bluuebunny/crossref_metadata_2025 huggingface dataset:
Microdissection and microcloning of the mouse X chromosome.
Authors: Fisher E M , Cavanna J S , Brown S D
Published: May 2006

A wild mouse (CD) karyotype in which all the chromosomes bar the X, 19, and Y, are fused as metacentrics has been used for the microdissection and microcloning of a specific mouse X chromosome region. Dissection of a proximal region of the X chromosome encompassing the genetic loci Hprt to Tfm and including mdx has yielded 650 clones. A number of the recovered clones containing sizable inserts have been confirmed as X chromosome specific. This X chromosome bank of clones provides a start point for the isolation of the mdx locus. It is now clear that microdissection and microcloning can be applied to all mouse chromosomes, including the X chromosome, yielding premapped banks of clones that will greatly aid in the isolation and characterization of important genetic loci.
Glucocorticoid regulation of mouse mammary tumor virus sequences in transgenic mice.
Authors: Ross S R , Solter D
Published: May 2006

We have introduced a chimeric plasmid, pLTR2TK, containing the mouse mammary tumor virus (MTV) long terminal repeat (LTR) linked to the herpes simplex virus type 1 thymidine kinase gene into the mouse germ line by microinjection. In one mouse line, the thymidine kinase gene is appropriately expressed in the lactating mammary glands of heterozygous females; expression also occurs in the ovaries of these mice. In heterozygous males of this line, and in a male derived from another microinjection, transcription of these new germ line genes occurs in the testes and is specifically stimulated by glucocorticoid hormones. Thus, the MTV LTR may contain elements that allow its expression both in lactating mammary glands and in testicular tissue.
Altered steady-state ratio of type I/III procollagen mRNAs correlates with selectively increased type I procollagen biosynthesis in cultured keloid fibroblasts.
Authors: Uitto J , Perejda A J , Abergel R P , Chu M L , Ramirez F
Published: May 2006

Regulation of collagen gene expression was studied in fibroblast cultures established from patients with keloids, fibrotic lesions of the skin. In selected keloid fibroblast cultures, an overproduction of type I procollagen was observed. This increase was accompanied by a parallel increase in type I procollagen-specific mRNA levels, as detected by dot-blot and RNA transfer hybridizations, without concomitant change in type I procollagen gene copy number. At the same time, type III procollagen mRNA levels were unaltered, resulting in markedly elevated type I/III procollagen mRNA ratios. Thus, keloid fibroblasts offer a unique model to study the independent regulation of the gene expression of two genetically distinct procollagens, type I and type III.
In vitro correction of the abnormal multimeric structure of von Willebrand factor in type IIa von Willebrand's disease.
Authors: Gralnick H R , Williams S B , McKeown L P , Maisonneuve P , Jenneau C , Sultan Y , Rick M E
Published: May 2006

Type IIa von Willebrand's disease (vWd) has been characterized by the absence of the largest and a reduction in the intermediate-sized multimers of the plasma and platelet von Willebrand factor (vWf) and by the diminished response of the platelet-rich plasma of these patients to ristocetin. Other recently demonstrated abnormalities include the presence of an abnormal triplet structure of vWf. We have studied the plasma and platelets from three patients with this form of vWd and have found that both their plasma and platelets manifest the previously described abnormalities. Because of the heterogeneity of the multimeric structure of the vWf in these patients, we considered the possibility that postsynthetic events may have modified the vWf. When blood was collected in 5 mM EDTA or 5 mM EDTA/leupeptin/N-ethylmaleimide, the abnormal multimeric structure of the plasma and platelet vWf was partially normalized in that the intermediate and the largest vWf multimers were increased, the abnormal multimer structure was no longer as apparent, and the fastest migrating band (an abnormality seen only in the type IIa vWd plasma and platelets) disappeared. The enzymatic activity responsible for this degradation can be classified as a calcium-dependent protease. Studies of normal radiolabeled vWf incubated with platelet lysates from normal subjects and these patients revealed that the patients' platelets did not contain increased amounts of calcium-dependent protease activity as assessed by degradation of normal vWf. These data suggest that patients with type IIa vWd synthesize an abnormal vWf protein that is susceptible to in vitro proteolytic degradation and that proteolytic degradation can play a significant role in the phenotypic expression of vWd by modifying the plasma and platelet vWf multimeric structure.
Transformation of prostaglandin D2 to 9 alpha, 11 beta-(15S)-trihydroxyprosta-(5Z,13E)-dien-1-oic acid (9 alpha, 11 beta-prostaglandin F2): a unique biologically active prostaglandin produced enzymatically in vivo in humans.
Authors: Liston T E , Roberts L J
Published: May 2006

