bluuebunny/crossref_metadata_2025 huggingface dataset:
Method for isolation of auxotrophs in the methanogenic archaebacteria: role of the acetyl-CoA pathway of autotrophic CO2 fixation in Methanococcus maripaludis.
Authors: Ladapo J , Whitman W B
Published: May 2006

A procedure was developed for the enrichment of auxotrophs in the antibiotic-insensitive archaebacterium Methanococcus. After mutagenesis with ethyl methanesulfonate, growing cells were selectively killed upon exposure to the base analogs 6-azauracil and 8-azahypoxanthine for 48 hr. Using this method, eight independent acetate autotrophs of Methanococcus maripaludis were isolated. Six of the auxotrophs had an absolute growth requirement for acetate and contained 1-16% of the wild-type levels of CO dehydrogenase. Three of these six also contained 14-29% of the wild-type levels of pyruvate oxidoreductase and 12-30% of the wild-type levels of pyruvate synthase. Two spontaneous revertants of these latter auxotrophs regained the ability to grow normally in the absence of acetate and wild-type levels of CO dehydrogenase, acetyl-CoA synthase, pyruvate oxidoreductase, and pyruvate synthase. Likewise, a spontaneous revertant of an auxotroph with reduced levels of CO dehydrogenase and wild-type levels of pyruvate oxidoreductase regained the ability to grow normally in the absence of acetate and wild-type levels of CO dehydrogenase and acetyl-CoA synthase. Two additional auxotrophs grew poorly in the absence of acetate but contained wild-type levels of CO dehydrogenase and pyruvate oxidoreductase. These results provide direct genetic evidence for the Ljungdahl-Wood pathway [Ljungdahl, L. G. (1986) Annu. Rev. Microbiol. 40, 415-450; Wood, H. G., Ragsdale, S. W. & Pezacka, E. (1986) Trends Biochem. Sci. 11, 14-18] of autotrophic acetyl-CoA biosynthesis in the methanogenic archaebacteria. Moreover, it suggests that the acetyl-CoA and pyruvate synthases may share a common protein or coenzyme component, be linked genetically, or be regulated by a common system.
HMf, a DNA-binding protein isolated from the hyperthermophilic archaeon Methanothermus fervidus, is most closely related to histones.
Authors: Sandman K , Krzycki J A , Dobrinski B , Lurz R , Reeve J N
Published: May 2006

Methanothermus fervidus grows optimally at 83 degrees C. A protein designated HMf (histone M. fervidus) has been isolated from this archaeal hyperthermophile that binds to double-stranded DNA molecules and increases their resistance to thermal denaturation. HMf binding to linear double-stranded DNA molecules of greater than 2 kilobase pairs also increases their electrophoretic mobilities through agarose gels. Visualization of this compaction process by electron microscopy has demonstrated the formation of quasispherical, macromolecular HMf-DNA complexes. HMf is a mixture of approximately equal amounts of two very similar polypeptides designated HMf-1 and HMf-2. Determination of the DNA sequence of the gene encoding HMf-2 (hmfB) has revealed that over 30% of the amino acid residues in HMf-2 are conserved in the consensus sequences derived for eucaryal histones H2A, H2B, H3, and H4. These archaeal polypeptides and eucaryal histones appear therefore to have evolved from a common ancestor and are likely to have related structures and functions.
Light regulates expression of a Fos-related protein in rat suprachiasmatic nuclei.
Authors: Aronin N , Sagar S M , Sharp F R , Schwartz W J
Published: May 2006

Mammalian circadian rhythmicity is endogenously generated by a pacemaker in the suprachiasmatic nuclei and precisely entrained to the 24-hr day/night cycle by periodic environmental light cues. We show that light alters the immunoreactive levels of a transcriptional regulatory protein, Fos, in the suprachiasmatic nuclei of albino rats. Photic regulation of Fos immunoreactivity does not occur in other retino-recipient brain areas except for the intergeniculate leaflet, which appears to be involved in mediating some of the complex effects of light on expressed circadian rhythms. Our results point to a promising new functional marker for the cellular effects of light and suggest that the expression of Fos or a related nuclear protein may be part of the mechanism for photic entrainment of the circadian clock to environmental light/dark cycles.
Molecular characterization of the GCN4-DNA complex.
Authors: Gartenberg M R , Ampe C , Steitz T A , Crothers D M
Published: May 2006

