bluuebunny/crossref_metadata_2025 huggingface dataset:
On some applications of diophantine approximations
Authors: Chudnovsky G. V.
Published: May 2006

Siegel's results [Siegel, C. L. (1929)
Abh. Preuss. Akad. Wiss. Phys.-Math. Kl.
1] on the transcendence and algebraic independence of values of
E
-functions are refined to obtain the best possible bound for the measures of irrationality and linear independence of values of arbitrary
E
-functions at rational points. Our results show that values of
E
-functions at rational points have measures of diophantine approximations typical to “almost all” numbers. In particular, any such number has the “2 + ε” exponent of irrationality: ǀΘ -
p
/
q
ǀ > ǀ
q
ǀ
-2-ε
for relatively prime rational integers
p,q
, with
q
≥
q
0
(Θ, ε). These results answer some problems posed by Lang. The methods used here are based on the introduction of graded Padé approximations to systems of functions satisfying linear differential equations with rational function coefficients. The constructions and proofs of this paper were used in the functional (nonarithmetic case) in a previous paper [Chudnovsky, D. V. & Chudnovsky, G. V. (1983)
Proc. Natl. Acad. Sci. USA
80, 5158-5162].
DNA binding activity of polyoma virus large tumor antigen.
Authors: Dilworth S M , Cowie A , Kamen R I , Griffin B E
Published: May 2006

Polyoma virus large tumor antigen from productively infected mouse cells has been purified to greater than 50% homogeneity by a simple immunoaffinity procedure using monoclonal antibodies. A radioimmunoreaction was devised for assaying purity. The purified large tumor antigen retained its antigenicity and its ability to bind DNA specifically. The regions on the polyoma virus genome recognized by the protein were characterized. Three binding regions were localized within the portion of the genome between the viral origin of DNA replication and the protein coding sequence, overlapping the early promoter and the sites of initiation of mRNAs that specify the viral tumor antigens. The binding regions each contain direct repeats of the pentanucleotide sequence G-R-G-G-C.
Specific amino acid substitutions in bacterioopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons
Authors: Lo Kin-Ming , Jones Simon S. , Hackett Neil R. , Khorana H. Gobind
Published: May 2006

To study the mechanism of light-dependent proton translocation by bacteriorhodopsin, we have introduced single-codon changes in the gene so as to produce the following specific amino acid substitutions in the protein: Tyr-185 to Phe, Pro-186 to Leu, Trp-189 to Phe, Ser-193 to Ala, and Glu-194 to Gln. The strategy involved replacement of a 62-base-pair restriction fragment by synthetic DNA duplexes containing the modified nucleotide sequences. This required a unique restriction site (
Xho
I) at Ile-203 which was created by oligonucleotide-directed point mutagenesis. The six DNA duplexes corresponding to the modified native and mutant restriction fragments were all prepared by DNA ligase-catalyzed joining of chemically synthesized deoxyribooligonucleotides. The bacterioopsin expression plasmids reconstructed by using the synthetic DNA fragments were characterized by restriction analysis and DNA sequence determination. An extremely rapid, efficient, and general method for purification of the synthetic oligonucleotides and of DNA fragments was developed.
Purification and properties of a pea chloroplast DNA polymerase
Authors: McKown R. L. , Tewari K. K.
Published: May 2006

A DNA polymerase has been purified >3,000-fold from the chloroplasts of pea plants by chromatography on DEAE-cellulose, phosphocellulose, single-stranded DNA-agarose, and sedimentation in a glycerol gradient. Electrophoretic analysis on polyacrylamide gels in the presence of sodium dodecyl sulfate indicates that the final fraction contained a single discernible protein band of 90,000 daltons. Gel filtration on Sephacryl S-200 and glycerol gradient sedimentation under nondenaturing conditions demonstrate that the chloroplast DNA polymerase has a native molecular mass of approximately 87,000 daltons. The purified polymerase lacks any associated nuclease activity. The enzyme activity is inhibited by
N
-ethylmaleimide (74% at 1.0 mM) and ethidium bromide (90% at 0.23 mM) and is resistant to aphidicolin. The purified enzyme is totally dependent on the presence of added DNA, has an absolute requirement for Mg
2+
(12 mM optimal), is stimulated by K
+
(120 mM optimal), and requires all four deoxynucleoside triphosphates for maximum activity. Native DNA which has been degraded to a limited extent with DNase I is the most efficient template.
X-ray evidence for two structural states of the actomyosin cross-bridge in muscle fibers.
Authors: Matsuda T , Podolsky R J
Published: May 2006

Biochemical data, stiffness measurements, and equatorial x-ray diffraction patterns provide evidence that actomyosin cross-bridges form in relaxed skinned rabbit fibers at low ionic strength (20 mM). In the present study we examined the structure of these cross-bridges by using two-dimensional x-ray diffraction. In contrast to rigor cross-bridges, which significantly weaken the myosin-based reflections characteristic of relaxed fibers at 120 mM ionic strength (notably the 86-A and 108-A layer lines and the 72-A and 143-A meridionals), the formation of low ionic strength cross-bridges produced only small changes in these reflections. In addition, these cross-bridges did not produce the additional intensity on the 59-A actin-based layer line near the meridian that is associated with rigor cross-bridges. However, the formation of low ionic strength cross-bridges caused the 215-A meridional reflection to decrease in intensity, as is also the case when rigor cross-bridges are formed. These observations show that the structure of the low ionic strength cross-bridge is significantly different from that of the rigor cross-bridge, and they raise the possibility that contractile force may be generated by a transition between these two actomyosin configurations.
Neurotrophic effects of hippocampal extracts on medial septal nucleus in vitro.
Authors: Ojika K , Appel S H
Published: May 2006

