bluuebunny/crossref_metadata_2025 huggingface dataset:
Exogenous expression of mouse interferon gamma cDNA in mouse neuroblastoma C1300 cells results in reduced tumorigenicity by augmented anti-tumor immunity.
Authors: Watanabe Y , Kuribayashi K , Miyatake S , Nishihara K , Nakayama E , Taniyama T , Sakata T
Published: May 2006

To examine the influence of interferon gamma (IFN-gamma) on tumorigenicity, we established constitutively IFN-gamma-producing cell lines from a malignant mouse neuroblastoma, C1300, by retroviral transfer of a mouse IFN-gamma cDNA. The gene-transferred cells generally showed an enhanced high-level expression of the major histocompatibility complex class I antigens at the cell surface and the transcription levels, irrespective of their IFN-gamma-producing potential. Although in vitro cell growth of these cells was unaffected by the IFN-gamma production, their s.c. tumor growth in syngeneic A/J mice was dependent upon levels of IFN-gamma production; tumors induced by a low-producer line grew well at a rate similar to those induced by the parental one, but tumor growth of a high-producer line was strongly suppressed. This apparent tumor suppression was abolished by simultaneous i.p. injection of anti-Lyt2.2 and/or anti-IFN-gamma monoclonal antibodies, and subsequently large tumors of the high producer were generated. Anti-asialoganglioside GM1 antibodies allowed the high-producer line to induce a substantial but only transient tumor growth, whereas other antibodies, such as anti-Lyt2.1, anti-IFN-beta, and anti-activated macrophage, had no such effect. The mice immunized with the high-producer line were resistant to tumor growth of the parental cells but permitted another kind of A/J tumor line, Sa-1, to induce remarkable tumors. These results indicate that the reduced tumorigenicity of the IFN-gamma high-producer line was due to the augmented specific anti-tumor immunity, in which cytotoxic T lymphocytes seemed to play a decisive role, probably as a result of the immunomodulatory effects of the IFN-gamma derived from the tumor.
Blocking of passive sensitization of human mast cells and basophil granulocytes with IgE antibodies by a recombinant human epsilon-chain fragment of 76 amino acids.
Authors: Helm B , Kebo D , Vercelli D , Glovsky M M , Gould H , Ishizaka K , Geha R , Ishizaka T
Published: May 2006

The recombinant peptide corresponding to residues 301-376 at the junction of constant regions 2 and 3 of the human IgE epsilon chain blocked the in vivo passive sensitization of human skin mast cells and in vitro sensitization of human basophil granulocytes with human IgE antibodies. An injection of the recombinant peptide or E myeloma protein into normal skin sites 1 hr before sensitization with an allergic serum blocked passive sensitization. In this system, approximately 10-fold higher molar concentration of the recombinant peptide than E myeloma protein was required for 50% inhibition of Prausnitz-Küstner reactions. When the mononuclear cells of two normal individuals were preincubated with the recombinant peptide or E myeloma protein for 15 min before passive sensitization with the same allergic serum and the cells were challenged with an optimal concentration of an antigen, approximately 11- to 13-fold higher concentration of the recombinant peptide than E myeloma protein was required for 50% inhibition of antigen-induced histamine release. Further studies with several recombinant peptides indicated that amino acid resides 363-376 in the Fc epsilon-chain fragment are not essential for binding of the peptide to Fc epsilon-chain receptor I.
Detection of "deleted" mitochondrial genomes in cytochrome-c oxidase-deficient muscle fibers of a patient with Kearns-Sayre syndrome.
Authors: Mita S , Schmidt B , Schon E A , DiMauro S , Bonilla E
Published: May 2006

Using in situ hybridization and immunocytochemistry, we studied a muscle biopsy sample from a patient with Kearns-Sayre syndrome (KSS) who had a deletion of mitochondrial DNA (mtDNA) and partial deficiency of cytochrome-c oxidase (COX; EC 1.9.3.1). We sought a relationship between COX deficiency and abnormalities of mtDNA at the single-fiber level. COX deficiency clearly correlated with a decrease of normal mtDNA and, conversely, deleted mtDNA was more abundant in COX-deficient fibers, especially ragged-red fibers. The distribution of mtRNA had a similar pattern, suggesting that deleted mtDNA is transcribed. Immunocytochemistry showed that the nuclear DNA-encoded subunit IV of COX was present but that the mtDNA-encoded subunit II was markedly diminished in COX-deficient ragged-red fibers. Because the mtDNA deletion in this patient did not comprise the gene encoding COX subunit II, COX deficiency may have resulted from lack of translation of mtRNA encoding all three mtDNA-encoded subunits of COX.
Identification of negative regulatory genes that govern the expression of early meiotic genes in yeast.
Authors: Strich R , Slater M R , Esposito R E
Published: May 2006

Mutations in Saccharomyces cerevisiae have been identified that derepress early meiotic genes functioning in separable pathways required for normal meiotic development. The phenotypes of these ume (unscheduled meiotic gene expression) mutations suggest that their wild-type alleles encode negative regulators acting downstream of both the cell-type and nutritional controls of meiosis. These newly defined loci do not affect either general transcription or transcription of meiotic genes expressed later in meiosis and spore formation.
Initiation of transcription on nucleosomal templates.
Authors: Wolffe A P , Drew H R
Published: May 2006

We describe a simple method that uses curved DNAs to move nucleosomes relative to a sequence of interest. With this method, small changes in the association of a T7 RNA polymerase promoter with a nucleosome are shown to lead to significant changes in transcription efficiency.
Specific disruption of intermediate filaments and the nuclear lamina by the 19-kDa product of the adenovirus E1B oncogene.
Authors: White E , Cipriani R
Published: May 2006