In vitro studies examining the metabolic transformation of prostaglandin D2 (PGD2) by human liver were conducted. PGD2 was found to be converted by a NADPH-dependent enzyme in the 100,000 X g supernatant of human liver exclusively to a single more polar compound that had a mass spectrum essentially the same as that of prostaglandin F2 alpha (PGF2 alpha). However, this compound could be chromatographically separated from PGF2 alpha and failed to form a butylboronate derivative. The structure of this compound was established as 9 alpha, 11 beta-(15S)-trihydroxyprosta-(5Z, 13E)-dien-1-oic acid (9 alpha, 11 beta-PGF2) by comparison of its chromatographic and mass spectral characteristics with authentic 9 alpha, 11 beta-PGF2. This compound was found to be biologically active by demonstrating increases in blood pressure in rats in a dose-related fashion following intravenous administration. By using a mass spectrometric assay, levels of this compound in plasma and urine from a normal volunteer were 6 pg/ml and 982 ng/24 hr, respectively. In a patient with systemic mastocytosis associated with overproduction of PGD2, urinary excretion of 9 alpha, 11 beta-PGF 2 was 6634 ng/24 hr and a circulating plasma level as high as 490 ng/ml was found during a severe episode of systemic mast cell activation. 9 alpha, 11 beta-PGF2 is structurally a unique prostaglandin, is enzymatically formed, is produced in vivo in humans, and is biologically active.
A putative Ca2+-binding protein: structure of the light subunit of porcine calpain elucidated by molecular cloning and protein sequence analysis.
Authors: Sakihama T , Kakidani H , Zenita K , Yumoto N , Kikuchi T , Sasaki T , Kannagi R , Nakanishi S , Ohmori M , Takio K
Published: May 2006

cDNA clones specific for the light subunit of porcine calpain I have been isolated from a porcine kidney cDNA library. The complete primary structure of the light subunit has been revealed by nucleotide sequence analysis of the cDNA clones isolated and amino acid sequence analysis of peptides isolated from the purified mature protein. We found that the light subunit contains two distinct domains. Domain I, the amino-terminal half, has two unusually long, paired polyglycyl sequences and may serve as a binding site to the heavy subunit. Domain II, the carboxyl-terminal half, is a region highly homologous to the putative Ca2+-binding domain of the heavy subunit of chicken calpain elucidated recently. This region has four potential Ca2+-binding sites, each having the "E-F hand" structure. Our results suggest that the Ca2+-mediated proteolytic activity of calpain is controlled through the cooperative and/or sequential actions of multiple Ca2+-binding sites present in both two-subunit molecules, heavy and light subunits of calpain.
Expression and assembly of active cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase in Escherichia coli containing stoichiometric amounts of large and small subunits.
Authors: Tabita F R , Small C L
Published: May 2006

The genes for the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase from the cyanobacterium Anacystis nidulans were subcloned into plasmid pUC9. After induction, both genes were expressed in Escherichia coli and the subunits were assembled into an active holoenzyme. The enzyme was purified from E. coli to high specific activity and was found to contain equimolar amounts of large and small subunits. The assembly of the hexadecameric ribulose bisphosphate carboxylase/oxygenase in E. coli should provide the basis for studies on the mechanism of assembly and the role of small subunits in catalysis.
Molecular mechanism of water oxidation in photosynthesis based on the functioning of manganese in two different environments
Authors: Kambara T. , Govindjee
Published: May 2006

We present a model of photosynthetic water oxidation that utilizes the property of higher-valent Mn ions in two different environments and the characteristic function of redox-active ligands to explain all known aspects of electron transfer from H
2
O to Z, the electron donor to P680, the photosystem II reaction center chlorophyll
a
. There are two major features of this model. (
i
) The four functional Mn atoms are divided into two groups of two Mn each: [Mn] complexes in a hydrophobic cavity in the intrinsic 34-kDa protein; and (Mn) complexes on the hydrophilic surface of the extrinsic 33-kDa protein. The oxidation of H
2
O is carried out by two [Mn] complexes, and the protons are transferred from a [Mn] complex to a (Mn) complex along the hydrogen bond between their respective ligand H
2
O molecules. (
ii
) Each of the two [Mn] ions binds one redox-active ligand (RAL), such as a quinone (alternatively, an aromatic amino acid residue). Electron transfer occurs from the reduced RAL to the oxidized Z. When the experimental data concerning atomic structure of the water-oxidizing center (WOC), electron transfer between the WOC and Z, the electronic structure of the WOC, the proton-release pattern, and the effect of Cl
-
are compared with the predictions of the model, satisfactory qualitative and, in many instances, quantitative agreements are obtained. In particular, this model clarifies the origin of the observed absorption-difference spectra, which have the same pattern in all S-state transitions, and of the effect of Cl
-
-depletion on the S states.
Mode of coaction between blue/UV light and light absorbed by phytochrome in light-mediated anthocyanin formation in the milo (
Sorghum vulgare
Pers.) seedling
Authors: Oelmüller Ralf , Mohr Hans
Published: May 2006