We report studies of the DNA complex formed by GCN4, a transcriptional activator of eukaryotic amino acid biosynthetic operons. The DNA thermodynamic binding domain, defined by primer extension analysis, spans at least 18 base pairs, a site much larger than the 9-base-pair consensus defined by homology with naturally occurring binding sites. Chemical modification experiments reveal multiple sites of protein-DNA contact: methylation of any guanine N-7 or adenine N-3, ethylation of any phosphate oxygen, or elimination of any nucleoside within a region spanning nearly one and a half turns of the double helix reduces the binding affinity of the complex measurably. Nevertheless, the protein yields no detectable hydroxyl radical footprint, implying that the minor groove is reagent-accessible in the protein-DNA complex. These chemical modification patterns indicate that GCN4 does not utilize any of the DNA-recognition motifs of paradigm DNA-binding proteins. Assays to detect DNA bending induced by truncated or intact GCN4 indicate that protein conformation and not a protein-induced bend is responsible for the anomalous electrophoretic behavior of GCN4-DNA complexes.
Juxtaposition of expressed variable antigen genes with a conserved telomere in the bacterium Borrelia hermsii.
Authors: Kitten T , Barbour A G
Published: May 2006

Borrelia hermsii, an agent of relapsing fever, survives in mammals through antigenic variation. Change in serotype-specific variable outer membrane proteins (Vmps) occurs when a Vmp gene at an expression site is replaced with a previously silent gene for another Vmp. Silent and active genes are on separate linear plasmids. The upstream site for a nonreciprocal recombination between two linear plasmids is near the 5' ends of the expressed and silent genes. In the present study we sought the downstream recombination sites in two serotypes, 7 and 21. Restriction fragments containing plasmid telomeres were identified by susceptibility to digestion with BAL-31 and rapid reannealment following denaturation. Whereas both silent genes and a minority population of both expression-linked genes were several kilobases from the telomeres, the predominant population of both expressed genes had 3' ends near plasmid telomeres. Sequence analysis of the predominant expression plasmids revealed that the telomeric sequences were the same in serotypes 7 and 21. Identical sequence was also downstream of silent Vmp genes. Switching of Vmp genes appears to occur by recombination that involves both upstream and downstream sites. The expression plasmid's telomere is preserved in the recombination event.
Single d(ApG)/cis-diamminedichloroplatinum(II) adduct-induced mutagenesis in Escherichia coli.
Authors: Burnouf D , Gauthier C , Chottard J C , Fuchs R P
Published: May 2006

The mutation spectrum induced by the widely used antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) showed that cisDDP[d(ApG)] adducts, although they account for only 25% of the lesions formed, are approximately 5 times more mutagenic than the major GG adduct. We report the construction of vectors bearing a single cisDDP[d(ApG)] lesion and their use in mutagenesis experiments in Escherichia coli. The mutagenic processing of the lesion is found to depend strictly on induction of the SOS system of the bacterial host cells. In SOS-induced cells, mutation frequencies of 1-2% were detected. All these mutations are targeted to the 5' base of the adduct. Single A----T transversions are mainly observed (80%), whereas A----G transitions account for 10% of the total mutations. Tandem base-pair substitutions involving the adenine residue and the thymine residue immediately 5' to the adduct occur at a comparable frequency (10%). No selective loss of the strand bearing the platinum adduct was seen, suggesting that, in vivo, cisDDP[d(ApG)] adducts are not blocking lesions. The high mutation specificity of cisDDP[d(ApG)]-induced mutagenesis is discussed in relation to structural data.
Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli.
Authors: Greenberg J T , Monach P , Chou J H , Josephy P D , Demple B
Published: May 2006

Escherichia coli responds to superoxide-generating agents by inducing approximately 40 proteins. We have identified a genetic locus, soxR (superoxide response), that positively regulates 9 of these proteins during superoxide stress. Induction under soxR control is at the transcriptional level, as shown with lac fusions to five paraquat-inducible promoters. Members of the soxR regulon include at least three proteins with demonstrable antioxidant roles: Mn-containing superoxide dismutase (which destroys superoxide radicals), endonuclease IV (which repairs radical-induced damages in DNA), and glucose-6-phosphate dehydrogenase (which produces NADPH). Induction of the soxR regulon also leads to diminished levels of the major outer membrane protein OmpF and alteration of the small-subunit ribosomal protein S6. These latter changes confer resistance to a variety of antibiotics. The soxR regulon may thus operate as an inducible defense against xenobiotics in general.
cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica.
Authors: Torian B E , Flores B M , Stroeher V L , Hagen F S , Stamm W E
Published: May 2006