The 19-kDa protein encoded within the adenovirus E1B gene is essential for transformation by adenovirus and for proper regulation of viral early gene transcription. In order to investigate the biological function of the 19-kDa E1B protein, vectors were constructed to produce the 19-kDa protein in mammalian cells under the direction of heterologous promoters. Surprisingly, during transient expression, the E1B 19-kDa protein specifically associated with and disrupted the organization of intermediate filaments and the nuclear lamina, without disturbing the organization of other cytoskeletal networks. These results directly demonstrate an effect of a viral transforming protein on the cytoskeleton and suggest a role for intermediate filaments and the nuclear lamina in modulation of viral gene expression and the process of oncogenic transformation.
An experimental approach to testing modular evolution: directed replacement of alpha-helices in a bacterial protein.
Authors: DuBose R F , Hartl D L
Published: May 2006

We have used oligonucleotide site-directed mutagenesis to ask whether certain structural motifs in proteins are determined mainly by local interactions among amino acids. Multiple consecutive amino acids in three alpha-helices in the alkaline phosphatase (EC 3.1.3.1) of Escherichia coli have been replaced with helical sequences from four other sources. Altogether, 12 distinct helical replacements were created, 9 of which retain enzymatic activity. Most short stretches of helical sequence can be replaced with unrelated helical sequences without eliminating enzyme activity. Replacements of the carboxyl half of an alpha-helix are less harmful than those of the amino half, and the two together are synergistic rather than additive. These results are consistent with the hypothesis that proteins originally evolved by the assembly of small functional folding units.
Organization of corticocortical connections in human visual cortex.
Authors: Burkhalter A , Bernardo K L
Published: May 2006

Clinical and psychophysical observations indicate that the visual cortex is critical for the perception of color, form, depth, and movement. Little, however, is known about the cortical circuitry that underlies these functions in humans. In an attempt to learn more about these connections, we have traced projections of primary (V1) and secondary (V2) visual cortex in the postmortem, fixed human brain, using the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate as an axonal marker. The results show that V1 makes a forward projection to layers 3 and 4 of V2, and V2 projects back to layers 1, 2, 3, 5, and 6 of V1. Some V2 injections also show an input to layer 4B of V1. Projections to 4B probably originate from cytochrome oxidase (CO)-reactive stripes that we have identified in V2. Differential connections between CO-rich (blobs) and CO-poor regions (interblobs) also exist within V1; blobs are connected to blobs and interblobs are connected to interblobs. The results show that the connections in human visual cortex are similar to those of nonhuman primates and that their organization is consistent with the concept of multiple processing streams in the visual system.
Altered sensitivity of system A amino acid transport to ouabain in normal and transformed C3H-10T1/2 cells during the cell cycle.
Authors: Leister K J , Schenerman M A , Racker E
Published: May 2006

Quiescent C3H-10T1/2 mouse fibroblasts that have not undergone any type of stress have a relatively low rate of 2-aminoisobutyrate (Aib) uptake by means of system A, which is primarily energized by the transmembrane Na+ chemical gradient potential. System A activity in these cells is not sensitive to ouabain or proton ionophores. In contrast, methylcholanthrene-transformed and confluent C3H-10T1/2 cells treated with 0.4 mM ouabain for 16-20 hr utilize the membrane potential generated by the Na+, K+-ATPase pump to drive Aib transport by means of system A as shown by the sensitivity of transport activity to ouabain and proton ionophores. Since glucose is present during the assay, the proton ionophores do not affect the availability of ATP, as indicated by the undiminished uptake of 86Rb+ by the Na+, K+-ATPase pump. As cells progress through the G1 phase of the cell cycle, they show an increased system A activity prior to entry into the S phase, which is also dependent on the electrogenicity of the Na+, K+-ATPase pump. There appears to be in all these cases a qualitative shift in the bioenergetic mechanism for the uptake of Aib as well as a marked quantitative increase in Aib uptake. The high activity after ouabain treatment was sustained in the transformed cells after removal of the ouabain, whereas in the confluent 10T1/2 cells the rate of uptake decayed rapidly, suggesting a difference in the mode of regulation. We conclude that transformed cells and normal cells in late G1 or under stress make use of the membrane potential generated by the Na+, K+-ATPase pump to drive amino acid uptake by means of system A.
Expression of human ferredoxin and assembly of the [2Fe-2S] center in Escherichia coli.
Authors: Coghlan V M , Vickery L E
Published: May 2006

A cDNA fragment encoding human ferredoxin, a mitochondrial [2Fe-2S] protein, was introduced into Escherichia coli by using an expression vector based on the approach of Nagai and Thøgersen [Nagai, K. & Thøgersen, M. C. (1984) Nature (London) 309, 810-812]. Expression was under control of the lambda PL promoter and resulted in production of ferredoxin as a cleavable fusion protein with an amino-terminal fragment derived from bacteriophage lambda cII protein. The fusion protein was isolated from the soluble fraction of induced cells and was specifically cleaved to yield mature recombinant ferredoxin. The recombinant protein was shown to be identical in size to ferredoxin isolated from human placenta (13,546 Da) by NaDodSO4/PAGE and partial amino acid sequencing. E. coli cells expressing human ferredoxin were brown in color, and absorbance and electron paramagnetic resonance spectra of the purified recombinant protein established that the [2Fe-2S] center was assembled and incorporated into ferredoxin in vivo. Recombinant ferredoxin was active in steroid hydroxylations when reconstituted with cytochromes P-450scc and P-450(11) beta and exhibited rates comparable to those observed for ferredoxin isolated from human placenta. This expression system should be useful in production of native and structurally altered forms of human ferredoxin for studies of ferredoxin structure and function.
Structural transitions in crystals of native aspartate carbamoyltransferase.
Authors: Gouaux J E , Lipscomb W N
Published: May 2006

