sera and to the development of serum-free media for studies on hormone-responsive cells in culture, little consideration has been given to the possibility that the media components themselves may have hormonal activity. We have found that phenol red, which bears a structural resemblance to some nonsteroidal estrogens and which is used ubiquitously as a pH indicator in tissue culture media, has significant estrogenic activity at the concentrations (15-45 microM) at which it is found in tissue culture media. Phenol red binds to the estrogen receptor of MCF-7 human breast cancer cells with an affinity 0.001% that of estradiol (Kd = 2 X 10(-5) M). It stimulates the proliferation of estrogen receptor-positive MCF-7 breast cancer cells in a dose-dependent manner but has no effect on the growth of estrogen receptor-negative MDA-MB-231 breast cancer cells. At the concentrations present in tissue culture media, phenol red causes partial estrogenic stimulation, increasing cell number to 200% and progesterone receptor content to 300% of that found for cells grown in phenol red-free media, thereby reducing the degree to which exogenous estrogen is able to stimulate responses. The antiestrogens tamoxifen and hydroxytamoxifen inhibit cell proliferation below the control level only when cells are grown in the presence of phenol red; in the absence of phenol red, the antiestrogens do not suppress growth. The estrogenic activity of phenol red should be considered in any studies that utilize estrogen-responsive cells in culture.
Pancreatic expression of human insulin gene in transgenic mice.
Authors: Bucchini D , Ripoche M A , Stinnakre M G , Desbois P , Lorès P , Monthioux E , Absil J , Lepesant J A , Pictet R , Jami J
Published: May 2006

We have investigated the possibility of obtaining integration and expression of a native human gene in transgenic mice. An 11-kilobase (kb) human chromosomal DNA fragment including the insulin gene (1430 base pairs) was microinjected into fertilized mouse eggs. This fragment was present in the genomic DNA of several developing animals. One transgenic mouse and its progeny were analyzed for expression of the foreign gene. Synthesis and release of human insulin was revealed by detection of the human C-peptide in the plasma and urine. Human insulin mRNA was found in pancreas but not in other tissues. These findings indicate that the 11-kb human DNA fragment carries the sequences necessary for tissue-specific expression of the insulin gene and the human regulatory sequences react to homologous signals in the mouse.
Prolactin as a modulator of lymphocyte responsiveness provides a possible mechanism of action for cyclosporine.
Authors: Hiestand P C , Mekler P , Nordmann R , Grieder A , Permmongkol C
Published: May 2006

Lymphocyte responsiveness in rats was found to depend on serum prolactin levels. Blocking pituitary prolactin release with bromocriptine severely reduces lymphocyte reactivity in vitro (mixed lymphocyte reaction) as well as in vivo (graft-versus-host reaction). In addition, evidence for a prolactin/growth hormone-related mRNA species produced in mitogen- and antigen-stimulated lymphocytes has been obtained. Prolactin was shown to compete in a dose-dependent fashion with the immunosuppressant cyclosporine (cyclosporin A) for a common binding site on the surface of T lymphocytes. Further, stimulation of prolactin secretion reversed the immunosuppression induced by cyclosporine. We conclude that prolactin is involved in the maintenance of T-cell immunocompetence and that the immunosuppressive effects of cyclosporine may be mediated by the displacement of prolactin from binding sites on lymphocytes.
Cloning and sequence analysis of complementary DNA encoding an aberrantly rearranged human T-cell gamma chain.
Authors: Dialynas D P , Murre C , Quertermous T , Boss J M , Leiden J M , Seidman J G , Strominger J L
Published: May 2006