Within peripheral sympathetic and sensory systems, target tissue provides diffusible factors such as nerve growth factor that influence neuronal survival, growth, and differentiation. To determine whether target tissue may exert retrograde effects within the central nervous system, we have used hippocampus as a source of neurotrophic factors and cultured medial septal nucleus explants as a source of neurons that may respond to such factors. Soluble extracts from rat hippocampus enhance cholinergic activity (choline acetyltransferase, choline uptake, acetylcholine synthesis) of the rat medial septal nucleus cultured in serum-free defined medium. The enhancement is dose dependent, relatively specific for hippocampus, and varies with age, reaching a peak in 2- to 3-week-old rat hippocampus. The enhancing activity of the hippocampal extract appears to be mediated by protease-sensitive polypeptide(s) that differ from nerve growth factor in biological and chemical characteristics.
Sequence of a complete chicken delta-crystallin cDNA.
Authors: Nickerson J M , Piatigorsky J
Published: May 2006

A full-length chicken delta-crystallin cDNA (p delta Cr17) was cloned and subjected to sequence analysis. The cDNA was shown to be full-length by both primer extension and S1 and mung bean nuclease experiments. Thus, the complete amino acid sequence of delta-crystallin is now available. The delta-crystallin polypeptide has a molecular mass of 48,542 daltons, as expected from its behavior on NaDodSO4/polyacrylamide gels. delta-Crystallin has one tryptophan, no cysteines, a prevalence of leucines (15%), and a paucity of aromatic residues. High alpha-helical content throughout the protein was predicted from the amino acid sequence. Nucleic acid sequence analysis suggests that delta-crystallin gene 1 encodes the mRNA that gave rise to the cDNA clone. These data provide a basis for the detailed analysis of the two delta-crystallin genes.
Assignment of the gene for methylthioadenosine phosphorylase to human chromosome 9 by mouse-human somatic cell hybridization.
Authors: Carrera C J , Eddy R L , Shows T B , Carson D A
Published: May 2006

The purine and polyamine metabolic enzyme methylthioadenosine (MeSAdo) phosphorylase is abundant in normal cells and tissues but is lacking from many human and murine malignant cell lines and from cells of some human leukemias in vivo. To explore the genetic control of MeSAdo phosphorylase expression, we measured levels of the enzyme in somatic cell hybrids prepared by fusing MeSAdo phosphorylase-deficient mouse L cell lines with human fibroblasts. In the hybrid clones, MeSAdo phosphorylase activity segregated concordantly with adenylate kinase 1, a marker for human chromosome 9, but not with enzyme markers for any other human chromosome. In hybrid clones derived from human fibroblasts with a reciprocal translocation between chromosomes 9 and 17, MeSAdo phosphorylase activity was confined to cells containing the 9pter----9q12 region. In every case, the enzyme-positive hybrid clones displayed bands of MeSAdo phosphorylase activity with isoelectric points characteristic of both the human and murine enzymes. These results indicate that the structural gene for human MeSAdo phosphorylase, designated MTAP, can be assigned to the 9pter----9q12 region of human chromosome 9. Furthermore, these studies with interspecies somatic cell hybrids show that the MeSAdo phosphorylase-deficient state is recessive in mouse L cell lines.
Transcription of human histone genes in extracts from synchronized HeLa cells.
Authors: Heintz N , Roeder R G
Published: May 2006

Nuclear extracts were prepared from synchronized HeLa cells at various times during the cell cycle and assayed for the ability to transcribe several cellular and viral genes. The efficiency of transcription of a human histone H4 gene is 3- to 10-fold greater in nuclear extracts from S phase nuclei than in extracts from non-S phase cells. In contrast, the adenovirus virus type 2 (Ad2) major late promoter is utilized 3- to 20-fold more efficiently in nuclear extracts from non-S phase cells. Transcription of other genes, including a human histone H3 and the simian virus 40 late transcription unit, is equally efficient in S and non-S phase extracts. Mixing experiments demonstrate that the rate-limiting activities for histone H4 and Ad2 major late transcription function independently and that the effects of these activities are additive. Competition studies suggest that the H4-specific transcription activity can be sequestered by preincubation with the H4 template DNA. These data support the concept that cell cycle regulation of human histone gene transcription may depend in part on soluble transcription activities that are modulated during the cell cycle. Further, in addition to the H4-specific transcription activity, there may exist other transcription factors whose activity can fluctuate according to the cell cycle or according to the growth state of the cells.
Complete amino acid sequence for human aldolase B derived from cDNA and genomic clones.
Authors: Rottmann W H , Tolan D R , Penhoet E E
Published: May 2006

Several aldolase B clones from a human liver cDNA library have been identified by using a rabbit aldolase A cDNA as a hybridization probe. The most complete of these, pHL413, is 1389 base pairs long and covers approximately equal to 80% of the length of the mRNA, including 90% of the translated region. The cDNA, pHL413, was used to identify a genomic clone, lambda HG313, which encoded the remaining amino acids of human aldolase B. We demonstrate that the amino acid and nucleotide sequences of aldolase are strongly conserved even between different isozymes. Furthermore, in the 3'-untranslated regions of the mRNAs for the B isozyme of human and rat there is an extensive stretch of homology. Aldolase B lacks a cysteine at positions 72 and 338 and lacks a histidine at position 361. These residues, which are present in rabbit aldolase A, have previously been proposed to take part in catalysis. Our findings suggest that this may not be the case.
Sequences of the S1 genes of the three serotypes of reovirus.
Authors: Cashdollar L W , Chmelo R A , Wiener J R , Joklik W K
Published: May 2006