Screened precession x-ray photographs of crystals of native aspartate carbamoyltransferase (EC 2.1.3.2, from Escherichia coli) ligated with L-aspartate and phosphate reveal the presence of a crystal unit-cell dimension that is intermediate between the T (tense) and R (relaxed) states. Characterizing the intermediate (I) crystal is a c-axis unit-cell dimension of 149 A, halfway between the c-axis length of the T (c = 142 A) and R (c = 156 A) states, in the space group P321. Preservation of the P321 space group indicates that the intermediate crystal form retains a threefold axis of symmetry, and therefore the enzyme has at minimum a threefold axis; however, we do not know whether the molecular twofold axis is conserved. The I crystals are formed by soaking T-state crystals with L-aspartate and phosphate. By raising the concentration of L-aspartate we can further transform the I crystals, without fragmentation, to a form that has the same unit-cell dimensions as R-state crystals grown in the presence of N-(phosphonoacetyl)-L-aspartate.
Iloprost-induced translocation of a 23-kDa protein that is recognized by a Gs alpha antiserum.
Authors: Molina Y Vedia L , Lapetina E G
Published: May 2006

We have observed that iloprost, a prostacyclin analog, causes the translocation of a 23-kDa protein from the membrane to the cytosol of human platelets. This 23-kDa protein is recognized by a Gs alpha (alpha subunit of stimulatory guanine nucleotide binding regulatory protein) antiserum but is not ADP-ribosylated by cholera toxin. The translocation of the Gs alpha-related protein might be an important part of the complex chain of events resulting in agonist-induced desensitization.
Inhibition of growth of nonbasal planes in ice by fish antifreezes.
Authors: Raymond J A , Wilson P , DeVries A L
Published: May 2006

Peptide and glycopeptide antifreezes from a variety of cold-water fishes cause ice single crystals grown from the melt to assume unusual and strikingly similar habits. The antifreezes inhibit growth on the prism faces but allow limited growth on the basal plane. As new layers are deposited on the basal plane, pyramidal surfaces develop on the outside of the crystal, and large hexagonal pits form within the basal plane. The pits are rotated 30 degrees with respect to the normal orientation of hexagonal ice crystals. Growth inhibition on the prism, pyramidal, and pit faces indicates that these faces contain sites of adsorption of the antifreeze molecules. Several properties of the antifreeze pits are consistent with (but do not prove) an origin of the pits at dislocations. The similarity of crystal habit imposed on ice by antifreezes with wide differences in composition and structure indicates a common mechanism.
Respiratory burst oxidase of fertilization.
Authors: Heinecke J W , Shapiro B M
Published: May 2006

Partially reduced oxygen species are toxic, yet sea urchin eggs synthesize H2O2 in a "respiratory burst" at fertilization, as an extracellular oxidant to crosslink their protective surface envelopes. To study the biochemical mechanism for H2O2 production, we have isolated an NADPH-specific oxidase fraction from homogenates of unfertilized Strongylocentrotus purpuratus eggs that produces H2O2 when stimulated with Ca2+ and MgATP2-. Concentrations of free Ca2+ previously implicated in regulation of egg activation modulate the activity of the oxidase. Inhibitors were used to test the relevance of this oxidase to the respiratory burst of fertilization. Procaine, two phenothiazines, and N-ethylmaleimide (but not iodoacetamide) inhibited H2O2 production by the oxidase fraction and oxygen consumption by activated eggs. The ATP requirement suggested that protein kinase activity might regulate the respiratory burst of fertilization; consonant with this hypothesis, H-7 and staurosporine were inhibitory. The respiratory burst oxidase of fertilization is an NADPH:O2 oxidoreductase that appears to be regulated by a protein kinase; although it bears a remarkable resemblance to the neutrophil oxidase, unlike the latter it does not form O2- as its initial product.
Divergent evolution of part of the involucrin gene in the hominoids: unique intragenic duplications in the gorilla and human.
Authors: Teumer J , Green H
Published: May 2006

The gene for involucrin, an epidermal protein, has been remodeled in the higher primates. Most of the coding region of the human gene consists of a modern segment of repeats derived from a 10-codon sequence present in the ancestral segment of the gene. The modern segment can be divided into early, middle, and late regions. We report here the nucleotide sequence of three alleles of the gorilla involucrin gene. Each possesses a modern segment homologous to that of the human and consisting of 10-codon repeats. The early and middle regions are similar to the corresponding regions of the human allele and are nearly identical among the different gorilla alleles. The late region consists of recent duplications whose pattern is unique in each of the gorilla alleles and in the human allele. The early region is located in what is now the 3' third of the modern segment, and the late, polymorphic region is located in what is now the 5' third. Therefore, as the modern segment expanded during evolution, its 3' end became stabilized, and continuing duplications became confined to its 5' end. The expansion of the involucrin coding region, which began long before the separation of the gorilla and human, has continued in both species after their separation.
Mitogenicity and down-regulation of high-affinity interleukin 2 receptor by YTA-1 and YTA-2, monoclonal antibodies that recognize 75-kDa molecules on human large granular lymphocytes.
Authors: Nakamura Y , Inamoto T , Sugie K , Masutani H , Shindo T , Tagaya Y , Yamauchi A , Ozawa K , Yodoi J
Published: May 2006