Complementary DNA (cDNA) encoding a human T-cell gamma chain has been cloned and sequenced. At the junction of the variable and joining regions, there is an apparent deletion of two nucleotides in the human cDNA sequence relative to the murine gamma-chain cDNA sequence, resulting simultaneously in the generation of an in-frame stop codon and in a translational frameshift. For this reason, the sequence presented here encodes an aberrantly rearranged human T-cell gamma chain. There are several surprising differences between the deduced human and murine gamma-chain amino acid sequences. These include poor homology in the variable region, poor homology in a discrete segment of the constant region precisely bounded by the expected junctions of exon CII, and the presence in the human sequence of five potential sites for N-linked glycosylation.
Regulation of interleukin 2 receptors on T cells from multiple sclerosis patients.
Authors: DeFreitas E C , Sandberg-Wollheim M , Schonely K , Boufal M , Koprowski H
Published: May 2006

Receptors for interleukin 2 (IL-2) are absent on resting T lymphocytes and are induced by antigenic and mitogenic stimulation. After a limited time (8-12 days), these receptors on normal T cells are down-regulated despite the presence of receptor-saturating concentrations of IL-2. We report here that both antigen- and mitogen-induced T-cell lines and clones obtained from peripheral blood and cerebrospinal fluid of multiple sclerosis patients show prolonged expression of IL-2 receptors. This expression is coincident with a prolonged responsiveness to the proliferative effects of IL-2. In addition, Leu 3+, IL-2 receptor-positive T-cell clone from the cerebrospinal fluid of a multiple sclerosis patient has been established and maintained for more than 1 year without IL-2. This clone has some morphologic and histochemical properties of T cells transformed or infected by human T-lymphotropic virus type I.
The role of lipoproteins and receptor-mediated endocytosis in the transport of bacterial lipopolysaccharide.
Authors: Van Lenten B J , Fogelman A M , Haberland M E , Edwards P A
Published: May 2006

The addition of bacterial lipopolysaccharide (LPS) from Escherichia coli 0111:B4 to human monocyte-macrophages cultured in serum results in suppression of scavenger receptor activity. The present studies were performed to examine if the effect on scavenger receptor activity was mediated by LPS alone or by LPS in association with lipoproteins. Radioiodinated LPS (125I-LPS) was added to human plasma in vitro and to normal and hyperlipidemic rabbit plasma in vitro and in vivo to determine the distribution of 125I-LPS among the lipoprotein classes. It was found that all lipoprotein classes bound LPS in direct proportion to their plasma cholesterol concentration. LPS alone was compared to LPS bound to low density lipoprotein (LDL), high density lipoprotein, or reductively-methylated LDL for their abilities to suppress scavenger receptor activity in monocyte-macrophages in lipoprotein-free serum. Only LPS bound to LDL (LPS-LDL) demonstrated an effect similar to that observed when LPS was added to cells in serum. Either unlabeled LDL or unlabeled LPS-LDL complexes competed with the uptake of 125I-LPS-LDL complexes, which appeared to proceed by receptor-mediated endocytosis. In contrast to the uptake of 125I-LDL, the uptake of 125I-LPS-LDL by cultured monocyte-macrophages was not followed by its hydrolysis and the release of its radioactive degradation products into the medium. The association of LPS with lipoproteins was very stable and appeared to be mediated by a lipid-lipid interaction. We hypothesize that LPS bound to lipoproteins may be transported into the artery wall and may initiate the atherosclerotic reaction.
Widespread occurrence of "87 kDa," a major specific substrate for protein kinase C.
Authors: Albert K A , Walaas S I , Wang J K , Greengard P
Published: May 2006