A gamma gt11 cDNA library was constructed from poly(U)-Sepharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically 35S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-125I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4 degrees C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.
Cooperation between two growth factors promotes extended self-renewal and inhibits differentiation of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells.
Authors: Bögler O , Wren D , Barnett S C , Land H , Noble M
Published: May 2006

Bipotential oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, which give rise to oligodendrocytes and type-2 astrocytes in cultures of rat optic nerve, are one of the few cell types in which most aspects of proliferation and differentiation can be manipulated in a defined in vitro environment. Previous studies have shown that O-2A progenitors exposed to platelet-derived growth factor (PDGF) divide as migratory bipolar cells a limited number of times, with a cell cycle time of 18 hr, before clonally related progenitors differentiate into nondividing oligodendrocytes with a timing similar to that seen in vivo. In contrast, O-2A progenitors grown in the absence of mitogen do not divide but instead differentiate prematurely into oligodendrocytes, and progenitors exposed to appropriate inducing factors differentiate into type-2 astrocytes. We now have found that O-2A progenitors can be induced to undergo continuous self-renewal in the absence of oligodendrocytic differentiation by exposure to a combination of PDGF and basic fibroblast growth factor (bFGF). With the exception of the inhibition of differentiation, the O-2A progenitors exposed to PDGF and bFGF behaved similarly to those exposed to PDGF alone. In contrast, progenitors exposed to basic bFGF alone were multipolar, had a cell-cycle length of 45 hr, showed little migratory behavior, underwent premature oligodendrocytic differentiation, and did not cease division upon expression of oligodendrocyte marker antigens. Thus, inhibition of differentiation required the presence of both mitogens. Our results demonstrate that PDGF and bFGF act on O-2A progenitors as both inducers of division and as regulators of differentiation that modulate multiple aspects of O-2A progenitor development and, additionally, reveal a previously unrecognized means of regulating self-renewal processes, wherein cooperation between growth factors promotes continuous division in the absence of differentiation.
Origins of structure in globular proteins.
Authors: Chan H S , Dill K A
Published: May 2006

The principal forces of protein folding--hydrophobicity and conformational entropy--are nonspecific. A long-standing puzzle has, therefore, been: What forces drive the formation of the specific internal architectures in globular proteins? We find that any self-avoiding flexible polymer molecule will develop large amounts of secondary structure, helices and parallel and antiparallel sheets, as it is driven to increasing compactness by any force of attraction among the chain monomers. Thus structure formation arises from the severity of steric constraints in compact polymers. This steric principle of organization can account for why short helices are stable in globular proteins, why there are parallel and anti-parallel sheets in proteins, and why weakly unfolded proteins have some secondary structure. On this basis, it should be possible to construct copolymers, not necessarily using amino acids, that can collapse to maximum compactness in incompatible solvents and that should then have structural organization resembling that of proteins.
Interleukin 1 is an autocrine regulator of human endothelial cell growth.
Authors: Cozzolino F , Torcia M , Aldinucci D , Ziche M , Almerigogna F , Bani D , Stern D M
Published: May 2006

Proliferation of endothelial cells is regulated through the autocrine production of growth factors and the expression of cognate surface receptors. In this study, we demonstrate that interleukin 1 (IL-1) is an inhibitor of endothelial growth in vitro and in vivo. IL-1 arrested growing, cultured endothelial cells in G1 phase; inhibition of proliferation was dose dependent and occurred in parallel with occupancy of endothelial surface IL-1 receptors. In an angiogenesis model, IL-1 could inhibit fibroblast growth factor-induced vessel formation. The autocrine nature of the IL-1 effect on endothelial proliferation was demonstrated by the observation that occupancy of cell-surface receptors by endogenous IL-1 depressed cell growth. The potential significance of this finding was emphasized by the detection of IL-1 in the native endothelium of human umbilical veins. A mechanism by which IL-1 may exert its inhibitory effect on endothelial cell growth was suggested by studies showing that IL-1 decreased the expression of high-affinity fibroblast growth factor binding sites on endothelium. These results point to a potentially important role of IL-1 in regulating blood vessel growth and suggest that autocrine production of inhibitory factors may be a mechanism controlling proliferation of normal cells.
Activation of murine c-abl protooncogene: effect of a point mutation on oncogenic activation.
Authors: Shore S K , Bogart S L , Reddy E P
Published: May 2006