A large number of interleukin 2 receptors lacking the Tac epitope (IL-2R/p75) were found to be constitutively expressed on the human large granular lymphocyte/natural killer cell line YT, which bears inducible IL-2R/p55 associated with Tac antigen. Two anti-YT IgG1 monoclonal antibodies, YTA-1 and YTA-2, recognizing different epitopes of the same 75- to 80-kDa molecule, were established. The 75-kDa antigen recognized by these monoclonal antibodies was strongly expressed on the large granular lymphocytes of normal peripheral blood mononuclear cells and on various lymphoid cell lines bearing IL-2R/p75. The YTA-1 and YTA-2 antibodies were mitogenic and were different from other mitogenic monoclonal antibodies such as anti-T3 (CD3), anti-T11 (CD2), and KOLT-2 (CD28). Further, they down-regulated the high-affinity IL-2R of peripheral blood mononuclear cells within 24 hr in culture. The relationship between the YTA-1/2 antigen and the IL-2R system is discussed.
Identity of human B-cell line cytotoxic lymphokine with tumor necrosis factor type beta.
Authors: Yamanaka H I , Karpas A
Published: May 2006

A humoral cytotoxic protein that is spontaneously produced by a cloned human lymphoblastoid cell line (K160b) was partially purified by a simple three-step column chromatography procedure and NaDodSO4/polyacrylamide gel electrophoresis. Proteins were electrically blotted onto a polyvinylidene difluoride membrane, and a band of the cytotoxic protein was excised after staining with Coomassie brilliant blue. Direct analysis of the amino acid sequence of this protein showed the primary structure of its N-terminal region was identical to that of natural tumor necrosis factor type beta (TNF-beta). The 24-kDa molecular mass of the cytotoxic protein, determined by NaDodSO4/PAGE, and its elution profiles from various types of columns correlated with those of natural TNF-beta. Specific activity of the cytotoxicity, standardized with recombinant TNF-beta, was comparable to that of the purified factor. However, polyclonal antiserum to recombinant TNF-beta failed to react with the purified factor. Since recombinant TNF-beta, when used in patients, causes unacceptable side effects, which may be due to absence of glycosylation, the cell line K160b could be a useful source of natural TNF-beta for clinical trials.
Helodermin-like peptides in thyroid C cells: stimulation of thyroid hormone secretion and suppression of calcium incorporation into bone.
Authors: Grunditz T , Persson P , Håkanson R , Absood A , Böttcher G , Rerup C , Sundler F
Published: May 2006

Helodermin is a vasoactive intestinal peptide-like peptide in the salivary gland venom of the lizard Heloderma suspectum. Helodermin-like immunofluorescence was observed in the parafollicular (C) cells in several mammals and in the C cell homologues of the chicken ultimobranchial gland. Thus, helodermin-like peptides coexist with calcitonin. The results of radioimmunoassay agreed with the immunocytochemical findings. HPLC of rat thyroid extracts revealed one major peak of helodermin-like immunoreactivity, which eluted in a position close to that of lizard helodermin. Helodermin stimulated basal thyroid hormone secretion and colloid droplet formation in conscious mice. The effect of large doses of helodermin was quite long-lasting and the maximal response occurred after 2-6 hr. In addition, helodermin suppressed the incorporation of calcium into bone in conscious rats. The findings suggest that helodermin-like peptides in C cells may be involved in the local regulation of thyroid hormone secretion and in the maintenance of calcium homeostasis.
Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease.
Authors: Fuller R S , Brake A , Thorner J
Published: May 2006

The KEX2-encoded endoprotease was overproduced in yeast several hundred-fold and further purified to achieve a 10,000-fold enrichment in specific activity. The enzyme was (i) membrane-bound, but solubilized by detergents; (ii) able to cleave peptide substrates at both Lys-Arg and Arg-Arg sites; (iii) inhibited by EDTA and EGTA (but not o-phenanthroline), but fully reactivated by Ca2+; (iv) unaffected by 5-10 mM phenylmethylsulfonyl fluoride, N alpha-(ptosyl)lysine chloromethyl ketone, or L-1-tosylamido-2-phenylethyl chloromethyl ketone, but inactivated by 1-2 microM Ala-Lys-Arg-chloromethyl ketone; (v) labeled specifically by 125I-labeled Tyr-Ala-Lys-Arg-chloromethyl ketone; and (vi) resistant to trans-epoxysuccinate compounds (which inactivate thiol proteases), but inactivated by diisopropyl fluorophosphate (a diagnostic serine protease inhibitor). Mutant enzyme molecules lacking as many as 200 C-terminal residues still retained Ca2+-dependent protease activity and were labeled by 125I-labeled Tyr-Ala-Lys-Arg-chloromethyl ketone.
Transforming growth factor alpha may be a physiological regulator of liver regeneration by means of an autocrine mechanism.
Authors: Mead J E , Fausto N
Published: May 2006