An 87-kDa phosphoprotein, identified previously as a major, specific substrate for Ca2+/phospholipid/diacylglycerol-dependent protein kinase (protein kinase C) in broken cell preparations from rat brain, has been characterized with respect to its species, tissue, and subcellular distribution. A similar protein was present in monkey, human, mouse, and bovine brain and in Torpedo californica electric organ. The protein was also identified in a variety of nonneuronal rat and bovine tissues. The rat protein had an apparent molecular mass 4-7 kDa lower, and was slightly more acidic, than the protein in bovine tissues. The 87-kDa proteins from various bovine tissues were identical by the following criteria: each was phosphorylated by exogenous protein kinase C, was of comparable molecular mass, generated multiple spots within the pH range of 4.4-4.9 upon isoelectric focusing, yielded identical patterns upon digestion with Staphylococcus aureus V8 protease, and was recognized by a specific 87-kDa antiserum. The relative concentrations of the 87-kDa protein in bovine tissues were highest in brain, spleen, and lung, moderate in testis, pancreas, adrenal, kidney, and liver, and lowest in heart and skeletal muscle. In the brain, the 87-kDa protein was concentrated in the synaptosomal membrane and in the cytosol. The membrane-bound protein was extractable with nonionic detergents but not with NaCl. This species, tissue, and subcellular distribution of the 87-kDa protein is similar to that of protein kinase C.
Mutational analysis of the signal-anchor domain of influenza virus neuraminidase.
Authors: Sivasubramanian N , Nayak D P
Published: May 2006

Influenza virus neuraminidase (NA; EC 3.2.1.18) possesses a signal-anchor hydrophobic domain at the amino terminus. To characterize the nature of this signal-anchor domain we have introduced single amino acid changes in this domain by oligonucleotide-directed mutagenesis. Three mutant NA proteins that were synthesized contained a single charged amino acid residue in place of a hydrophobic amino acid residue at position 11, 17, or 26 of the signal-anchor domain. When the altered NA proteins were expressed in CV-1 cells, two phenotypes were observed: substitution of arginine in place of glycine at position 11 and substitution of aspartic acid for valine at position 17 did not abolish the signal, the anchor, or the transport functions. On the other hand, substitution of arginine for isoleucine at position 26 blocked the migration of the NA protein from the Golgi complex to the cell surface. Double mutants were constructed from these single point mutations and they exhibited two phenotypes: one double mutant (aspartic acid at position 17 and arginine at position 26) was present mostly in the cytoplasm and the other (arginine at positions 11 and 26) was present mostly in the rough endoplasmic reticulum. These results indicate that the hydrophobic amino acids at positions 11, 17, and 26 are required for intracellular transport. Furthermore, the accumulation of the mutant proteins in the rough endoplasmic reticulum or the Golgi apparatus suggests the existence of putative intracellular transport (or traffic) signals in the signal-anchor domain of NA.
Organization of the neural cell adhesion molecule (N-CAM) gene: alternative exon usage as the basis for different membrane-associated domains.
Authors: Owens G C , Edelman G M , Cunningham B A
Published: May 2006

The neural cell adhesion molecule, N-CAM, is expressed as at least three polypeptide chain, (ld, sd, and ssd chains) specified by a single gene and derived by alternative splicing and polyadenylation-site selection during RNA processing. We describe here the characterization of seven overlapping genomic phage clones reactive with N-CAM cDNA, indicating that the chicken N-CAM gene is more than 50 kilobases long. Analysis of the gene shows that there are at least 19 exons and that the coding sequences for the ld, sd, and ssd chains are assembled from 18, 17, and 15 exons, respectively. The first 14 exons appear to be common to all three chains and encode the amino-terminal portion of N-CAM, which contains five tandem homologous repeats resembling those seen in the immunoglobulin gene superfamily. In contrast to other genes containing such domains, each of these segments in N-CAM is specified by two exons. The carboxyl-terminal portion of each N-CAM chain is different as a result of the alternative use of exons. A single exon encodes the carboxyl-terminal 26 amino acids of the ssd chain and the 3' untranslated region of its mRNA, ending with a poly(A)-addition site. Two exons encode the transmembrane and cytoplasmic sequences common to the ld and sd chains, and another exon encodes the additional 261 amino acids found in the cytoplasmic domain of the ld chain. The carboxyl-terminal 21 amino acids common to the ld and sd chains and the 3' untranslated region common to their mRNAs are encoded by a single large exon of 3475 base pairs that ends with a second poly(A)-addition site. Sequences from the 13-kilobase intron that separates the exons encoding the amino-terminal and carboxyl-terminal regions of the molecule hybridize to a 2-kilobase poly(A)+ RNA transcript of unknown identity. This description of the chicken N-CAM gene provides a basis for determining the mechanisms that regulate the differential expression of the N-CAM polypeptide chains during development.
Sequences from a prokaryotic genome or the mouse dihydrofolate reductase gene can restore the import of a truncated precursor protein into yeast mitochondria.
Authors: Baker A , Schatz G
Published: May 2006