Anthocyanin formation in milo (
Sorghum vulgare
Pers.) seedlings (coleoptile, mesocotyl, taproot) occurs only in white light and blue/UV light (BL/UV), while red light (RL) and far-RL are totally ineffective. However, after a BL/UV pretreatment, the participation of phytochrome can be demonstrated. With a short-wavelength light source [peak emission in longwave UV (UV-A)], the mode of coaction between BL/UV and light absorbed by phytochrome (RL) was studied with the following principal results. (
i
) As soon as the seedling becomes competent to respond to UV-A (with regard to anthocyanin formation), the involvement of phytochrome can be detected. (
ii
) A 5-min pulse of UV-A has a strong effect on the anthocyanin synthesis in the milo mesocotyl. This effect is fully reversible if a long-wavelength far-RL pulse (RG9 light) is given immediately after the UV-A light pulse. (
iii
) When seedlings treated with 5 min of UV-A and 5 min of RG9 light are kept in darkness for 3 hr and then transferred to RL, anthocyanin appears. (
iv
) In continuous UV-A treatment, anthocyanin accumulation starts after a lag phase of 3.5 hr (25°C). A RL pretreatment prior to the onset of UV-A treatment strongly increases anthocyanin accumulation in UV-A, though the lag phase is not affected. Moreover, a RL pretreatment does not affect the time course for escape from reversibility in UV-A. It is concluded from these data that BL/UV cannot mediate induction of anthocyanin synthesis in the absence of P
fr
, the active form of phytochrome that absorbs maximally in the far-red. Rather, the action of BL/UV must be considered to establish responsiveness of the anthocyanin-producing mechanism to P
fr
. P
fr
operates in this system via two different channels. As the effector of the terminal response, it sets in motion the signal-response chain that eventually leads to the appearance of anthocyanin. This is a slow process with a lag phase of the order of 3.5 hr. The second function of P
fr
is to determine the responsiveness to the effector P
fr
in mediating anthocyanin synthesis. This is a very fast and highly sensitive phytochrome action that can be detected readily within 1 min. However, as long as the plant has not received BL/UV, the strong effect of RL on the effectiveness of P
fr
remains cryptic. The effect of a RL pretreatment and the effect of a UV-A pretreatment on responsiveness towards P
fr
(or, effectiveness of P
fr
) were found to be totally independent of each other, even though it is the UV-A that permits operation of P
fr
.
(Glu62, Ala30, Tyr8)n serves as high-affinity substrate for tyrosylprotein sulfotransferase: a Golgi enzyme.
Authors: Lee R W , Huttner W B
Published: May 2006

Tyrosylprotein sulfotransferase, the enzyme catalyzing the sulfation of proteins on tyrosine residues, was characterized by using the acidic polymer containing tyrosine (Glu62, Ala30, Tyr8)n (referred to as Glu,Ala,Tyr) as exogenous "protein" substrate. After subcellular fractionation of a bovine adrenal medulla homogenate, tyrosylprotein sulfotransferase activity was found to be highest in fractions enriched in Golgi membrane vesicles. Tyrosylprotein sulfotransferase required the presence of a nonionic detergent for sulfation of exogenous Glu,Ala,Tyr, indicating an orientation of the catalytic site of the enzyme toward the Golgi lumen. Tyrosylprotein sulfotransferase was solubilized by Triton X-100, suggesting that the enzyme was tightly associated with the Golgi membrane, possibly as an integral membrane protein. The apparent Golgi localization of tyrosylprotein sulfotransferase was supported by the observation that tyrosine sulfation of proteins in intact cells was blocked by monensin and was in line with previous observations that all tyrosine-sulfated proteins known so far are secretory. Glu,Ala,Tyr was found to have a very high affinity for tyrosylprotein sulfotransferase (apparent Km, 300 nM), similar to that reported for certain tyrosylprotein kinases. While this may suggest some similarity between these enzymes, the Golgi localization of tyrosylprotein sulfotransferase segregates tyrosine sulfation from the sites of tyrosine phosphorylation of proteins in the intact cell. If, however, tyrosylprotein sulfotransferase was allowed to react with cytoplasmic proteins by using a nonionic detergent, tyrosine sulfation of tubulin was observed.
Change of karyoskeleton during spermatogenesis of Xenopus: expression of lamin LIV, a nuclear lamina protein specific for the male germ line.
Authors: Benavente R , KrohneG
Published: May 2006

Lamins are the major constituent proteins of the nuclear lamina. In the frog, Xenopus laevis, they are the products of a multigene family whose expression can be correlated to certain routes of cell differentiation. For example, lamins LI (Mr, 72,000) and LII (Mr, 68,000) is expressed, together with LI/LII, in certain highly differentiated cell types such as neurons and muscle cells and is the only lamin present in diplotene oocytes. Here we report the identification by means of two monoclonal antibodies of a fourth lamin (LIV) of Mr 75,000, which is expressed specifically during the later stages of spermatogenesis. In the seminiferous tubules, Sertoli cells contain LI/LII and LIII whereas, among the spermatogenic cells, spermatogonia contain only LI and LII. In contrast, in spermatids and sperm cells these lamins are completely replaced by lamin LIV. Primary spermatocytes are negative with both antibodies, indicating that a switch in the expression of lamins occurs early in spermatogenesis. Lamin LIV is distributed in patches along the nuclear envelopes of elongated spermatids and sperm cells rather than in the characteristic continuous lamina pattern found in most other cells. We hypothesize that the specific expression of lamin LIV is related to the conspicuous changes of nuclear architecture and chronmatin composition that are known to take place during the late stages of sperm development.
Comparative statistics for DNA and protein sequences: multiple sequence analysis.
Authors: Karlin S , Ghandour G
Published: May 2006

Concepts and methods [Karlin, S. & Ghandour, G. (1985) Proc. Natl. Acad. Sci. USA 82, 5800-5804] for the analysis of patterns and relationships are extended to multiple DNA and protein sequences. Functionals include multiple sequence common word occurrence distributions, characterizations of high frequency shared words, and ascertainment of long block identities. Various comparisons of sequences using natural alphabets obtained from grouping nucleotides or amino acids by their chemical and functional characteristics are described. Specific applications are given to globin genes, mitochondrial genomes, and a variety of mammalian viruses.
DNA-damage-inducible (din) loci are transcriptionally activated in competent Bacillus subtilis.
Authors: Love P E , Lyle M J , Yasbin R E
Published: May 2006