We investigated whether transforming growth factor alpha (TGF-alpha) is involved in hepatocyte growth responses both in vivo and in culture. During liver regeneration after partial hepatectomy in rats, TGF-alpha mRNA increased; it reached a maximum (approximately 9-fold higher than normal) at the peak of DNA synthesis. The message and the peptide were localized in hepatocytes and found in higher amounts in hepatocytes obtained from regenerating liver. TGF-alpha caused a 13-fold elevation of DNA synthesis in hepatocytes in primary culture and was slightly more effective than epidermal growth factor. TGF-beta blocked TGF-alpha stimulation when added either simultaneously with TGF-alpha or a day later. TGF-alpha message increased in hepatocytes stimulated to undergo DNA synthesis by TGF-alpha or epidermal growth factor, and the peptide was detected in the culture medium by RIA. In the regenerating liver, the increase in TGF-alpha mRNA during the first day after partial hepatectomy coincided with an increase in epidermal growth factor/TGF-alpha receptor mRNA and a decrease (already reported) in the number of these receptors. We conclude that TGF-alpha may function as a physiological inducer of hepatocyte DNA synthesis during liver regeneration by means of an autocrine mechanism and that its stimulatory effects in this growth process are balanced by the inhibitory action of TGF-beta 1.
Immunological function of the blood-cerebrospinal fluid barrier.
Authors: Nathanson J A , Chun L L
Published: May 2006

Because the brain lacks a true lymphatic system, it is unclear how peripheral lymphocytes recognize foreign antigens present in the central nervous system. This report demonstrates that the choroid plexus, which constitutes the blood-cerebrospinal fluid barrier, is able to present foreign antigen to, and stimulate the proliferation of, peripheral helper T lymphocytes through an Ia-dependent, major histocompatibility complex-restricted mechanism. Furthermore, in vivo, choroid plexus epithelial cells have access to, and are capable of taking up, virus-sized particles injected elsewhere into the cerebrospinal fluid. Thus these data suggest that the blood-cerebrospinal fluid barrier may play a role in immunological communication between the central nervous system and periphery, a function relevant to the initiation of immunological responses to central nervous system infections and autoimmune processes and for the surveillance of tumor cells in the cerebrospinal fluid.
Stimulus-specific neuronal oscillations in orientation columns of cat visual cortex.
Authors: Gray C M , Singer W
Published: May 2006

In areas 17 and 18 of the cat visual cortex the firing probability of neurons, in response to the presentation of optimally aligned light bars within their receptive field, oscillates with a peak frequency near 40 Hz. The neuronal firing pattern is tightly correlated with the phase and amplitude of an oscillatory local field potential recorded through the same electrode. The amplitude of the local field-potential oscillations are maximal in response to stimuli that match the orientation and direction preference of the local cluster of neurons. Single and multiunit recordings from the dorsal lateral geniculate nucleus of the thalamus showed no evidence of oscillations of the neuronal firing probability in the range of 20-70 Hz. The results demonstrate that local neuronal populations in the visual cortex engage in stimulus-specific synchronous oscillations resulting from an intracortical mechanism. The oscillatory responses may provide a general mechanism by which activity patterns in spatially separate regions of the cortex are temporally coordinated.
Optical imaging of the spatial distribution of beta-particles emerging from surfaces.
Authors: Charpak G , Dominik W , Zaganidis N
Published: May 2006

The multiplication in gases of ionization electrons, by the effect of the electric fields between parallel electrodes, leads to the emission of light from the molecules excited in the avalanche process. The optical imaging of this light, with intensifiers, on charge-coupled devices permits the localization, in the gaseous volume, of the entrance points of the beta-particles emitted by radioactive compounds placed close to or at the cathode electrode. Thin slices of anatomical samples labeled with 3H show detailed structures 30 microns in size. Gels carrying 32P or 35S are imaged with accuracies of the order of 0.5 mm (full width at half maximum). In comparison with photographic emulsion, the gain in time for data taking is close to a factor of 100, with the advantage of linearity and wider dynamic range in the intensity measurement and a greatly improved signal-to-noise ratio.
Molecular cloning of rat type 2C (IA) protein phosphatase mRNA.
Authors: Tamura S , Lynch K R , Larner J , Fox J , Yasui A , Kikuchi K , Suzuki Y , Tsuiki S
Published: May 2006

A full-length cDNA encoding rat type 2C (IA) protein phosphatase was isolated from a kidney cDNA library. The cDNA was identified by screening the library with oligonucleotides based on a partial amino acid sequence determined from purified rat liver phosphatase. This clone is 2.35 kilobase pairs long and has a single extended translation reading frame that predicts a 382-amino acid protein of 42,416 daltons. The deduced amino acid sequence contains segments corresponding to three peptides from rat liver type 2C protein phosphatase and two peptides from rabbit skeletal muscle type 2C phosphatase. Rat kidney type 2C protein phosphatase is distantly related to yeast adenylate cyclase but is not related to the catalytic subunits of two other protein phosphatases (types 1 and 2A).
Rate of evolution of a character without epistasis
Authors: Nagylaki Thomas
Published: May 2006

It is shown that the rate of change of the mean of a character under selection is [Formula: see text] where
C, ¯W, Δ̄Z
, and
B
denote the genic (additive genetic) covariance of the character and fitness (i.e., the covariance of the average effect on the character and the average excess for fitness of every allele that affects the character), the mean fitness, the average of the changes in the genotypic values of the character over the next generation, and a correction term, respectively. Generations are discrete and nonoverlapping; the monoecious population mates at random. The character is determined by arbitrarily many multiallelic loci without epistasis; the linkage map is also arbitrary. If there is no dominance, then
B
= 0; if, in addition, the genotypic values of the character are constant, then Δ
¯Z
=
C/¯W
. Even if dominance is present, for weak selection and slowly varying genotypic values, Δ
¯Z
≈
C/¯W
. If the character is fitness itself, then
C
is the genic variance in fitness.
Isolation of a transposable element from Neurospora crassa.
Authors: Kinsey J A , Helber J
Published: May 2006