Sequences that are capable of restoring mitochondrial targeting to a truncated yeast cytochrome c oxidase subunit IV presequence are encoded within the genome of Escherichia coli and within the gene for a higher eukaryotic cytosolic protein, mouse dihydrofolate reductase. These sequences, which resemble authentic presequences in their overall amino acid composition and degree of hydrophobicity, are rather frequent; greater than 2.7% of clones generated from E. coli DNA and greater than 5% of clones from the dihydrofolate reductase gene were functional in our screening system. These results suggest that, during evolution, mitochondrial precursor proteins could arise as a result of DNA rearrangements that place potential mitochondrial presequences at the amino terminus of existing open reading frames. Primitive eukaryotic cells may have used this mechanism to target proteins to their endosymbiotic protomitochondria.
Synchrotron x-ray diffraction studies of actin structure during polymerization.
Authors: Matsudaira P , Bordas J , Koch M H
Published: May 2006

Synchrotron x-ray diffraction was used to identify the oligomers that formed during the earliest stages of actin polymerization. Solution diffraction patterns from G-actin (monomer) and from F-actin (polymer) contain information about the size and shape of actin monomers and the length, width, and subunit organization of filaments. Comparison of patterns collected during polymerization reveals an increase in scatter at spacings greater than 9.0 nm; formation of scattering bands at 5.4,4.9, and 3.4 nm; formation of a scattering minimum at 6.5 nm; and the presence of an isosbestic point at 9.0 nm. These scattering bands arise from the formation of, and organization of subunits in, filaments. At various actin concentrations (0.37-5 mg/ml), the change in scatter in these regions follows simple exponential kinetics with no detectable lag. Our analysis of the x-ray patterns shows that by 0.4 sec after mixing, most of the actin has formed dimers, which then rapidly incorporate into oligomers. At 4 mg/ml the early oligomers increase in length to greater than 30.0 nm within 10 sec. These results suggest that under our conditions actin molecules condense into filaments without the rate-limiting formation of nuclei.
Detection of human DNA polymorphisms with a simplified denaturing gradient gel electrophoresis technique.
Authors: Noll W W , Collins M
Published: May 2006

Single base pair differences between otherwise identical DNA molecules can result in altered melting behavior detectable by denaturing gradient gel electrophoresis. We have developed a simplified procedure for using denaturing gradient gel electrophoresis to detect base pair changes in genomic DNA. Genomic DNA is digested with restriction enzymes and hybridized in solution to labeled single-stranded probe DNA. The excess probe is then hybridized to complementary phage M13 template DNA, and the reaction mixture is electrophoresed on a denaturing gradient gel. Only the genomic DNA probe hybrids migrate into the gel. Differences in hybrid mobility on the gel indicate base pair changes in the genomic DNA. We have used this technique to identify two polymorphic sites within a 1.2-kilobase region of human chromosome 20. This approach should greatly facilitate the identification of DNA polymorphisms useful for gene linkage studies and the diagnosis of genetic diseases.
Resistance of cytotoxic T lymphocytes to lysis by a clone of cytotoxic T lymphocytes.
Authors: Kranz D M , Eisen H N
Published: May 2006