DNA damage-inducible (din) operon fusions were generated in Bacillus subtilis by transpositional mutagenesis. These YB886(din::Tn917-lacZ) fusion isolates produced increased beta-galactosidase when exposed to mitomycin C, UV radiation, or ethyl methanesulfonate, indicating that the lacZ structural gene had inserted into host transcriptional units that are induced by a variety of DNA-damaging agents. One of the fusion strains was DNA-repair deficient and phenotypically resembled a UV-sensitive mutant of B. subtilis. Induction of beta-galactosidase also occurred in the competent subpopulation of each of the din fusion strains, independent of exposure to DNA-damaging agents. Both the DNA-damage-inducible and competence-inducible components of beta-galactosidase expression were abolished by the recE4 mutation, which inhibitS SOS-like (SOB) induction but does not interfere with the development of the competent state. The results indicate that gene expression is stimulated at specific loci within the B. subtilis chromosome both by DNA-damaging agents and by the development of competence and that this response is under the control of the SOB regulatory system. Furthermore, they demonstrate that at the molecular level SOB induction and the development of competence are interrelated cellular events.
Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules.
Authors: Leigh J A , Signer E R , Walker G C
Published: May 2006

By screening with the fluorescent stain Calcofluor, we have isolated 26 independent transposon Tn5 insertion mutants of Rhizobium meliloti that are deficient in the production of a known extracellular polysaccharide (Exo-). The mutants belonged to six distinct genetic groups based on the ability of their Exo- phenotype to be complemented by different recombinant plasmids from a R. meliloti clone bank. With few exceptions, all of the mutants formed ineffective (non-nitrogen-fixing) nodules on alfalfa. For all but one group, the complementing plasmids restored effective nodulation. These results establish a firm and extensive correlation between the ability of Rhizobium to produce a particular polysaccharide and symbiotic proficiency. The ineffective nodules appeared to contain no bacteroids and to form without shepherds' crooks or infection threads; this symbiotic phenotype matches that described for a set of independently isolated mutants that belong phenotypically and genetically to the group B exopolysaccharide mutants described previously [Finan et al. (1985) Cell 40, 869-877]. Apparently the exopolysaccharide, although not required for nodule formation, is involved in wild-type nodule invasion.
Cloning, sequence, and expression of a human granulocyte/macrophage colony-stimulating factor.
Authors: Cantrell M A , Anderson D , Cerretti D P , Price V , McKereghan K , Tushinski R J , Mochizuki D Y , Larsen A , Grabstein K , Gillis S
Published: May 2006

Human granulocyte/macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is essential for the in vitro proliferation and differentiation of precursor cells into mature granulocytes and macrophages. In this report we have used a mouse GM-CSF cDNA clone to isolate human GM-CSF clones from libraries made from HUT-102 messenger RNA and mitogen-stimulated T-lymphocyte messenger RNA. The human cDNA clones contained a single open-reading frame encoding a protein of 144 amino acids with a predicted molecular mass of 16,293 daltons and showed 69% nucleotide homology and 54% amino acid homology to mouse GM-CSF. One of these cDNA clones was shown to direct the synthesis of biologically active GM-CSF using a yeast expression system. The gene for human GM-CSF appears to exist as a single-copy gene.
Apolipoprotein multigene family: tandem organization of human apolipoprotein AI, CIII, and AIV genes.
Authors: Karathanasis S K
Published: May 2006

The genes for two of the proteins of the plasma lipid transport system, apolipoprotein AI (apoAI) and CIII (apoCIII) are closely linked in the human genome. An approximately equal to 30-kilobase (kb) DNA segment containing these genes and their flanking sequences has been cloned and extensively characterized. Hybridization studies revealed that a DNA fragment located 12 kb 3' to the apoAI gene contains sequences homologous to a 1.8-kb mRNA transcript in human fetal intestine and adult liver but not in fetal liver, kidney, heart, brain, or muscle. This DNA fragment was used as a probe to isolate a clone from an adult human liver cDNA library. The nucleotide sequence of this clone is 74.8% homologous to the cDNA sequence of rat apolipoprotein AIV (apoAIV), another protein of the lipid transport system, and codes for a protein that is 58.6% identical to rat apoAIV. These results indicate that apoAI, apoCIII, and apoAIV genes are closely linked in the human genome and suggest that all three of them are derived from a common ancestral precursor.
Mutagenesis of avian carcinoma virus MH2: only one of two potential transforming genes (delta gag-myc) transforms fibroblasts.
Authors: Zhou R P , Kan N , Papas T , Duesberg P
Published: May 2006