The S1 genes of the three serotypes of reovirus have been cloned and sequenced. The S1 genes encode protein sigma 1, the protein against which serotype-specific neutralizing antibodies are directed; it is also the reovirus hemagglutinin and cell-attachment protein and is a major determinant of host range/tissue specificity and of the nature of the interaction of reovirus with cells of the immune system. The S1 genes of serotypes 1, 2, and 3 are 1458, 1442, and 1416 nucleotides long, respectively. They possess untranslated regions 13, 13, and 12 nucleotides long at their 5' termini and 188, 229, and 36 nucleotides long at their 3' termini. They possess two open reading frames. The first starts with a "weak" initiation codon and extends for 418, 399, and 455 codons, respectively; this is the size expected for the sigma 1 proteins. The other reading frame starts at a "strong" initiation codon about 70 residues downstream from the 5' terminus but extends for only about 120 codons, being terminated by 3 in-phase termination codons in all three genes. The proteins encoded by these short open reading frames are quite basic. The serotype 1 and 2 S1 genes are much more closely related to each other (28% homology) than to the serotype 3 S1 gene (5% and 9% homology, respectively). These figures are based on direct homology calculations, adjusted for 25% random coincidence. Serologic evidence and hydrophobicity profiles agree that the sigma 1 proteins of serotypes 1 and 2 are much more closely related to each other (about 40% homology) than to that of serotype 3 (only about 20% homology). The fact that the serotype 1 and 2 S1 genes are much more closely related to each other than to the serotype 3 S1 gene is remarkable since for all other nine reovirus genes the serotype 1 and 3 genes are much more closely related to each other than to the serotype 2 gene. Mechanisms that may effect this remarkable evolutionary pattern are discussed.
Transmissible spongiform encephalopathy in the gray tremor mutant mouse.
Authors: Sidman R L , Kinney H C , Sweet H O
Published: May 2006

Gray tremor (gt) is an autosomal recessive mutation in the mouse linked to caracul (Ca) on chromosome 15. The complex mutant phenotype includes pigmentation defects, tremor, seizures, hypo- and dysmyelination in central and peripheral nervous systems, spongiform encephalopathy, and early death. The heterozygote (+/gt) is phenotypically normal but develops a mild spongiform encephalopathy from 2 months of age onward. The pigmentation and myelination disorders indicate that the gt genetic locus is active neonatally and probably earlier. This report focuses mainly on the later-expressed vacuolating disorder, which most closely mimics in tissue distribution, histopathology, and ultrastructure the spongiform encephalopathies caused by unconventional transmissible agents. This lesion was produced in genetically normal mice in a transmission experiment: of 99 neonatal mice inoculated intracerebrally with gt/gt brain homogenate, all 7 mice of three strains (BALB/cBy, C3HeB/FeJ, and C57BL/6J) allowed to survive for the unusually long interval of 682-721 days after inoculation, developed spongiform changes distributed as in the mutant phenotype. The gray tremor mutant presents a naturally occurring spongiform encephalopathy whose expression is determined by the interaction of genetic factors and a transmissible agent.
Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase.
Authors: Blumenthal D K , Takio K , Edelman A M , Charbonneau H , Titani K , Walsh K A , Krebs E G
Published: May 2006

In the course of determining the primary structure of rabbit skeletal muscle myosin light chain kinase (MLCK; ATP:protein phosphotransferase, EC 2.7.1.37) a peptide fragment was obtained that appears to represent the calmodulin-binding domain of this enzyme. Low concentrations of the peptide inhibited calmodulin activation of MLCK (Ki congruent to 1 nM). The peptide was not associated with a catalytically active, calmodulin-independent form of MLCK that was obtained by limited proteolysis. The peptide is 27 residues in length and represents the carboxyl terminus of MLCK. The sequence of the peptide shows no significant homology with any known protein sequence. The peptide contains one tryptophanyl residue and a high percentage of basic and hydrophobic residues, but no acidic or prolyl residues. Much of the sequence has a high probability of forming alpha helix. A chemically synthesized peptide has been prepared to study the interactions of the peptide and calmodulin in more detail. The intrinsic tryptophan fluorescence of the synthetic peptide shows a significant enhancement (approximately equal to 45%) in the presence of Ca2+ and calmodulin; fluorescence enhancement is maximal at a peptide:calmodulin stoichiometry of 1:1. Calmodulin-Sepharose affinity chromatography in the presence of 2 M urea indicates that the interaction of peptide and calmodulin is Ca2+-dependent. The results of these studies indicate that the catalytic and calmodulin-binding domains of MLCK represent distinct and separable regions of the protein. In addition, the results provide a basis for future studies of the molecular and evolutionary details of calmodulin-dependent enzyme regulation.
Intra- and interspecies transfer and expression of
Rhizobium japonicum
hydrogen uptake genes and autotrophic growth capability
Authors: Lambert Grant R. , Cantrell Michael A. , Hanus F. Joe , Russell Sterling A. , Haddad Karen R. , Evans Harold J.
Published: May 2006