To investigate how cytotoxic T lymphocytes (CTL) avoid killing themselves when they destroy target cells, we compared 20 different cell lines as target cells, including several CTL cell lines, for their susceptibility to lysis by CTL. Variations in recognition of this diverse set of target cells was circumvented by attaching to all of them a monoclonal antibody to the antigen-specific receptor of a cloned CTL cell line (clone 2C) and using the 2C cell line as the standard aggressor or effector cell. All of the nine tumor cell lines and the four noncytolytic T-helper cell lines tested as targets were highly susceptible to lysis by the aggressor CTL, but seven cytotoxic T-cell lines (six CTL and one T-helper cell line with cytotoxic activity) were largely resistant. These results, and the use of the lectin Con A as an alternative means for triggering CTL activity, point clearly to a level of resistance that could enable CTL to avoid their own destruction when they lyse target cells. The resistance of the cytolytic T cells did not appear to be accompanied by a similar resistance to complement-mediated lysis, indicating that mechanisms of CTL-mediated and complement-mediated lysis are not identical.
gamma-Aminobutyric acid exerts a local inhibitory action on the axon terminal of bipolar cells: evidence for negative feedback from amacrine cells.
Authors: Tachibana M , Kaneko A
Published: May 2006

It is well-established morphologically that bipolar cells, the second-order neurons in the vertebrate retina, make reciprocal synapses with amacrine cells in the inner plexiform layer. However, neither the property nor the physiological function of the feedback synapse is understood. Autoradiographic and immunohistochemical studies suggest the presence of gamma-aminobutyric acid (GABA)-ergic amacrine cells, and therefore the bipolar cells are thought to receive GABAergic inputs from amacrine cells. This possibility was investigated in the present study, in which we used solitary bipolar cells dissociated from the goldfish retina enzymatically. Dissociated solitary bipolar cells showed a large variety in morphology. In the present study, we selected the bipolar cells with a huge bulbous axon terminal. Bipolar cells of this subtype were identical in morphology to the on-center cells with rod-dominant inputs as revealed in earlier studies by intracellular staining. Membrane currents were measured under voltage clamp with a patch pipette in the whole cell configuration. In some experiments, GABA-sensitive membrane was excised as an outside-out patch from the axon terminal bulb of solitary bipolar cells. All cells of this type responded to GABA. The highest sensitivity was located at the axon terminal. The minimal effective dose was on the order of 10(-7) M. GABA increased the chloride conductance and evoked a membrane hyperpolarization. Partial desensitization was observed during the application of GABA. The bipolar cells had GABA type A receptors. These results are consistent with the idea that the rod-dominant on-center bipolar cells receive negative feedback inputs from GABAergic amacrine cells.
Tunicamycin tightens the glucose- and D-allose-mediated control of hexose transport in a metabolic fibroblast mutant.
Authors: Ullrey D B , Kalckar H M
Published: May 2006

The hexose transport system of a fibroblast mutant, DS7, unable to convert glucose 6-phosphate to fructose 6-phosphate ("the phosphoglucose isomerase mutant"), is subject to a specific down-regulation ("curb") evoked by only glucose or D-allose. Neither fructose nor mannose has a curbing effect on this mutant. Further addition of tunicamycin intensified the transport curb on the mutant mediated by glucose or allose. Mannose added to the parental cell line 023 seems able to mimic a glucose-mediated transport curb. In this line, but not the mutant, tunicamycin also intensifies a mannose-mediated curb. It seems that the tightening of the allose-mediated curb is a function of a specific type of transport regulation and perhaps too of interference with glycosylation of the hexose transporter. Furthermore, this type of curb can be strikingly reversed by shifting the cultures to medium containing fructose.
The T-cell receptor gamma chain-CD3 complex: implication in the cytotoxic activity of a CD3+ CD4- CD8- human natural killer clone.
Authors: Alarcon B , De Vries J , Pettey C , Boylston A , Yssel H , Terhorst C , Spits H
Published: May 2006