Avian carcinoma virus MH2 contains two potential transforming genes, delta gag-mht and delta gag-myc. Thus, MH2 may be a model for two-gene carcinogenesis in which transformation depends on two synergistic genes. Most other directly oncogenic viruses contain single, autonomous transforming (onc) genes and are models for single-gene carcinogenesis. To determine which role each potential onc gene of MH2 plays in oncogenesis, we have prepared deletion and frameshift mutants of each of the two MH2 genes by in vitro mutagenesis of cloned proviral DNA and have tested transforming function and virus production in cultured primary quail cells. We have found that mht deletion mutants and wild-type virus transform primary cells and that myc deletion and frameshift mutants do not. The morphologies of cells transformed by the mht deletion mutants and by wild-type MH2 are similar yet vary considerably. Nevertheless, typical mutant transformed cells can often be distinguished from cells transformed by wild-type MH2. We conclude that the delta gag-myc gene transforms primary cells by itself, without the second potential onc gene. This myc-related gene is the smallest that has direct transforming function. delta gag-mht is without detectable transforming function but may affect transformation by delta gag-myc. Thus, MH2 behaves like a virus with a single onc gene, although it expresses two potential onc genes, and it appears not to be a model for two-gene carcinogenesis. Further work is necessary to determine whether the delta gag-mht gene possibly enhances oncogenic function of delta gag-myc or has independent oncogenic function in animals.
Crystallographic structure of an active, sequence-engineered ribonuclease.
Authors: Taylor H C , Komoriya A , Chaiken I M
Published: May 2006

X-ray diffraction methods were used to test a synthetic-modeling approach to the sequence engineering of bovine pancreatic ribonuclease. A model of RNase S-peptide (residues 1-20), having a simplified amino acid sequence but retaining elements deduced to be essential for conformation and function, was previously synthesized and found to form a catalytically active and stable complex with native S-protein (residues 21-24). We have now obtained a 3-A-resolution electron density map of this semisynthetic complex which reveals that the conformation of model peptide closely mimics that of native S-peptide, as intended by sequence design. Some small differences from the native structure are observed: Glu-2 and Arg-10 of the model complex are not close enough to form a salt bridge, the position of the His-12 imidazole ring is slightly shifted in the active site, and the peptide's amino terminus is reoriented. Nonetheless, the major structural features predicted to be essential by computer-aided peptide-design analysis are preserved in the model peptide portion of the complex. These include (i) the alpha-helical framework involving residues 3-13, (ii) the catalytically competent orientation of His-12, and (iii) complex-stabilizing non-bonding interactions involving Phe-8 and Met-13 of S-peptide and hydrophobic residues in the cleft region of S-protein. Further, sequence simplification has not introduced any non-native, potentially stabilizing contacts between the model peptide and S-protein. The results emphasize the usefulness, in redesigning native proteins, of categorizing sequence into residues providing conformational framework and those determining intra-and intermolecular surface recognition.
Aflatoxin metabolism in humans: detection of metabolites and nucleic acid adducts in urine by affinity chromatography.
Authors: Groopman J D , Donahue P R , Zhu J Q , Chen J S , Wogan G N
Published: May 2006

A high-affinity IgM monoclonal antibody specific for aflatoxins was covalently bound to Sepharose 4B and used as a preparative column to isolate aflatoxin derivatives from the urine of people and experimental animals who had been exposed to the carcinogen environmentally or under laboratory conditions. Aflatoxin levels were quantified by radioimmunoassay and high-performance liquid chromatography after elution from the affinity column. In studies on rats injected with [14C]aflatoxin B1, we identified the major aflatoxin-DNA adduct, 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (AFB1-N7-Gua), and the oxidative metabolites M1 and P1 as the major aflatoxin species present in the urine. When this methodology was applied to human urine samples obtained from people from the Guangxi Province of China exposed to aflatoxin B1 through dietary contamination, the aflatoxin metabolites detected were also AFB1-N7-Gua and aflatoxins M1 and P1. Therefore, affinity chromatography using a monoclonal antibody represents a useful and rapid technique with which to isolate this carcinogen and its metabolites in biochemical epidemiology and for subsequent quantitative measurements, providing exposure information that can be used for risk assessment.
A v-erbB-related protooncogene, c-erbB-2, is distinct from the c-erbB-1/epidermal growth factor-receptor gene and is amplified in a human salivary gland adenocarcinoma.
Authors: Semba K , Kamata N , Toyoshima K , Yamamoto T
Published: May 2006

From a human genomic library, we obtained six v-erbB-related DNA clones. A DNA probe prepared from one of the clones, lambda 107, hybridized to EcoRI fragments of 6.4 and 13 kilobase pairs of human DNA. Neither of these fragments was amplified in A431 vulva carcinoma cells, in which the gene encoding the epidermal growth factor receptor is amplified. In addition, the probe from lambda 107 hybridized with a single, 4.8-kilobase poly(A)+ RNA species and did not react with EGF receptor mRNA. Thus, we conclude that clone lambda 107 represents a v-erbB-related gene (c-erbB-2) that is distinct from the EGF receptor gene. In contrast, the other five clones were shown to represent the EGF receptor gene (c-erbB-1). Partial nucleotide sequence analysis of the lambda 107 insert showed that this clone contained at least seven putative exons and that six of them could encode the kinase domain characteristic of protein products of the src oncogene family. Southern blot analysis showed close similarity of the restriction patterns of the rat c-erbB-2 gene and the rat neu oncogene, suggesting possible involvement of c-erbB-2 in human cancer. In fact, approximately 30-fold amplification of c-erbB-2 was observed in a human adenocarcinoma of the salivary gland.
Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection
Authors: Showalter Allan M. , Bell John N. , Cramer Carole L. , Bailey John A. , Varner Joseph E. , Lamb Chris J.
Published: May 2006