A Neurospora crassa strain from Adiopodoumé, Ivory Coast, contains multiple copies of a transposable element, Tad. The element was detected as a 7-kilobase insertion in two independently isolated spontaneous forward mutants of the am (glutamate dehydrogenase) gene. Laboratory strains do not contain Tad. All progeny from crosses of the Adiopodoumé strain to laboratory strains contain multiple copies. When the element was inserted in am, target sequences of 14 and 17 base pairs were duplicated in the two cases analyzed. One mutation, caused by the insertion of Tad at the beginning of the am coding sequence, is genetically stable. The other mutation, caused by insertion upstream of the transcriptional start site, has a reversion frequency of 2.5 x 10(-3). Precise excisions of Tad have not been found.
Immunoglobulin receptor signaling depends on the carboxyl terminus but not the heavy-chain class.
Authors: Webb C F , Nakai C , Tucker P W
Published: May 2006

To examine the isotypic and structural requirements involved in signaling through the immunoglobulin (Ig) receptor on B lymphocytes, we established a panel of T15 idiotype-positive transfectants that expressed wild-type IgM, wild-type IgD, or hybrid IgM molecules. Growth inhibition of the transfected lymphoma cells in response to anti-idiotype antibodies was used to measure signaling. Hybrid IgM molecules were constructed so that the membrane-spanning region of the mu heavy chain was replaced by that of delta, gamma 2b, or alpha heavy chains or that of the I-Ab class II (Ia) alpha chain. All transfectants that expressed IgM or hybrid IgM molecules with membrane-spanning regions from another Ig isotype underwent signaling in response to anti-idiotype antibodies, whereas the IgM-Ia hybrid transfectants did not. Transfectants that expressed wild-type IgD molecules also underwent signaling, although this response was particularly sensitive to serum concentrations. These results imply that signaling occurs in a similar manner through heavy-chain receptors of any isotype and suggest that conserved amino acid sequences in the transmembrane regions are important in this process.
Promotion of wound repair in old mice by local injection of macrophages.
Authors: Danon D , Kowatch M A , Roth G S
Published: May 2006

Application of peritoneal macrophages to experimentally induced cutaneous wounds of old mice accelerates healing to levels almost comparable to those of untreated young animals. Slightly greater acceleration is observed when macrophages are obtained from young as opposed to old donors. These findings are consistent with a defect in macrophage function as a cause of impaired wound healing in senescence and suggest a possible therapeutic strategy.
Interleukin 1 potentiates the secretion of beta-endorphin induced by secretagogues in a mouse pituitary cell line (AtT-20).
Authors: Făgăraşan M O , Eskay R , Axelrod J
Published: May 2006

Previous work has shown that corticotropin releasing factor, vasoactive intestinal peptide, phorbol ester, and forskolin cause the secretion of adrenocorticotropic hormone and beta-endorphin from the AtT-20 mouse pituitary cell line. Human recombinant interleukin 1 alpha and 1 beta also stimulated adrenocorticotropic hormone and beta-endorphin secretion from AtT-20 cells in a time- and dose-related manner. The effect appeared only after pretreatment with interleukin 1 (IL-1) for at least 18 hr and was maximum at 24 hr. After pretreatment of the cells over a period of time with IL-1, the secretion induced by corticotropin releasing factor and vasoactive intestinal peptide was increased in more than an additive manner. The enhancement of corticotropin releasing factor-induced beta-endorphin release produced by IL-1 was apparent after 12 hr and reached a maximum at 24 hr. IL-1 did not affect forskolin-induced cAMP generation but enhanced the effect of forskolin on beta-endorphin secretion. This suggests that IL-1 does not induce adenylate cyclase and that forskolin causes the secretion of beta-endorphin by a mechanism independent of cAMP. IL-1 enhanced phorbol ester-induced beta-endorphin secretion. After prolonged treatment with phorbol ester (an activator of protein kinase C), the secretion induced by phorbol ester was abolished as well as the enhancement induced by IL-1. However, prolonged treatment with phorbol ester had no effect on IL-1-induced beta-endorphin secretion. These observations suggest that IL-1 enhances peptide-generated secretion of beta-endorphin by inducing protein kinase C.
Cellular localization of 1B236/myelin-associated glycoprotein mRNA during rat brain development.
Authors: Higgins G A , Schmale H , Bloom F E , Wilson M C , Milner R J
Published: May 2006

The protein encoded by the rat brain cDNA 1B236 has been shown to be identical to myelin-associated glycoprotein (MAG). In this report we describe the cellular distribution of 1B236/MAG mRNA transcripts in rat brain by using in situ hybridization. At postnatal day 20, large numbers of 1B236/MAG mRNA-containing oligodendrocytes are concentrated in myelinated fiber tracts and throughout gray matter regions. The presence of high levels of 1B236/MAG mRNA within oligodendrocytes at postnatal day 20 is consistent with the proposed role of MAG in formation of the myelin sheath during development. In the adult brain, our results suggest that not only is 1B236/MAG mRNA expressed at reduced levels within oligodendrocytes but also 1B236/MAG or a 1B236/MAG-like mRNA is present within neurons. This localization is consistent with the results of previous immunocytochemical studies using antibodies against the 1B236/MAG mRNA with different cell-type-specific patterns of expression suggests that oligodendrocytes and neurons employ different mechanisms for regulating the same gene. Thus, different cell types may use a similar cell adhesion molecule both during myelinogenesis and in the mature nervous system.
Visualization of chandelier cell axons by parvalbumin immunoreactivity in monkey cerebral cortex.
Authors: DeFelipe J , Hendry S H , Jones E G
Published: May 2006