A subset of human T cells has recently been described. These cells express the CD3 complex but they do not carry the classical T-cell receptor (TCR)-alpha/-beta heterodimer on their surface (WT31- CD3+). Instead, they express a TCR-gamma chain associated with another type of polypeptide termed TCR-delta. We report here that a T-cell clone with natural killer (NK)-like activity, WM-14, had a disulfide bridged TCR-gamma homodimer associated with CD3 on its surface. The TCR-gamma chains of WM-14 cells were present in three different glycosylation forms of 43, 40, and 38 kDa, but they appeared to contain the same polypeptide backbone. Since cytotoxicity by WM-14 could be inhibited by anti-CD3 antibodies, we concluded that the TCR-gamma-CD3 complex was involved in the NK-like unrestricted killer activity. Although normal CD3-gamma, CD3-delta, and CD3-epsilon chains were present in this clone, the association with the TCR-gamma homodimer may be the cause of a complete processing of the N-linked oligosaccharides attached to the CD3-delta chain.
A topological concept of smallness
Authors: Pfeffer W. F. , Prikry K.
Published: May 2006

We introduce a concept of smallness yielding topological spaces intuitively smaller than those that are simultaneously of universal measure zero and perfectly meager. Without any set-theoretic assumptions we show that there is a small uncountable algebraic subfield of the reals.
Proximal and distal regulatory elements that influence in vivo expression of a cell cycle-dependent human H4 histone gene.
Authors: Kroeger P , Stewart C , Schaap T , van Wijnen A , Hirshman J , Helms S , Stein G , Stein J
Published: May 2006

We have examined the sequences required in vivo to promote transcription of a cell cycle-regulated human H4 histone gene. Deletion mutants of the 5' flanking region were assayed in mouse cells or fused with the chloramphenicol acetyltransferase (CAT) gene for assay in HeLa cells. The functional limits of the regulatory sequences were shown to extend at least 6.5 kilobases (kb) upstream. Sequences sufficient for correctly initiated transcription were found in the 70 base pairs (bp) immediately 5' to the cap site. A proximal element located 200-400 bp upstream increased the level of transcription several times above the basal level, although not to maximal levels. Maximal levels of expression were achieved with 6.5 kb of 5' flanking sequence adjacent to the proximal promoter sequences or when a distal enhancer element with both position- and orientation-independent function was moved proximal to the promoter. Our results indicate that a series of 5' cis-acting sequences are functionally related to the fidelity and level of expression of this human H4 histone gene.
Expression of P1-450 and P3-450 DNA coding sequences as enzymatically active cytochromes P-450 in mammalian cells.
Authors: Battula N , Sagara J , Gelboin H V
Published: May 2006

Two cDNA clones representing the mRNA coding sequences for mouse cytochromes P1-450 and P3-450 were inserted into the thymidine kinase gene of the wild-type vaccinia virus under the control of the vaccinia virus promoter. Murine and human cells infected with each of the resulting infectious recombinant viruses efficiently expressed their respective P-450 proteins. The newly synthesized protein products are translocated into the microsomes, and their characterization by immunochemical analysis indicates that the sizes of the polypeptides expressed were indistinguishable from their cytochrome P-450 counterparts found in mammalian liver microsomes. Functional analysis of each of the proteins by spectral and enzymatic analysis indicates that the expressed proteins have incorporated heme, and the holoenzymes displayed catalytic activities characteristic of their respective cytochrome P-450 enzymes. Thus, this system can be used to produce properly processed and catalytically active P-450 gene products in a wide variety of cells. The remarkable fidelity of expression and processing of these enzymes suggests that the vaccinia virus recombinants can be used for a wide variety of studies, including analysis of the effects of defined mutations produced in vitro, and directly correlate the structure/activity relationships of the cytochrome P-450 enzymes.
Morphological constraint on egg size: a challenge to optimal egg size theory?
Authors: Congdon J D , Gibbons J W
Published: May 2006