Activation of the c-abl protooncogene occurs in Abelson murine leukemia virus, in Hardy-Zuckerman 2 feline sarcoma virus, and during the chromosomal translocations that generate BCR-ABL gene fusion products. To study the molecular mechanism involved in the c-abl activation, we have created a series of modifications in murine c-abl and assayed these constructs for oncogenic activity using the NIH 3T3 cell transformation assay. Our results show that amino-terminal deletions are sufficient for oncogenic activation of c-abl and high levels of oncogenic activities were generated by a deletion of 114 codons from the 5' end that deleted the SH3 region. A deletion of 53 codons from the 5' end (inclusive of deletions seen in Hardy-Zuckerman 2 feline sarcoma virus and BCR-ABL gene products) that retains the SH3 region of c-abl resulted in the generation of low levels of transforming activity. This transforming potential was substantially increased with the introduction of a G----A point mutation in codon 832 that is present in v-abl. The point mutation was found to affect the secondary structure and the tyrosine kinase activity of the mutant gene products.
Role of 1,25-dihydroxycholecalciferol in growth-plate cartilage: inhibition of terminal differentiation of chondrocytes in vitro and in vivo.
Authors: Kato Y , Shimazu A , Iwamoto M , Nakashima K , Koike T , Suzuki F , Nishii Y , Sato K
Published: May 2006

The effects of vitamin D metabolites on alkaline phosphatase [ALPase; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] activity, a marker of terminal differentiation, in chondrocyte cultures and growth plates in vivo were examined. In cultures of pelleted rabbit growth-plate chondrocytes, 1,25-dihydroxycholecalciferol (1,25-dihydroxyvitamin D3) increased the contents of DNA and macromolecules containing uronic acid (proteoglycans). It also decreased ALPase activity with an ED50 of less than 1 nM. Other vitamin D3 metabolites, such as 24,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol, had little effect on these biochemical parameters. In rachitic growth plates, the uronic acid content was half that in normal growth plates, whereas ALPase activity was 2.5 times that in normal growth plates. Administration of 1,25-dihydroxycholecalciferol at a low dose (0.1 micrograms per kg of body weight) to rachitic rats increased the uronic acid content 1.4-fold and decreased ALPase activity by 40%. This compound, like 24,25-dihydroxycholecalciferol (10 micrograms per kg of body weight), increased the calcium level of the blood. However, administration of 24,25-dihydroxycholecalciferol had little effect on the uronic acid and ALPase contents in growth plates. These observations suggest that 1,25-dihydroxycholecalciferol is a bioactive form of vitamin D that plays an important role in the control of chondrocyte terminal differentiation.
Immunoglobulin heavy-chain-associated amyloidosis.
Authors: Eulitz M , Weiss D T , Solomon A
Published: May 2006

Immunoglobulin- or multiple myeloma-associated amyloidosis has been distinguished by the tissue deposition of Congophilic, fibrillar protein consisting of light chains or light-chain fragments (AL amyloidosis). We now report the isolation and characterization of another form of immunoglobulin-associated amyloid obtained from a patient who had extensive systemic amyloidosis and in whom the amyloid deposits consisted not of light chains but rather of an unusual form of heavy chain. This component, isolated from splenic amyloid extracts, represented an internally deleted IgG1 heavy chain as evidenced by immunochemical, electrophoretic, and amino acid sequence analyses. A comparable immunoglobulin-related monoclonal protein, consisting only of IgG heavy chains, was present in the patient's urine. Based on serologic reactivity with a battery of anti-immunoglobulin antisera, these two immunoglobulin-related components were antigenically identical; however, when compared to normal IgG, both were deficient in Fc-associated gamma-chain determinants. The structural abnormality of the amyloid gamma-chain protein was further evidenced by SDS/PAGE and immuno-blotting analyses: An unusually low molecular mass of approximately 22 kDa was found for this material vs. the expected value of approximately 55 kDa for a normal gamma heavy chain. Despite the lack of certain Fc determinants, the amyloid and urinary heavy-chain proteins expressed the IgG1 subclass allotype marker G1m(a) located on the third constant region (CH3) domain of the internally deleted IgG1 heavy chains. That the amyloid protein contained an intact CH3 domain was established through amino acid sequence analyses of cyanogen bromide fragments and peptides generated by a lysine-specific protease. These studies also revealed that the gamma-chain amyloid protein contained the complete heavy-chain variable (VH) domain [including the diversity (DH) and joining (JH) segments] that was contiguous with the CH3 domain. The low molecular mass of the protein resulted from the total absence of the first (CH1), hinge, and second (CH2) heavy-chain constant regions. Such extensive CH deletions and the presence of a complete VH distinguish this amyloid-associated heavy chain from all other heretofore characterized gamma-heavy-chain disease proteins. This heavy-chain-related form of immunoglobulin-associated amyloidosis is tentatively designated AH amyloidosis.
The spatial distribution of exposed nuclear DNA in normal, cancer, and reverse-transformed cells.
Authors: Krystosek A , Puck T T
Published: May 2006