Antibodies directed against the calcium-binding protein parvalbumin label a subpopulation of gamma-aminobutyric acid-releasing neurons in the cerebral cortex that is thought to have particular metabolic and physiological properties. The chandelier cell is a well-characterized morphological type of gamma-aminobutyric acid-releasing cortical interneuron, the axon of which possesses very distinctive terminal portions located around the initial axon segments of pyramidal cells. In the pre- and postcentral gyri of the monkey, we found that these distinctive terminal portions of chandelier cell axons were immunocytochemically stained for parvalbumin in a manner that reveals their complete structure. The chandelier cell axons were identified light-microscopically as short, vertically oriented rows of parvalbumin-positive puncta (PV-Rs). The PV-Rs varied in both length and complexity and were located beneath unstained pyramidal cells. PV-Rs were very numerous in layers II-III, where most pyramidal cells appeared to have a PV-R beneath them. Fewer PV-Rs were found in deeper layers, and in layer VI PV-Rs were rare. With EM all PV-Rs could be seen to form multiple synaptic contacts of the symmetrical type on the initial segments of pyramidal cell axons. Parvalbumin immunoreactivity can therefore be used as a reliable marker for chandelier cell axons.
Resistance to axonal degeneration after nerve compression in experimental diabetes.
Authors: Dyck P J , Engelstad J K , Giannini C , Lais A C , Minnerath S R , Karnes J L
Published: May 2006

To determine the effect of diabetes on the development of axonal degeneration after acute nerve compression, the mobilized peroneal nerves of rats with streptozotocin-induced diabetes and of control rats were compressed at 150 mmHg (1 mmHg = 133 Pa) for 30 min by using specially devised cuffs. At three intervals after compression--3 days, rats diabetic for 31 wk; 14 days, diabetic for 6 wk; and 24 days, diabetic for 31 wk--groups of nerves were studied to assess numbers and sizes of fibers above, at, and below the cuff and to assess frequency of fiber degeneration in teased fibers from nerve distal to the cuff. Teased fibers with pathologic abnormalities were more frequent in nerves from controls than in nerves from diabetic rats in all three groups but the difference was statistically significant only at 3 and 14 days after compression. The lack of significant difference at 24 days may be explained by higher rates of disappearance of degenerating products and of fiber regeneration at 24 than at 3 and 14 days. This study provides evidence that in addition to delaying the reported functional deficit of vibratory detection threshold and conduction block during nerve compression, diabetes also may partially prevent axonal injury. Low nerve myo-inositol concentration did not predispose diabetic nerve to acute compression injury. If these results also apply to human diabetes and if repeated acute compression is involved in the genesis of fiber degeneration in entrapment, then a higher frequency of entrapment neuropathy among diabetics might be due to mechanisms other than increased susceptibility of fibers to acute compression--e.g., possibly to greater constriction of nerve due to pathologic alterations of the carpal ligament.
Analyzing variation in egg-to-adult viability in experimental populations of Drosophila melanogaster.
Authors: Wallace B
Published: May 2006

Selective culling in populations of most organisms is rank-order: individuals of low rank on a scale of potential fitnesses tend to be eliminated during early development, whereas surviving adults (whose number reflects the carrying capacity of the environment) are generally drawn from the distribution's upper end. Haldane pointed out [Haldane, J.B.S. (1932) The Causes of Evolution (Harper & Row, New York)] that selection which favors individuals in the upper tail of a composite distribution curve tends to favor members of the more variable of two populations, rather than members of a less variable one, even though the latter may possess the higher mean. In addition to reviewing earlier observations bearing on Haldane's argument, the present report describes an analysis of the comparative egg-to-adult viabilities of flies (Drosophila melanogaster) carrying combinations of second chromosomes obtained from one or another of eight experimental populations. Overall, the viabilities of flies carrying combinations of chromosomes one of which is shared (i/j vs. j/k) are as different as those of flies carrying combinations of independently sampled chromosomes (i/j vs. k/l). Episodes seemingly occurred within the populations during which flies carrying combinations that shared a chromosome differed more in their viabilities than flies carrying unrelated combinations. Such episodes could reflect the occurrence of selection of the sort described by Haldane.
Replacement of aspartic acid-96 by asparagine in bacteriorhodopsin slows both the decay of the M intermediate and the associated proton movement.
Authors: Holz M , Drachev L A , Mogi T , Otto H , Kaulen A D , Heyn M P , Skulachev V P , Khorana H G
Published: May 2006