Cosmids containing hydrogen uptake genes have previously been isolated in this laboratory. Four new cosmids that contain additional
hup
gene(s) have now been identified by conjugal transfer of a
Rhizobium japonicum
122DES gene bank into a Tn5-generated Hup
-
mutant and screening for the acquisition of Hup activity. The newly isolated cosmids, pHU50-pHU53, contain part of the previously isolated pHU1 but extend as far as 20 kilobases beyond its border. pHU52 complements five of six Hup
-
mutants and confers activity on several Hup
-
wild-type
R. japonicum
strains in the free-living state and where tested in nodules. Transconjugants obtained from interspecies transfer of pHU52 to
Rhizobium meliloti
102F28, 102F32, and 102F51 and
Rhizobium leguminosarum
128C53 showed hydrogen-dependent methyleneblue reduction, performed the oxyhydrogen reaction, and showed hydrogen-dependent autotrophic growth by virtue of the introduced genes. The identity of the presumptive transconjugants was confirmed by antibiotic-resistance profiles and by plant nodulation tests.
Cytochrome P-450 metabolic activity in embryonic and extraembryonic tissue lineages of mouse embryos.
Authors: Pedersen R A , Meneses J , Spindle A , Wu K , Galloway S M
Published: May 2006

Mouse morulae, blastocysts, and embryonic and extraembryonic tissue layers were examined for benzo[a]-pyrene metabolism by cytochrome P-450, using the sister chromatid exchange assay. Benzo[a]pyrene exposure in vitro increased sister chromatid exchanges in blastocysts of all genetically responsive mice examined [BALB/cDub, C3H/AnfCum, and outbred Dub:(ICR) strains] but not blastocysts of the nonresponsive AKR/J strain. Benzo[a]pyrene treatment of responsive 7 1/2- and 8 1/2-day (postimplantation-stage) embryos, either intact or as separate tissue layers, increased sister chromatid exchanges in tissues of both embryonic and extraembryonic lineages--i.e., in the embryo proper, in isolated embryonic ectoderm, and in yolk sac, chorion, extraembryonic ectoderm, and extraembryonic endoderm layers. These results indicate that cytochrome P-450 is active in most or all tissues of the early mammalian embryo. It could metabolize xenobiotic molecules reaching the conceptus near the onset of morphogenesis and organogenesis, or it could have another as yet undefined role in normal development.
Dual role for Escherichia coli RecA protein in SOS mutagenesis.
Authors: Ennis D G , Fisher B , Edmiston S , Mount D W
Published: May 2006

Induction of the Escherichia coli SOS system increases the ability of the cells to perform DNA repair and mutagenesis. Previous work has shown that this increased mutagenesis is the result of derepression of specific genes through a complex regulatory mechanism controlled by LexA and RecA proteins. One role of RecA protein in this process is to facilitate proteolytic cleavage of LexA protein (the repressor) in response to an inducing signal that reversibly activates RecA protein to perform this function. We show that activated RecA protein plays a second role in SOS mutagenesis, as revealed by analyzing repair of UV-damaged phage lambda in host mutants with alterations in the SOS regulatory system. First, phage mutagenesis was not expressed constitutively in a mutant that is derepressed through lack of functional LexA protein; activated RecA protein was still required. Second, phage mutagenesis was constitutively expressed in the presence of recA mutations that alter RecA protein so that it is activated in normally growing cells. There was also RecA-dependent constitutive expression of SOS mutagenesis in host mutants that lack functional LexA protein and carry plasmids. We discuss several possible biochemical mechanisms for this second role of activated RecA protein in SOS mutagenesis.
Maass cusp forms
Authors: Deshouillers J.-M. , Iwaniec H. , Phillips R. S. , Sarnak P.
Published: May 2006

It is shown that, under certain standard assumptions, such as extended Riemann hypotheses, the scattering matrix ϕ(
s
) for generic Γ ≤ SL(2, R) is unexpectedly of order 2. This leads to the conjecture that the generic cofinite Γ has very few Maass cusp forms.
Overproduction of the protein product of a nonselected foreign gene carried by an adenovirus vector.
Authors: Yamada M , Lewis J A , Grodzicker T
Published: May 2006

We have constructed a recombinant adenovirus that carries the herpes simplex virus type I gene for thymidine kinase (EC 2.7.1.21) and expresses thymidine kinase under control of adenovirus major late promoter. A DNA fragment carrying thymidine kinase coding sequences but lacking the thymidine kinase promoter was sandwiched between a piece of adenoviral DNA and simian virus 40 early DNA on a plasmid. The aligned fragment was then inserted into the adenoviral genome, replacing internal adenoviral DNA. Hybrid viruses carrying the thymidine kinase gene were obtained by selecting for viruses that express simian virus 40 tumor antigen (T antigen) in monkey cells. The thymidine kinase gene was positioned in the third segment of the adenovirus tripartite leader downstream from the major late promoter by in vivo DNA recombination between the duplicated adenoviral sequences present in the plasmid insert and the viral vector. Levels of thymidine kinase activity in human or monkey cells infected with this hybrid virus were several times higher than in cells infected with herpes simplex virus. Infected cells produced thymidine kinase protein at very high levels, similar to those found for adenovirus late major capsid proteins. The thymidine kinase protein represented 10% of the newly synthesized protein in late infected cells and accumulated to represent 1% of total cell protein under optimal conditions. This vector system offers a procedure by which a variety of gene products that are biologically active and properly modified can be produced at high levels in mammalian cells.
Larval settlement rate: A leading determinant of structure in an ecological community of the marine intertidal zone
Authors: Gaines S. , Roughgarden J.
Published: May 2006