The malignant CHO-K1 cell is reverse-transformed by cAMP, regaining the phenotype of a normal fibroblast. During this reaction, much of its DNA re-acquires sensitivity to hydrolysis by DNase I in a way characteristic of the normal fibroblast. Exposed DNA forms a rim about the nucleus in both the normal and reverse-transformed cell but not in the malignant CHO-K1. Reacquisition of the nuclear rim requires an organized cytoskeleton. Sequestered DNA forms families of different degrees of sequestration. In accordance with previous theoretical developments it is proposed that (i) genes specific to a given differentiation state are stored in the nuclear rim, whereas genes specific to other states are sequestered within the nucleus; (ii) only exposed genes are active, and their activity is modulated by regulatory molecules in the fluid medium; (iii) exposure and sequestration are regulated by cytoskeletal and nuclear protein structures; (iv) in at least several types of cancer the regulatory defect lies in the genome exposure process so that the specific DNA sequences and their associated growth regulatory loci have been transferred from the exposed to the sequestered condition with consequent loss of the nuclear rim of exposed DNA. The methodology described should be generally applicable to examining the accessibility state of subsets of DNA during various physiological modulations of cell function.
General colorimetric method for DNA diagnostics allowing direct solid-phase genomic sequencing of the positive samples.
Authors: Wahlberg J , Lundeberg J , Hultman T , Uhlén M
Published: May 2006

A system for rapid colorimetric detection of specific genome DNA fragments amplified by the polymerase chain reaction (PCR) is described that has been designed to allow direct solid-phase sequencing of positive samples. The amplified material is immobilized on magnetic beads by using the biotin streptavidin system. An Escherichia coli lac operator DNA sequence is incorporated in the amplified material during the second step of a nested primer procedure. This 21-base-pair sequence is used for a general colorimetric detection with a fusion protein consisting of the E. coli Lac repressor and beta-galactosidase. Positive samples can be treated subsequently with alkali to obtain a single-stranded DNA template suitable for direct genomic sequencing. This method to detect immobilized amplified nucleic acids (DIANA) is well adapted for automated or semiautomated clinical assays. Here, we show that it can be used to detect and sequence Chlamydia trachomatis genomic DNA in clinical samples.
Inter- and intraspecies spread of Escherichia coli in a farm environment in the absence of antibiotic usage.
Authors: Marshall B , Petrowski D , Levy S B
Published: May 2006

The spread of wild-type Escherichia coli bearing a transferable plasmid was studied in a farm environment. E. coli of bovine and porcine origin were marked by resistance to nalidixic acid (Nar) or rifampicin (Rfr), and a transferable, multiple resistance plasmid (pSL222-1 derivative of plasmid R222) was introduced by conjugation. In separate experiments, the two mutant derivatives were fed back to the respective host animals, which were housed adjacent to, but separate from, one or more "recipient" animals. No antibiotic was given. Although the Rfr derivatives declined rapidly to undetectable levels within 1 week, the Nar mutants of bovine and porcine origin persisted in the original hosts and in their bedding throughout most of each 4-month test period. Test bacteria were isolated from mice residing in the same pen as the donor animals and from multiple secondary hosts having direct or indirect contact with the inoculated donors, but not from neighboring animals maintained in isolation. The bovine mutant was excreted by two caretakers for greater than 4 weeks and was recovered for 4-6 weeks from pigs, fowl, and flies. Although the porcine mutant appeared to colonize less effectively, it spread rapidly to flies and mice and was recovered transiently from humans and fowl. Despite high transfer rates of plasmid pSL222-1 from E. coli K-12 in vitro, transfer of the plasmid from the animal E. coli host was very low and transfer in vivo was not detected among indigenous gut or environmental bacteria. E. coli of animal origin can spread rapidly and can colonize the intestinal tract of humans and of other animals in the absence of antibiotic selection.
A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin.
Authors: Good D J , Polverini P J , Rastinejad F , Le Beau M M , Lemons R S , Frazier W A , Bouck N P
Published: May 2006