Hydroxyproline-rich glycoproteins (HRGPs) are important structural components of plant cell walls and also accumulate in response to infection as an apparent defense mechanism. Accumulation of HRGP mRNA in biologically stressed bean (
Phaseolus vulgaris
L.) cells was monitored by blot hybridization with
32
P-labeled tomato genomic HRGP sequences. Elicitor treatment of suspension-cultured cells caused a marked increase in hybridizable HRGP mRNA. The response was less rapid but more prolonged than that observed for mRNAs encoding enzymes of phytoalexin biosynthesis. HRGP mRNA also accumulated during race:cultivar-specific interactions between bean hypocotyls and the partially biotrophic fungus
Colletotrichum lindemuthianum
, the causal agent of anthracnose. In an incompatible interaction (host resistant) there was an early increase in HRGP mRNA correlated with expression of hypersensitive resistance; whereas, in a compatible interaction (host susceptible), marked accumulation of HRGP mRNA occurred as a delayed response at the onset of lesion formation. In both interactions, mRNA accumulation was observed in uninfected cells distant from the site of fungal inoculation, indicating intercellular transmission of an elicitation signal.
In vivo prevention of thyroid and pancreatic autoimmunity in the BB rat by antibody to class II major histocompatibility complex gene products.
Authors: Boitard C , Michie S , Serrurier P , Butcher G W , Larkins A P , McDevitt H O
Published: May 2006

Evidence is accumulating that the development of insulin-dependent diabetes mellitus involves autoimmune phenomena, both in the human and in the BB rat model. A strong association is observed in both cases with alleles of the class II major histocompatibility complex (MHC). Results of the present study show that autoimmune phenomena, as assessed by the presence of clinical diabetes or histological thyroiditis, are prevented by the injection of monoclonal antibodies to class II gene products in the BB rat. Immunosuppression was specifically obtained with a monoclonal antibody to the murine I-E equivalent, as opposed to the murine I-A equivalent, of the rat major histocompatibility complex. This represents indirect evidence for I-E subregion control of immune responses to islet cell and thyroid antigens in the BB rat model. The frequent occurrence of anaphylactic type deaths in young (1 month old) animals receiving more than six weekly injections of partially purified homologous (rat) monoclonal antibodies to rat class II gene products underscores the potential risks of this type of immunotherapy. The presumed immunologic mechanism (IgE antibody) and its specificity (anti-allotype, anti-idiotype, or anti-impurity) must be clarified to assess the risks and feasibility of this type of therapy.
Purification and partial amino acid sequence of asialo murine granulocyte-macrophage colony stimulating factor.
Authors: Sparrow L G , Metcalf D , Hunkapiller M W , Hood L E , Burgess A W
Published: May 2006

A procedure utilizing reversed-phase high-performance liquid chromatography is described for the purification of asialo granulocyte-macrophage colony stimulating factor (asialo-GM-CSF) from mouse lung-conditioned medium. In the purification, the partially purified factor was treated with neuraminidase to reduce charge heterogeneity due to variable degrees of sialation. Three active forms of the asialo factor were separated by the final reversed-phase liquid chromatography step. These each gave a single major band and several minor bands on polyacrylamide gel electrophoresis and had similar amino acid compositions. The specific activity of purified murine asialo-GM-CSF was approximately 8 X 10(9) colonies per mg of protein. Amino acid sequence determination of the major form gave a single amino-terminal sequence, which has been used to develop oligonucleotide probes for the isolation of two cDNA clones encoding GM-CSF. The nucleotide sequence of these two clones gave a deduced amino acid sequence almost identical with that determined for the amino terminus of asialo-GM-CSF and an amino acid composition very similar to that for asialo-GM-CSF.
An enhancer-like element in the adenovirus E2 promoter contains sequences essential for uninduced and E1A-induced transcription.
Authors: Imperiale M J , Hart R P , Nevins J R
Published: May 2006

Using a chloramphenicol acetyltransferase gene under the control of a series of adenovirus E2 promoter mutants, we have determined the sequence requirements for E1A-induced as well as uninduced expression of the E2 promoter. Fully induced expression requires 79 nucleotides of upstream sequence, and the same sequences are required for uninduced expression. A promoter containing 70 nucleotides of upstream sequence is inducible, although to a lower level than wild type, while a promoter containing 59 nucleotides of upstream sequence is not inducible. The sequences required for induction as well as for uninduced promoter activity map between 21 and 79 nucleotides upstream of the transcriptional initiation site and function in either orientation and at a distance. We suggest that E1A-mediated induction involves the effective increase in a factor utilizing this upstream site and that the factor can functionally interact with the site irrespective of its location relative to the start of transcription.
Cation binding by bacteriorhodopsin
Authors: Chang C.-H. , Chen J.-G. , Govindjee R. , Ebrey T.
Published: May 2006