Field studies demonstrate that the population structure of the barnacle
Balanus glandula
differs between locations of high and low larval settlement rate. These observations, together with results from a model for the demography of an open, space-limited population, suggest that the settlement rate may be a more important determinant of rocky intertidal community structure than is presently realized. Locations with a low larval settlement rate exhibit a generally low abundance of barnacles that varies slightly within years and greatly between years, reflecting yearly differences in settlement. Locations with a high-settlement rate exhibit a generally high abudance of barnacles. However, the abundance varies greatly within years with a significant oscillatory component (period, 30 weeks) and only slightly between years regardless of yearly differences in settlement. At the low-settlement location mortality of barnacles is independent of the area occupied by barnacles. At the high-settlement location mortality is cover-dependent due to increased predation by starfish on areas of high barnacle cover. In both locations the cover-independent component of mortality does not vary with age during the first 60 weeks. As assumed in the demographic model, the kinetics of larval settlement can be described as a process in which the rate of settlement to a quadrat is proportional to the fraction of vacant space within the quadrat. Generalizations that the highest species diversity in a rocky intertidal community is found at locations of intermediate disturbance, and that competition causes zonation between species of the barnacle genera
Balanus
and
Chthamalus
, seem to apply only to locations with high-settlement rates.
Identification of an IgE-binding protein by molecular cloning.
Authors: Liu F T , Albrandt K , Mendel E , Kulczycki A , Orida N K
Published: May 2006

The synthesis and function of IgE are dependent on IgE-binding proteins, which include cell surface IgE receptors and IgE-binding lymphokines. To further our understanding of the IgE system, we have engaged in the molecular cloning of genes for some of these proteins. In studying the in vitro translation products of mRNA from rat basophilic leukemia (RBL) cells, we have identified a Mr 31,000 polypeptide that binds IgE and is also reactive with antibodies to proteins affinity-purified from RBL cells with IgE immunoadsorbent. For the molecular cloning, double-stranded cDNA was synthesized from sucrose gradient-fractionated RBL mRNA, inserted into plasmid pBR322, and used to transform Escherichia coli. By screening transformants with a hybridization-selection/in vitro translation procedure, we identified one clone containing cDNA that hybridized to mRNA coding for a Mr 31,000 IgE-binding protein. The DNA sequence of this cloned cDNA (571 base pairs) was determined and the amino acid sequence corresponding to part of the protein was deduced. In RNA blot analysis, the cDNA hybridized with a mRNA of 1100 nucleotides found in RBL cells but absent in cells not expressing IgE receptors. This cloned cDNA most likely codes for the Mr 31,000 IgE-binding protein identified in RBL cells, which appears to be related to the IgE-binding phenotype of the cells and which may have a significant role in the IgE-mediated activation of basophils and mast cells.
Caenorhabditis elegans DNA that directs segregation in yeast cells.
Authors: Stinchcomb D T , Mello C , Hirsh D
Published: May 2006

We have isolated seven DNA fragments from Caenorhabditis elegans that enhance the mitotic segregation of autonomously replicating plasmids in the yeast Saccharomyces cerevisiae. These segregators, designated SEG1-SEG7, behave like isolated yeast chromosomes: they increase the stability and simultaneously lower the copy number of circular plasmids during mitotic growth in yeast. During meiosis, plasmids containing the C. elegans segregators show higher levels of precocious or aberrant disjunction than do plasmids bearing isolated yeast centromeres. Yet one of the segregators improved the meiotic segregation of the parental plasmid. We estimate that there may be as many as 30 segregator sequences in the C. elegans genome, a value that is consistent with the polycentric nature of C. elegans chromosomes. Five of the seven segregators are linked to sequences that are repeated in the worm genome, and four of these five segregators cross-hybridize. Other members of this family of repetitive DNA do not contain segregator function. Segregator sequences may prove useful for probing the structure of centromeres of both C. elegans and S. cerevisiae chromosomes.
Antigen induces chronic idiotype suppression.
Authors: Tokuhisa T , Rajewsky K
Published: May 2006

Hapten coupled to isologous gamma globulin can substitute for anti-idiotypic antibody in the induction of neonatal chronic idiotype suppression. The immunologically suppressed animals fail to express an idiotypically defined subset of anti-hapten antibodies in later life and harbor regulatory T cells that prevent the expression of this same antibody subset by B cells. Suppression ceases after several months, but memory of suppression persists and can be recalled by small doses of hapten-carrier conjugate. These data show that an antigen present in a mouse at birth is able to generate a T-cell-dependent suppressive mechanism that controls expression of antigen-specific antibodies through the recognition of antibody idiotypes. The idiotypic network is thus involved in the control of tolerance and the available antibody repertoire.
Sympathetic neuron density differentially regulates transmitter phenotypic expression in culture.
Authors: Adler J E , Black I B
Published: May 2006