Some freshwater turtles appear unable to produce eggs large enough to achieve the balance between size and number of eggs predicted by optimal egg size theory. We present evidence that pelvic girdle structure constrains egg size and thus offspring size in females of smaller-bodied species (Chrysemys picta and Deirochelys reticularia). The constraint is demonstrated by the correspondence of slopes of the increase of the pelvic aperture and egg width with increasing body size. This constraint appears to be relaxed in a larger-bodied species (Pseudemys scripta), in which the increase in pelvic aperture relative to body size is greater than the increase in egg width. This type of structural constraint on a reproductive trait should not occur unless there is strong selection on pelvic architecture for other functions, such as locomotion, support, and limb retraction, that prevent expansion of the pelvic aperture. Although other explanations may exist for other groups of organisms that appear to vary egg size, the large variation in egg size associated with body size in some species of turtles can be reconciled with optimal egg size theory only if a pelvic constraint model is accepted.
Arginine decarboxylase inhibitors reduce the capacity of Trypanosoma cruzi to infect and multiply in mammalian host cells.
Authors: Kierszenbaum F , Wirth J J , McCann P P , Sjoerdsma A
Published: May 2006

The capacity of blood (trypomastigote) forms of Trypanosoma cruzi to infect mouse peritoneal macrophages or rat heart myoblasts in vitro was inhibited by treatment of the trypomastigotes with DL-alpha-difluoromethylarginine (F2Me Arg), monofluoromethylagmatine, or (E)-alpha-monofluoromethyl-3-4-dehydroarginine--all irreversible inhibitors of arginine decarboxylase. Similar results were obtained when F2MeArg-treated parasites were incubated with rat heart myoblasts. The inhibitory effects were characterized by marked reductions in both the proportion of infected cells and the number of parasites per 100 host cells. The concentrations of the arginine decarboxylase inhibitors that affected infectivity had no detectable effect on either the concentration or motility of the parasite and, therefore, could not have affected the collision frequency. F2MeArg appeared to inhibit the ability of T. cruzi to penetrate the host cells since the drug had no significant effect on the extent of parasite binding to the surface of the host cells. The inhibitory effect of F2MeArg was markedly reduced or abrogated in the presence of either agmatine or putrescine, as would have been expected if F2MeArg acted by inhibiting arginine decarboxylase. Addition of F2MeArg to macrophage or myoblast cultures immediately after infection or at a time when virtually all of the intracellular parasites had transformed into the multiplicative amastigote form, resulted in a markedly reduced parasite growth rate. This effect was also prevented by exogenous agmatine. These results indicate the importance of polyamines and polyamine biosynthesis in the following two important functions of T. cruzi: invasion of host cells and intracellular multiplication. Furthermore, concentrations of the inhibitors tested that affected the parasite did not alter the viability of the host cells, the cellular density of the cultures, or the ability of uninfected myoblasts to grow. Thus, arginine decarboxylase inhibitors may have a potential application in chemotherapy against T. cruzi infection.
A monoclonal antibody-Pseudomonas toxin conjugate that specifically kills multidrug-resistant cells.
Authors: FitzGerald D J , Willingham M C , Cardarelli C O , Hamada H , Tsuruo T , Gottesman M M , Pastan I
Published: May 2006

One form of multidrug resistance is due to the expression of a 170-kDa energy-dependent drug efflux pump called P-glycoprotein in the plasma membranes of human cancer cells. We have prepared conjugates of Pseudomonas toxin with the anti-P-glycoprotein monoclonal antibody MRK-16. These anti-P-glycoprotein-toxin conjugates specifically kill multidrug-resistant human KB cells. Similar conjugates could be useful in cancer therapy to reduce or eliminate multidrug-resistant tumor populations in tumors intrinsically resistant to chemotherapy or in populations that become resistant during combination chemotherapy.
Ontogeny of the serotonergic projection to rat neocortex: transient expression of a dense innervation to primary sensory areas.
Authors: D'Amato R J , Blue M E , Largent B L , Lynch D R , Ledbetter D J , Molliver M E , Snyder S H
Published: May 2006