The photocycle, electrical charge translocation, and release and uptake of protons from the aqueous phase and release and uptake of protons from the aqueous phase were investigated for bacteriorhodopsin mutants with aspartic acid-96 replaced by asparagine or glutamic acid. At neutral pH the main effect of the Asp-96----Asn mutation is to slow by 2 orders of magnitude the decay of the M intermediate and the concomitant charge displacement associated with the reprotonation of the Schiff base from the cytoplasmic side of the membrane. The proton uptake measured with the indicator dye pyranine is likewise slowed without affecting the stoichiometry of proton pumping. The corresponding results for the Asp-96----Glu mutant, on the other hand, are very close to those for the wild-type protein. These results provide a kinetic explanation for the fact that at pH 7 and saturating light intensities the steady-state proton pumping is almost abolished in the Asp-96----Asn mutant but is close to normal in the Asp-96----Glu mutant. Thus, the pump is simply turning over much more slowly in the Asp-96----Asn mutant. The time constants of the decay of M and the associated charge translocation increase strongly with increasing pH for the Asp-96----Asn mutant but are virtually pH-independent for the Asp-96----Glu mutant and wild-type bacteriorhodopsin. At pH 5 the M decay of the Asp-96----Asn mutant is as fast as for wild type. These results suggest that Asp-96 serves as an internal proton donor in the proton-uptake pathway from the cytoplasm to the Schiff base.
Ca2+-channel blockers inhibit the action of recombinant platelet-derived growth factor in vascular smooth muscle cells.
Authors: Block L H , Emmons L R , Vogt E , Sachinidis A , Vetter W , Hoppe J
Published: May 2006

Human platelet-derived growth factor (PDGF) is mainly composed of two polypeptide chains (PDGF-AB). All three possible dimeric forms of PDGF--i.e., PDGF-AA, PDGF-BB and PDGF-AB--exist in nature. We have used two recombinant PDGF homodimers to determine the roles of each isoform in the activation of phosphatidylinositol turnover in vascular smooth muscle cells (VSMC) isolated from rat thoracic aorta, their mitogenic effect on VSMC, and their vasoconstrictor effect on intact strips of aortic vascular tissue. Three Ca2+-channel blockers, nifedipine, verapamil, and diltiazem, were used as antagonists for investigating the PDGF-dependent changes mediated by the homodimers. PDGF-BB had a greater efficacy than PDGF-AA on inositol 1,4,5-trisphosphate release, on the formation of diacylglycerol, and on Ca2+ mobilization, which was also associated with vasoconstrictor activity and effective mitogenicity. PDGF-AA, on the other hand, was more potent than PDGF-BB in stimulating protein kinase C. In all instances, the activation of the phosphatidylinositol turnover by the two homodimers was inhibited by the Ca2+-channel blockers.
Molecular and biological characterization of a replication competent human immunodeficiency type 2 (HIV-2) proviral clone.
Authors: Franchini G , Fargnoli K A , Giombini F , Jagodzinski L , De Rossi A , Bosch M , Biberfeld G , Fenyo E M , Albert J , Gallo R C
Published: May 2006

We obtained complete genomic clones of human immunodeficiency virus type 2 (HIV-2) from the DNA of the neoplastic human cell line HUT 78 freshly infected with a HIV-2 isolate, strain SBL6669. The recombinant phage DNA was transfected into the lymphocytes of CD4-positive HUT 78 cell line to test the replication competence of the proviral DNA. One genomic clone, designated HIV-2SBL/ISY, yielded retroviral particles after a few weeks of culture of the transfected cells. The HIV-2SBL/ISY clone contained a complete provirus and cellular flanking sequence. We obtained the DNA sequence of the provirus and compared it with the published sequence of two other HIV-2 isolates. The degree of variability among HIV-2 isolates is comparable to that observed among African HIV-1 isolates sequenced to date. Immunologically, HIV-2SBL/ISY is similar to the parental virus (HIV-2SBL6669) but differs in the envelope transmembrane protein that is truncated (gp32-34) in the parental virus and not in HIV-2SBL/ISY (gp41). Both the parental and the cloned viruses are infectious and cytopathic for some human T-cell lines, induce syncytia, and infect a human macrophage cell line (U937) in vitro. The availability of a biologically active HIV-2 clone provides the means to study the role and interaction of HIV-2 genes in vitro as well as to assess the functional similarities among HIV-1 and HIV-2 genes. Since HIV-2SBL/ISY cloned virus infects fresh peripheral blood T cells from Rhesus macaques in vitro and infects the same animal in vivo, its use in animals may represent a model for functional study of viral genes in vivo as well as for development of experimental approaches to prevent and cure retroviral infection in humans.
Standard basis in supersymplectic algebras
Authors: Rota Gian-Carlo , Stein Joel A.
Published: May 2006

An integral standard basis is given for products of Pfaffians containing positively and negatively signed variables. Applications to invariant theory are derived.
Molecular cloning and analysis of cDNA encoding a plant tryptophan decarboxylase: comparison with animal dopa decarboxylases.
Authors: De Luca V , Marineau C , Brisson N
Published: May 2006

The sequence of a cDNA clone that includes the complete coding region of tryptophan decarboxylase (EC 4.1.1.28, formerly EC 4.1.1.27) from periwinkle (Catharanthus roseus) is reported. The cDNA clone (1747 base pairs) was isolated by antibody screening of a cDNA expression library produced from poly(A)+ RNA found in developing seedlings of C. roseus. The clone hybridized to a 1.8-kilobase mRNA from developing seedlings and from young leaves of mature plants. The identity of the clone was confirmed when extracts of transformed Escherichia coli expressed a protein containing tryptophan decarboxylase enzyme activity. The tryptophan decarboxylase cDNA clone encodes a protein of 500 amino acids with a calculated molecular mass of 56,142 Da. The amino acid sequence shows a high degree of similarity with the aromatic L-amino acid decarboxylase (dopa decarboxylase) and the alpha-methyldopa-hypersensitive protein of Drosophila melanogaster. The tryptophan decarboxylase sequence also showed significant similarity to feline glutamate decarboxylase and mouse ornithine decarboxylase, suggesting a possible evolutionary link between these amino acid decarboxylases.