The effects of cell density and aggregation on expression of transmitter traits were examined in dissociated, pure sympathetic neuron cultures, grown in fully defined, serum-free medium. After 1 week at a density of 7-8 X 10(3) neurons per 35-mm dish, moderate levels of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) activity and substance P were detected. When neuron density was increased 4-fold, a 4-fold increase in tyrosine hydroxylase activity was observed; i.e., there was no change in tyrosine hydroxylase activity per neuron. In contrast, substance P increased 30-fold, corresponding to a 7-fold increase in substance P per neuron. Choline O-acetyltransferase (EC 2.3.1.6) activity, not detected at low cell densities, was first detectable at a concentration of 15,000 neurons per dish and increased 6-fold when this cell concentration was doubled. Medium conditioned by high-density cultures failed to reproduce these effects on low-density cultures, suggesting that diffusible factors are not involved in the density-dependent differential regulation. Time-lapse phase-contrast microscopy of high-density cultures showed neuronal migration and progressive aggregation, which did not occur in low-density cultures. Our observations suggest that cell contact may mediate differential expression of transmitter traits.
Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs.
Authors: Brinster R L , Chen H Y , Trumbauer M E , Yagle M K , Palmiter R D
Published: May 2006

Microinjection of foreign DNA into fertilized mammalian eggs is a convenient means of introducing genes into the germ line. Some of the more important parameters that influence successful integration of foreign DNA into mouse chromosomes are described. The effects of DNA concentration, size, and form (supercoiled vs. linear with a variety of different ends) are considered as well as the site of injection (male pronucleus, female pronucleus, or cytoplasm) and buffer composition. The optimal conditions for integration entail injection of a few hundred linear molecules into the male pronucleus of fertilized one-cell eggs. Under these conditions about 25% of the mice that develop inherit one or more copies of the microinjected DNA. The overall efficiency also depends on the choice of mouse strains; for example, generating transgenic mice that express foreign growth hormone genes is about eight times easier with C57/BL6 X SJL hybrid mice than with inbred C57/BL6 mice.
Amino-terminal amino acid sequence of murine colony-stimulating factor 1.
Authors: Ben-Avram C M , Shively J E , Shadduck R K , Waheed A , Rajavashisth T , Lusis A J
Published: May 2006

We report the amino acid composition, the NH2-terminal sequence, and the tryptic map by HPLC for murine L-cell colony-stimulating factor 1 (CSF-1). CSF-1 purified by affinity chromatography was S-carboxymethylated under denaturing conditions and subjected to sequence microanalysis. CSF-1 contains four or five cysteine residues per subunit and approximately 30-40% carbohydrate by weight. Three contiguous cysteine residues were located in the NH2-terminal sequence. An additional two cysteine residues were located on the tryptic map. Previous studies as well as immunoblotting studies reported here suggest that sulfhydryl bridging plays a major role in maintaining the conformation of the protein. Carbohydrate was located on two tryptic fragments. The findings of a single NH2-terminal sequence in high yield and a relatively simple tryptic map (15 major peptides) suggest that CSF-1 contains two identical subunits. The NH2-terminal sequence of CSF-1 has only limited homology to insulin or insulin-like hormones but no homology with granulocyte/macrophage-CSF or interleukin 3.
Generation and regulation of autocytotoxicity in mixed lymphocyte cultures: evidence for active suppression of autocytotoxic cells.
Authors: Rosenkrantz K , Dupont B , Flomenberg N
Published: May 2006

Using limiting dilution analysis, we have detected both the generation and suppression of autocytotoxic cells following autologous or allogeneic stimulation in vitro. Assay conditions were established in which the cytotoxic response toward an allogeneic sensitizing cell was consistent with a traditional single-hit kinetic model. Under identical conditions, cytolytic activity toward autologous phytohemaglutinin-activated lymphoblasts exhibited a distinct biphasic response. At low responder cell doses, a clear autocytotoxic response was observed. However, at higher responder cell numbers, this autocytotoxic reaction disappeared. This biphasic pattern of autocytotoxicity developed after stimulation with allogeneic or autologous peripheral blood mononuclear leukocytes (PBL) or Epstein-Barr virus-transformed B cells. This pattern of response is consistent with the counterpoised actions of two distinct cell populations, an autoaggressive population and a lower frequency autosuppressor population. Autocytotoxicity was not the result of mitogenic or xenogeneic antigenic stimulation, as it was observed after stimulation with autologous PBL in autologous serum and an autologous interleukin 2 preparation. Thus, cells capable of autocytotoxicity are present in peripheral blood but at a lower frequency than allocytotoxic T lymphocytes. Furthermore, autoaggressive cells are down-regulated by an autologous suppressor population. These findings suggest that immunologic self-tolerance is, at least in part, an actively maintained condition. Disturbances in this autoregulatory network may have relevance to the pathogenesis of some autoimmune diseases and graft-versus-host disease.
Neural cell adhesion molecule (N-CAM) accumulates in denervated and paralyzed skeletal muscles.
Authors: Covault J , Sanes J R
Published: May 2006

We have used immunofluorescence and immunoblotting methods to study the amount and distribution of the neural cell adhesion molecule (N-CAM) in rat skeletal muscle; this molecule is thought to mediate adhesion of neurons to cultured myotubes. N-CAM is present on the surface of embryonic myotubes, but it is lost as development proceeds and is nearly absent from adult muscle. However, denervation of adult muscle results in the reappearance of N-CAM. In denervated muscle, N-CAM is associated both with muscle fibers and with cells in interstitial spaces between fibers. The N-CAM in interstitial spaces is concentrated near denervated endplates, which are known to be preferential sites for reinnervation. Paralysis of innervated muscle, known to mimic denervation in many respects, also induces the accumulation of N-CAM. Axons that regenerate to reinnervate muscle bear N-CAM on their terminals, and reinnervation results in the disappearance of N-CAM from muscle. Denervation induces accumulation of N-CAM in mouse and chicken, as well as in rat muscles. Thus, the expression of N-CAM in muscle is regulated by the muscle's state of innervation. In that N-CAM-rich muscles (embryonic, denervated, and paralyzed) are known to be competent to accept synapses, while N-CAM-poor muscles (normal adult and reinnervated) are refractory to hyperinnervation, N-CAM might, in turn, participate in regulating muscle's susceptibility to innervation.
Transferrin receptors in rat brain: neuropeptide-like pattern and relationship to iron distribution.
Authors: Hill J M , Ruff M R , Weber R J , Pert C B
Published: May 2006