We have found that extensively washed purple membrane has about 1 calcium and 3-4 magnesium ions bound per bacteriorhodopsin molecule. When these divalent cations are removed by any of a variety of means, the pigment changes its color from purple to blue (λ
max
≈ 600 nm). This blue pigment, which can be formed at near neutral pH, is probably very similar to blue species formed when the pH of a purple membrane sample is lowered to ≈2. When any of a wide variety of cations are added to a blue membrane preparation, the characteristic purple color of bacteriorhodopsin returns. Divalent and trivalent cations are much more efficient than monovalent cations in restoring the purple color and are effective at a ratio approaching one cation per pigment molecule. Besides shifting the absorption spectrum, removal of the divalent cations drastically alters the photochemical cycle of bacteriorhodopsin, including abolishing the unprotonated Schiff base (M-type) intermediate. Finally, lanthanum not only displaces the divalent cations normally bound to the purple membrane but also greatly reduces both the rate of decay of the M412 intermediate and proton uptake.
Spin-label oximetry: kinetic study of cell respiration using a rapid-passage T1-sensitive electron spin resonance display.
Authors: Froncisz W , Lai C S , Hyde J S
Published: May 2006

An unusual ESR display has been developed that exhibits sensitivity to bimolecular collisions of dissolved oxygen in water with nitroxide radical spin probes at oxygen concentrations as low as 0.1 microM, requiring only 1 microliter of sample. The method involves observation of the ESR rapid-passage signal when tuned to the dispersion using a loop-gap resonator. The bimolecular collision rate determines the phase of the signal. The method has been used in a closed-chamber geometry to study respiration of asynchronous populations of Chinese hamster ovary (CHO) cells. An integral of the Michaelis-Menten equation permits direct comparison with experiment and is shown to be incompatible with the data. The theory of diffusion limitation also is developed and shown to be inconsistent with experiment. The average oxygen concentration is found to decrease as Vmaxt, where t is the time after sealing the chamber, to a critical oxygen concentration of 5.2 microM. Below 5.2 microM, the concentration can be fitted to an exponential form, exp(-t/tau), where tau = 15 sec for 4000 cells per microliter. It is believed that this experimental behavior is determined by complex enzyme kinetics.
Unmodified low density lipoprotein causes cholesteryl ester accumulation in J774 macrophages.
Authors: Tabas I , Weiland D A , Tall A R
Published: May 2006

Cholesteryl ester (CE)-loaded macrophages (foam cells) are a prominent feature of atherosclerotic plaques. Previous studies have shown that human monocytes or resident mouse peritoneal macrophages accumulate CE in response to low density lipoprotein (LDL) only when the LDL has been appropriately chemically modified. By contrast, we report here that J774 macrophages accumulate large amounts of CE when incubated with unmodified LDL. The CE is stored in oil red O-positive droplets, which have the typical appearance of foam cell inclusions by electron microscopy. The fatty acid moieties of the cellular CE are enriched in oleate unlike those of LDL-CE, which are enriched in linoleate, indicating that the LDL-CE undergoes hydrolysis and reesterification by acyl CoA:cholesterol acyltransferase. Studies with 125I-labeled LDL at both 4 degrees C and 37 degrees C indicate that the LDL is internalized by a specific receptor that has several characteristics in common with the apolipoprotein B/E (apo B/E) receptor. However, in comparison with fibroblasts, the LDL receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity in J774 cells are relatively resistant to down-regulation by LDL or 25-hydroxycholesterol, leading to receptor-mediated CE storage. In addition, J774 cells appear to accumulate CE from LDL internalized by nonspecific means. Thus, macrophage-like cells can accumulate CE in response to unmodified LDL by both nonspecific and receptor-mediated processes.
Genetic control of chromosome length in yeast.
Authors: Walmsley R M , Petes T D
Published: May 2006

The chromosomes of the yeast Saccharomyces cerevisiae terminate with sequences that have the form poly(C1-3-A). In this paper, we show that within an individual yeast strain all chromosomes end with tracts of poly(C1-3-A) of similar lengths; however, different strains can have tracts that vary in length by a factor of two. By a genetic analysis, we demonstrate that yeast cells have a mechanism that allows them to change rapidly the length of their chromosomes by altering the length of the poly(C1-3-A) tract.
Binding of a possible transition state analogue to the active site of carboxypeptidase A.
Authors: Christianson D W , Lipscomb W N
Published: May 2006

The mode of binding of the competitive inhibitor 2-benzyl-3-formylpropanoic acid to the active site of carboxypeptidase A has been studied by x-ray diffraction methods to a resolution of 1.7 A. The actual species bound to the enzyme was determined to be the gem-diol resulting from covalent hydration at the aldehyde carbonyl. Details relating to the process of association of inhibitor with enzyme are unknown at this time: the free aldehyde could initially bind to the enzyme and subsequently undergo catalytic hydration; or, the hydrate itself could be the species initially binding to the enzyme, because it does exist to a high degree (25%) in aqueous solution. Nevertheless, the structure of the complex reported is reminiscent of a possible tetrahedral intermediate that would be encountered in a general base hydrolytic mechanism. Of course, other mechanistic proposals, such as the anhydride pathway, cannot be ruled out simply on the basis of the structure of this enzyme-inhibitor complex.
Expression sequences of cell adhesion molecules.
Authors: Crossin K L , Chuong C M , Edelman G M
Published: May 2006