A secreted inhibitor of angiogenesis that is controlled by a tumor suppressor gene in hamster cells has been found to be similar to a fragment of the platelet and matrix protein thrombospondin. The two proteins were biochemically similar and immunologically crossreactive and could substitute for one another in two functional assays. Human thrombospondin inhibited neovascularization in vivo and endothelial cell migration in vitro, as does the hamster protein, gp140. gp140 sensitized smooth muscle cells to stimulation by epidermal growth factor, as does human thrombospondin. The thrombospondin gene has been localized on human chromosome 15. These results demonstrate a function for the ubiquitous adhesive glycoprotein thrombospondin that is likely to be important in the normal physiological down-regulation of neovascularization. In addition, they raise the possibility that thrombospondin may be one of a number of target molecules through which a tumor suppressor gene could act to restrain tumor growth.
DNA polymerases alpha, delta, and epsilon: three distinct enzymes from HeLa cells.
Authors: Syväoja J , Suomensaari S , Nishida C , Goldsmith J S , Chui G S , Jain S , Linn S
Published: May 2006

DNA polymerases alpha, delta, and epsilon have been purified and characterized from the same HeLa cell extract in order to determine their relationship by comparing them from the same cell type. The catalytic properties and the primary structures of the large subunits of the DNA polymerases as compared by partial peptide mapping with N-chlorosuccinimide are different. Likewise, the small subunit of DNA polymerase epsilon appears to be distinct from the large subunit of the same polymerase and from the smaller subunits of DNA polymerase alpha. HeLa DNA polymerase delta is processive only when HeLa proliferating cell nuclear antigen is present, whereas DNA polymerase epsilon is quite processive in its absence. Inhibitor and activator spectra of DNA polymerases alpha, delta, and epsilon also distinguish the three enzymes. These results and immunologic comparisons published elsewhere support the premise that HeLa DNA polymerases alpha, delta, and epsilon are distinct enzymes that have common properties with yeast DNA polymerases I, III, and II, respectively.
Reconstituted basement membrane (matrigel) and laminin can enhance the tumorigenicity and the drug resistance of small cell lung cancer cell lines.
Authors: Fridman R , Giaccone G , Kanemoto T , Martin G R , Gazdar A F , Mulshine J L
Published: May 2006

Small cell lung cancer (SCLC) is a fatal malignancy due to its propensity to metastasize widely and to reoccur after chemotherapy in a drug-resistant form. While most SCLC cell lines are anchorage independent for growth, laminin induced the attachment of five of six SCLC cell lines tested (NCI-N417, NCI-H345, NCI-H146, NCI-H187, NCI-H510, and NCI-H209). NCI-N417 SCLC cells adopted a flattened morphology on laminin, and a classic SCLC cell line (NCI-H345) demonstrated a neuron-like appearance while the other SCLC cell lines except NCI-H187 cells, attached but did not spread. Adhesion to laminin was associated with increased resistance to several cytotoxic drugs. Matrigel, an extract of basement membrane proteins, greatly accelerated tumor growth when coinjected with SCLC cells in athymic mice. A synthetic peptide from the B1 chain of laminin, cyclic-YIGSR (Tyr-Ile-Gly-Ser-Arg), inhibited laminin-induced SCLC cell adhesion and migration in vitro and reduced the size of the tumors they formed when coinjected with matrigel and YIGSR. These results suggest that the interaction of SCLC cells with laminin and possibly with other basement membrane proteins can enhance their tumorigenicity and drug resistance.
Circulating human peripheral blood granulocytes synthesize and secrete tumor necrosis factor alpha.
Authors: Dubravec D B , Spriggs D R , Mannick J A , Rodrick M L
Published: May 2006