We have characterized and visualized the binding of 125I-labeled transferrin to sections of rat brain. This saturable, reversible, high-affinity (Kd = 1 X 10(-9) M) binding site appears indistinguishable from transferrin receptors previously characterized in other tissues. Moreover, a monoclonal antibody raised to rat lymphocyte transferrin receptors could immunoprecipitate recovered intact transferrin solubilized from labeled brain slices, indicating that labeling was to the same molecular entity previously characterized as the transferrin receptor. The pattern of transferrin receptor distribution visualized in brain with both 125I-labeled transferrin and an anti-transferrin receptor monoclonal antibody are almost indistinguishable but differ from the pattern of iron distribution. Iron-rich brain areas generally receive neuronal projections from areas with abundant transferrin receptors, suggesting that iron may be transported neuronally. However, many brain areas with a high density of transferrin receptors appear unrelated to iron uptake and neuronal transport and form a receptor distribution pattern similar to that of other known neuropeptides. This "neuropeptide-like" distribution pattern suggests that transferrin may have neuromodulatory, perhaps behavioral, function in brain.
Hb Long Island: a hemoglobin variant with a methionyl extension at the NH2 terminus and a prolyl substitution for the normal histidyl residue 2 of the beta chain.
Authors: Barwick R C , Jones R T , Head C G , Shih M F , Prchal J T , Shih D T
Published: May 2006

Hb Long Island was found in a diabetic man and his nondiabetic mother as the result of a routine clinical measurement of Hb AIc. It is present in amounts approximately equal to Hb A. Its alpha chains are normal but its beta chains have two alterations compared to the normal. A methionyl residue is attached to the usual NH2-terminal valyl residue. This valyl residue is followed by prolyl residue in place of the usual histidyl residue 2. The remaining sequence of the beta chain is normal. No hemoglobin or abnormal beta chain containing only the prolyl substitution could be detected by several different electrophoretic and HPLC procedures. We postulate that Hb Long Island is the result of a mutation in which a single nucleotide change causes the substitution of a prolyl residue for the normal histidyl residue at position 2 of the beta chain. We further postulate that this abnormal prolyl residue inhibits enzymatic cleavage of the initiator methionyl residue from the abnormal beta chain during posttranslational processing. Although the oxygen affinities of the whole blood, suspended cells, and hemolysate are normal, the affinity of the isolated Hb Long Island is slightly decreased and the effects of organic phosphates are reduced compared to normal. These changes are consistent with the loss of the normal histidyl residue 2 and the extension of the NH2-terminal end of the beta-chain molecule.
Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins.
Authors: Schatten G , Maul G G , Schatten H , Chaly N , Simerly C , Balczon R , Brown D L
Published: May 2006

Nuclear structural changes during fertilization and embryogenesis in mice and in sea urchins have been followed by using antibodies against the nuclear lamins A/C and B and against antigens at the periphery of nuclei and chromosomes. Lamins are found on all pronuclei and nuclei during mouse fertilization, but with a diminished intensity on the second polar body nucleus. On sperm in both systems, lamins are reduced and detected only at the acrosomal and centriolar fossae. In sea urchin eggs, lamins are found on both pronuclei. Unlike in other dividing cells, the mitotic chromosomes of sea urchin eggs and embryos retain an association with lamins. The peripheral antibodies delineate each chromosome and nucleus except the mature mouse sperm nucleus. A dramatic change from the expected lamin distribution occurs during early development. In mouse morulae or blastocysts, lamins A/C are no longer recognized, although lamin B remains. In sea urchins both lamins A/C and lamin B, as detected with polyclonal antibodies, are lost after the blastula stage, although a different lamin A/C epitope emerges as recognized by a monoclonal antibody. These results demonstrate that pronucleus formation in both systems involves a new association or exposure of lamins, that the polar body nucleus is largely restricted from the cytoplasmic pool of lamins, and that mitotic chromosomes in the rapidly proliferating sea urchin egg retain associated lamins. They also suggest that changes in the expression or exposure of different lamins are a common feature of embryogenesis.
Precursor forms of substance P (SP) in nervous tissue: detection with antisera to SP, SP-Gly, and SP-Gly-Lys.
Authors: Kream R M , Schoenfeld T A , Mancuso R , Clancy A N , el-Bermani W , Macrides F
Published: May 2006