The development of serotonergic innervation to rat cerebral cortex was characterized by immunohistochemical localization of serotonin combined with autoradiographic imaging of serotonin-uptake sites. In neonatal rat, a transient, dense, serotonergic innervation appears in all primary sensory areas of cortex. In somatosensory cortex, dense patches of serotonergic innervation are aligned with specialized cellular aggregates called barrels. The dense patches are not apparent after 3 weeks of age, and the serotonergic innervation becomes more uniform in adult neocortex. This precocious neonatal serotonergic innervation may play a transient physiologic role in sensory areas of cortex or may exert a trophic influence on the development of cortical circuitry and thalamocortical connections.
Characterization of cDNA for nodulin-75 of soybean: A gene product involved in early stages of root nodule development
Authors: Franssen Henk J. , Nap Jan-Peter , Gloudemans Ton , Stiekema Willem , Van Dam Hans , Govers Francine , Louwerse Jeanine , Van Kammen Albert , Bisseling Ton
Published: May 2006

Establishment of a nitrogen-fixing root nodule is accompanied by a developmentally regulated expression of nodulin genes, only some of which, the so-called early nodulin genes, are expressed in stages preceding actual nitrogen fixation. We have isolated soybean cDNA clones representing early nodulin genes and have studied clone pENOD2 in detail. The cDNA insert of this clone hybridizes to nodule-specific RNA of 1200 nucleotides in length. The RNA that was hybrid-selected by the cloned ENOD2 DNA was
in vitro
translated to produce two nodulins with an apparent
M
r
of 75,000, the N-75 nodulins. These two nodulins differ slightly in charge and one does not contain methionine. The amino acid sequence deduced from the DNA sequence shows that proline accounts for 45% of the 240 residues in these nodulins and the sequence contains at least 20 repeating heptapeptide units. The amino acid composition of none of the (hydroxy)proline-rich (glyco)proteins described in plants resembles the composition of the N-75 nodulins, especially with respect to the high glutamic acid and the low serine content. This suggests that the N-75 nodulins belong to a hitherto unidentified class of presumably structural proteins. The genes encoding the N-75 nodulins were found to be expressed in nodule-like structures devoid of intracellular bacteria and infection threads, indicating that these nodulins do not function in the infection process but more likely function in nodule morphogenesis.
Cell autonomy of expression of neurogenic genes of Drosophila melanogaster.
Authors: Technau G M , Campos-Ortega J A
Published: May 2006

In embryos of Drosophila melanogaster the development of a pluripotent cell in the neurogenic ectoderm as a member of either a neural or an epidermal lineage depends on its interactions with neighboring cells. Certain genes, designated neurogenic, participate in this process in that there is a deficiency of epidermal histotypes in mutant embryos lacking neurogenic gene functions. To test the cell autonomy of expression of the neurogenic phenotype, individual cells were transplanted from the neurogenic ectoderm of mutant donor embryos into wild-type host embryos. Cells transplanted from donors homozygous for any of several mutant alleles of the neurogenic genes amx, N, bib, mam, neu, and Dl were found to give rise to clones exhibiting a distribution of neural and epidermal histotypes similar to that of the wild type. By contrast, cells transplanted from donors homozygous for loss of the neurogenic E(spl) gene gave rise exclusively to clones of neural histotypes. Thus, only the expression of E(spl) is autonomous, with that of all of the other tested neurogenic genes being nonautonomous. These results are consistent with the inference that the nonautonomous genes provide a source and the autonomous gene provides a receptor of a hypothetical intercellular regulatory signal that is necessary for cell commitment to an epidermal rather than neural fate.