A reexamination of the expression of cell adhesion molecules (CAMs) during the development of the chicken embryo was carried out using more sensitive immunocytochemical techniques than had been used previously. While the previously determined sequence of CAM expression was confirmed, neural CAM (N-CAM) was also detected on endodermal structures such as the lung epithelium, gut epithelium, and pancreas and on budding structures such as the pancreatic duct and gall bladder. It was also found on ectodermal derivatives of the skin. In most of these sites, N-CAM expression was transient, but in the chicken embryo lung, the epithelium remained positive for N-CAM and liver CAM (L-CAM) into adult life. Thus, at one time or another, both of these primary CAMs can be expressed on derivatives of all three germ layers. At sites of embryonic induction, epithelial cells expressing both L-CAM and N-CAM, or L-CAM only, were apposed to mesenchymal cells expressing N-CAM. Examples included epiblast (NL) and notochord (N); endodermal epithelium (NL) and lung mesenchyme (N); Wolffian duct (NL) and mesonephric mesenchyme (N); apical ectodermal ridge (NL) and limb mesenchyme (N); and feather placode (L) and dermal condensation (N). The cumulative observations indicate that cell surface modulation of the primary CAMs at induction sites can be classified into two modes. In mode I, expression of N-CAM (or both CAMs) in mesenchyme decreases to low amounts at the cell surface, and then N-CAM is reexpressed. In mode II, one or the other CAM disappears from epithelia expressing both CAMs. As a result of the primary processes of development, collectives of cells linked by N-CAM and undergoing modulation mode I are brought into the proximity of collectives of cells linked by L-CAM plus N-CAM or by L-CAM undergoing modulation mode II. Such adjoining cell collectives or CAM couples were found at all sites of embryonic induction examined.
Is human X chromosome inactivation a sex-determining device?
Authors: Chandra H S
Published: May 2006

The evolutionary function of X chromosome inactivation is thought to be dosage compensation. However, there is, at present, little evidence to suggest that most X chromosome-linked genes require such compensation. Another view--that X chromosome inactivation may be related to sex determination--is examined here. Consider a hypothetical DNA sequence regulating a major structural gene concerned with the determination of maleness. If this regulatory sequence occurs in both X and Y chromosomes and if its copy number in the Y chromosome is significantly greater than in the X chromosome, then the male-determining properties of the Y chromosome could be attributed to this higher copy number. On the other hand, if the Y chromosome has the same copy number of this sequence as the X chromosome, it is difficult to see how determination of two sexes would occur under such circumstances because XX and XY genomes would then be indistinguishable in this regard. Such a situation seems to occur in the human species with respect to the banded krait minor satellite, a repetitious DNA sequence associated with sex determination. This apparent difficulty may be resolved if X chromosome inactivation renders regulatory as well as structural genes nonfunctional and thereby brings about a significant reduction in the effective copy number of X chromosome-linked DNA sequences concerned with sex determination. It is suggested that X chromosome inactivation brings about, in this manner, a critical inequality between XX and XY embryos and that sex determination in humans is a consequence of this inequality. An analogous situation appears to exist in certain insects in which inactivation of a haploid set of chromosomes (and presumably, therefore, a 50% reduction in the effective copy number of most genes) is associated with maleness. If this line of reasoning is correct, it would suggest that sex determination may be the primary function of X chromosome inactivation.
Cerebellin: a quantifiable marker for Purkinje cell maturation.
Authors: Slemmon J R , Danho W , Hempstead J L , Morgan J I
Published: May 2006

The cerebellum-specific hexadecapeptide cerebellin has been localized by immunocytochemical means to the perikarya and dendrites of cerebellar Purkinje cells. Biochemical analysis using ion-pairing HPLC shows cerebellin to first appear 5 days after birth, whereafter levels rise to a maximum at 25 days postpartum, and then decline to stable adult values. This same pattern of development occurs with a lag of approximately 5 days for the major metabolite of cerebellin, des-Ser1-cerebellin. The immunocytochemical picture of cerebellin in developing Purkinje cells mirrors the biochemical data. These results show that cerebellins represent unique quantifiable markers for the investigation of Purkinje cell maturation and lend support to the feasibility of using unique endogenous peptides to chart neurodevelopment.
Limited proteolysis of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania tropica.
Authors: Garvey E P , Santi D V
Published: May 2006

The structure and activity of the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from the protozoan parasite Leishmania tropica were examined by limited proteolysis with five different endopeptidases. Each reaction resulted in a rapid, time-dependent loss of TS activity and no effect on DHFR activity. The proteolytic products were examined by NaDodSO4/PAGE; each digestion produced a fragment of apparent Mr approximately 35,000, and three of the five digestions generated a fragment of Mr approximately 20,000. Attempts to separate the fragments under nondenaturing conditions failed, suggesting that the proteolyzed protein remains a dimer with the gross structure of the subunits more or less undisturbed. In contrast, kinetic data indicate that some aspects of higher-order structure in the native protein are affected by proteolysis. The fragments (Mr 36,600 and 20,000) generated by Staphylococcus aureus V8 protease were subjected to sequence analysis. Whereas neither the native protein nor the Mr 36,600 fragment yielded an NH2-terminal amino acid, we obtained the sequence of the first 28 amino acids of the Mr 20,000 fragment. This sequence bore strong homology with sequences situated within TS of human, Lactobacillus casei, Escherichia coli, and bacteriophage T4. These and other data indicate that the TS-DHFR polypeptide consists of a DHFR sequence at the blocked NH2-terminal and a TS sequence at the COOH-terminal end of the protein. The region that is the target of the five proteases corresponds to a highly variable region within the sequences of the other four TSs. We suggest that an insertion occurs within the TS-DHFR sequence, positioned on the surface of the protein and quite vulnerable to the action of endopeptidases.