Antisera generated to substance P-Gly (SP-G) and substance P-Gly-Lys (SP-G-K), the likely unamidated COOH-terminally extended forms of substance P, were used to quantify and localize substance P precursor forms in hamster brain stem and spinal cord. The precursor determinant SP-G-K was liberated from larger heterogeneous forms by mild trypsinization of tissue extracts and was converted into the second precursor determinant, SP-G, by subsequent treatment with carboxypeptidase B. The basal levels of SP-G-K in brain stem and spinal cord were approximately equal to 0.5 pg/mg of tissue and rose 43- to 64-fold after trypsinization. Basal levels of SP-G were comparable to those of SP-G-K and rose 10- to 29-fold after combined enzyme treatments. Immunohistochemical labeling of axons and somata with anti-SP-G-K increased dramatically after trypsinization. This labeling was eliminated by preadsorption with authentic SP-G-K but not substance P or SP-G. Gel-permeation chromatography revealed SP-G-K-like immunoreactivity in fractions corresponding to considerably higher molecular weight than mature substance P. Collectively, these results support the hypothesis that substance P is synthesized from larger precursors and demonstrate that extended precursor forms are normally present in the axons and somata of neural systems that synthesize substance P.
Geometry and topology of isoparametric submanifolds in euclidean spaces
Authors: Hsiang W. Y. , Palais R. S. , Terng C. L.
Published: May 2006

Restrictions are given on the possible marked Dynkin diagrams of isoparametric submanifolds, and their homology and cohomology are computed by an extension of the techniques used by Bott and Samelson [Bott, R. & Samelson, H. (1958)
Am. J. Math.
80, 964-1029] and by Borel [Borel, A. (1953)
Ann. Math.
57 (2), 115-207] for G/T.
Kinetics of carbon monoxide binding to phenobarbital-induced cytochrome P-450 from rat liver microsomes: a simple bimolecular process.
Authors: Oertle M , Richter C , Winterhalter K H , Di Iorio E E
Published: May 2006

The kinetics of carbon monoxide binding to phenobarbital-induced cytochrome P-450 (P-450PB) and to its enzymatically inactive form P-420PB have been investigated by both stopped-flow and flash-photolysis spectrophotometry. When the simultaneous presence of both forms of the enzyme is taken into account, the binding of CO to these two proteins can be described in terms of two bimolecular processes with rate constants of 4.5 X 10(6) M-1.S-1 and 4.7 X 10(5) M-1.S-1 for P-450PB and 1.7 X 10(7) M-1.S-1 and 1.5 X 10(6) M-1.S-1 for P-420PB. From kinetic studies of the binding of CO to P-450PB under different experimental conditions, investigations of the homogeneity of our P-450PB preparations, and comparative kinetic investigations of P-450s from different sources, we conclude that CO binding to reduced P-450PB is a simple bimolecular process and that the observed biphasic traces are due to heterogeneity of the proteins. This conclusion is in contrast with previous reports of complex reaction mechanisms for the binding of CO to P-450PB. Optical spectroscopy studies indicate the existence of an equilibrium between P-450PB and P-420PB, at least for the reduced carbonyl derivatives of the enzymes. The interconversion is strongly influenced by the aggregation state of the protein. Large differences between the CO binding properties of P-450PB and those of P-420PB are found. These are discussed in terms of possible effects of the proximal ligation state of the iron on heme reactivity.
Ectopic peptides released by a human melanoma cell line that modulate the transformed phenotype.
Authors: De Larco J E , Pigott D A , Lazarus J A
Published: May 2006

Cells derived from a human melanoma strain by low-serum selection in monolayer were found to be capable of growth in semisolid medium, forming colonies ranging from tight to loose or dispersed. These phenotypes were found to be stable after cloning and retesting. Examination of the biological activities of ectopic peptides produced by a clone that gives rise to loose dispersed colonies revealed the presence of a transforming growth factor beta (TGF-beta; apparent Mr 14,700) and a single TGF-alpha species produced at an unusually high concentration (2.2 ng/ml). Coeluted with the TGF-alpha at approximately 22,500 was a mitogenic activity and a previously undescribed activity capable of modulating the phenotype of induced normal rat kidney fibroblast (NRK-49F) colonies in semisolid medium. This activity can modulate the usual tight colony to express a loose or dispersed phenotype characteristic of the producer cells. Both the possible role these ectopic peptides play in the expression of the transformed phenotype by the tumor cells producing them and the possible correlation between the Mr 22,500 epidermal growth factor-like peptide released by this particular tumor line and high molecular weight epidermal growth factor-like peptides found in the urine of cancer patients are discussed.
Transformation of salivary gland secretion protein gene Sgs-4 in Drosophila: stage- and tissue-specific regulation, dosage compensation, and position effect.
Authors: Krumm A , Roth G E , Korge G
Published: May 2006

The Sgs-4 gene of Drosophila melanogaster encodes one of the larval secretion proteins and is active only in salivary glands at the end of larval development. This gene lies in the X chromosome and is controlled by dosage compensation--i.e., the gene is hyperexpressed in males. Therefore, males with one X chromosome produce nearly as much Sgs-4 products as females with two X chromosomes. We used a 4.9-kilobase-pair (kb) DNA fragment containing the Sgs-4d coding region embedded in 2.6 kb of upstream sequences and 1.3 kb of downstream sequences for P-element-mediated transformation of the Sgs-4h underproducer strain Kochi-R. Sgs-4d gene expression was found in all 15 transformed lines analyzed, varying with the site of chromosomal integration. The transposed gene was subject to tissue- and stage-specific regulation. At X-chromosomal sites, the levels of gene expression were similar in both sexes, signifying dosage compensation. At autosomal sites, it was on average 1.5 times higher in males than in females. The results indicate that the transforming DNA fragment contains all sequences necessary for tissue- and stage-specific regulation and for hyperexpression in males.