Circulating peripheral blood polymorphonuclear neutrophils (PMNs) have long been considered terminally differentiated cells that do not synthesize or secrete protein. However, work of others and ourselves has shown that PMNs can secrete the cytokine interleukin 1. In the present study we investigated whether circulating PMNs are capable of synthesizing and secreting another cytokine, tumor necrosis factor alpha (TNF-alpha). Highly purified (greater than 99% granulocytes) PMNs were isolated from normal human volunteer blood and cultured with or without bacterial lipopolysaccharide (LPS) for up to 24 hr. Cell culture supernatants were collected and tested for TNF-alpha, and total RNA was isolated from cells at various times after stimulation and assessed for TNF-alpha mRNA by Northern blot techniques. The results showed that message for TNF-alpha was produced after 60 min of in vitro stimulation with LPS and was maximal at about 4 hr. TNF-alpha was secreted into the supernatant of unstimulated PMNs from two different donors during 24 hr of culture (35-50 pg/ml), but significantly more (160-190 pg/ml) was secreted by PMNs when stimulated with LPS. PMNs from six other normal volunteers showed significant LPS-stimulated secretion of TNF at 60-180 min of culture. The secreted product also had biological activity against the TNF-sensitive L-M cell line, confirming that PMNs can make and secrete immunologically and biologically active TNF. Since it is also possible for monocytes to synthesize and secrete TNF, the amount of TNF secreted by a monocyte population equal to 20% of the PMNs cultured was measured. The results showed that monocytes at a concentration 20 times that potentially contaminating the PMN populations cultured could not produce as much TNF (unstimulated, 26-65 pg/ml; stimulated, 32-87 pg/ml). The PMN must now be considered a cell capable of altering the acute inflammatory response and modulating the immune response through the synthesis and release of cytokines.
Characterization of a beta subunit of the gastric H+/K(+)-transporting ATPase.
Authors: Reuben M A , Lasater L S , Sachs G
Published: May 2006

The catalytic subunit of the H+/K(+)-transporting ATPase (EC 3.6.1.3) has 62% identity to the alpha, or catalytic subunit, of the Na+/K(+)-transporting ATPase (EC 3.6.1.37); however, a homologous beta subunit was unknown until recently. Removal of the carbohydrate from purified hog H+/K(+)ATPase vesicles reveals a 35-kDa peptide that, when fragmented with protease V8, gives sequences homologous to both beta 1 and beta 2 subunits of the Na+/K(+)-ATPase. cDNA clones for a beta subunit of the gastric H+/K(+)-ATPase were isolated from a rabbit stomach cDNA library by using degenerate 17-mer oligonucleotide probes made to the protease V8-treated peptides. An open reading frame (54-926) encodes a predicted 291-amino acid peptide with Mr = 33,320, which exhibits 31% and 44% homologies to the Na+/K+)-ATPase beta 1 and Na+/K(+)-ATPase beta 2 proteins, respectively. A Kyte-Doolittle hydropathy plot predicts a single N-terminal transmembrane domain with a small hydrophobic region near the C terminus. The presumed extracytosolic domain contains seven potential N-linked glycosylation sites and six out of nine cysteines. Northern (RNA) blot analysis of stomach RNA with the rabbit H+/K(+)-ATPase beta probe identifies a single mRNA of 1.3-1.5 kilobases, similar in concentration to the alpha subunit mRNA. The presence of a defined gastric H+/K(+)-ATPase beta subunit extends the homology between H+/K(+)-ATPase and the Na+/K(+)-ATPase subclass of phosphoenzyme transport ATPases and distinguishes them from the monomeric Ca2+ and proton pump subclasses.
Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells.
Authors: Stern A S , Podlaski F J , Hulmes J D , Pan Y C , Quinn P M , Wolitzky A G , Familletti P C , Stremlo D L , Truitt T , Chizzonite R
Published: May 2006

A cytokine that can synergize with interleukin 2 to activate cytotoxic lymphocytes was purified to homogeneity. The protein, provisionally called cytotoxic lymphocyte maturation factor (CLMF), was isolated from a human B-lymphoblastoid cell line that was induced to secrete lymphokines by culture with phorbol ester and calcium ionophore. The purification method, utilizing classical and high-performance liquid chromatographic techniques, yielded protein with a specific activity of 8.5 x 10(7) units/mg in a T-cell growth factor assay. Analysis of the purified protein by sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated that CLMF is a 75-kDa heterodimer composed of disulfide-bonded 40-kDa and 35-kDa subunits. Determination of the N-terminal amino acid sequences of the two subunits revealed that both subunits are not related to any previously identified cytokine. Purified CLMF stimulated the proliferation of human phytohemagglutinin-activated lymphoblasts by itself and exerted additive effects when used in combination with suboptimal amounts of interleukin 2. Furthermore, the purified protein was shown to synergize with low concentrations of interleukin 2 in causing the induction of lymphokine-activated killer cells.
A major endothelial plasmalemmal sialoglycoprotein, gp60, is immunologically related to glycophorin.
Authors: Schnitzer J E , Ulmer J B , Palade G E
Published: May 2006

Glycophorins, the major sialoglycoproteins of red blood cells in many species, are generally considered to be specific to erythroid cells. Using polyclonal antibodies directed against mouse glycophorin (alpha gp), we have identified a glycoprotein antigenically related to glycophorin on the surface of bovine and rat cultured endothelial cells. Immunoblotting with alpha gp identified a single 60-kDa polypeptide on transfers of SDS/polyacrylamide gels of solubilized confluent endothelial monolayers. In addition, a 60-kDa polypeptide was immunoprecipitated by alpha gp from lysates of 125I-labeled intact endothelial cells. Controls with preimmune serum were negative. This antibody interaction was inhibited by murine erythrocyte ghosts and purified glycophorins. Our past work identified several endothelial surface sialoglycoproteins including a 60-kDa glycoprotein (gp60) that (i) interacts with albumin, (ii) binds Limax flavus, Ricinus communis, and Triticum vulgare agglutinins but not other lectins, (iii) is sequentially precipitated from 125I-labeled cell lysates by using R. communis agglutinin followed by T. vulgare agglutinin, and (iv) is sensitive to sialidase digestion. Immunoblotting of such precipitates with alpha gp demonstrates that lectins recognize the same glycoprotein, namely gp60. These results indicate that gp60, a major endothelial surface sialoglycoprotein, shares antigenic epitope(s) with glycophorin.
Allozyme and restriction fragment length polymorphism analyses confirm Entomophaga maimaiga responsible for 1989 epizootics in North American gypsy moth populations.
Authors: Hajek A E , Humber R A , Elkinton J S , May B , Walsh S R , Silver J C
Published: May 2006

In 1989, populations of North American gypsy moth, Lymantria dispar, in seven contiguous northeastern states were severely reduced by a fungal pathogen. Based on morphology, development, and pathology, this organism appeared to be Entomophaga maimaiga. We have now used allozyme and restriction fragment length polymorphism analyses to confirm this identification. Previously, this mycopathogen had been reported only from gypsy moth populations in Japan. During 1989, E. maimaiga occurred only in areas that had been initially defoliated by gypsy moth >10 years ago. E. maimaiga caused 60-88% mortality in late instar larvae on research sites in central Massachusetts.
Ligand-affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor.
Authors: Kiefer M C , Stephans J C , Crawford K , Okino K , Barr P J
Published: May 2006

Expression cloning of cDNAs encoding a basic fibroblast growth factor (FGF) binding protein confirms previous hypotheses that this molecule is a cell-surface heparan sulfate proteoglycan. A cDNA library constructed from a hamster kidney cell line rich in FGF receptor activity was transfected into a human lymphoblastoid cell line. Clones expressing functional basic FGF binding proteins at their surfaces were enriched by panning on plastic dishes coated with human basic FGF. The amino acid sequence deduced from the isolated cDNAs revealed several interesting features, including hydrophobic signal and transmembrane domains that flank an extracellular region containing six potential attachment sites for glycosaminoglycan side chains. The structure also contains a short hydrophilic cytoplasmic tail sequence homologous to previously reported actin binding domains. Binding of basic FGF to cells expressing the binding protein could be inhibited by heparin and heparan sulfate but not by chondroitin sulfate, dermatan sulfate, or keratan sulfate. In addition to binding basic FGF, this protein or related surface proteins may function as an initial cellular attachment site for other growth factors and for viruses, such as herpes simplex virus.
Expression of muscarinic acetylcholine and dopamine receptor mRNAs in rat basal ganglia.
Authors: Weiner D M , Levey A I , Brann M R
Published: May 2006

Within the basal ganglia, acetylcholine and dopamine play a central role in the extrapyramidal control of motor function. The physiologic effects of these neurotransmitters are mediated by a diversity of receptor subtypes, several of which have now been cloned. Muscarinic acetylcholine receptors are encoded by five genes (m1-m5), and of the two known dopamine receptor subtypes (D1 and D2) the D2 receptor gene has been characterized. To gain insight into the physiological roles of each of these receptor subtypes, we prepared oligodeoxynucleotide probes to localize receptor subtype mRNAs within the rat striatum and substantia nigra by in situ hybridization histochemistry. Within the striatum, three muscarinic (m1, m2, m4) receptor mRNAs and the D2 receptor mRNA were detected. The m1 mRNA was expressed in most neurons (greater than 80%); the m2 mRNA, in neurons which were both very large and rare; and the m4 and D2 mRNAs, in 40-50% of the neurons, one-third of which express both mRNAs. Within the substantia nigra, pars compacta, only the m5 and D2 mRNAs were detected, and most neurons expressed both mRNAs. These data provide anatomical evidence for the identity of the receptor subtypes which mediate the diverse effects of muscarinic and dopaminergic drugs on basal ganglia function.
The myosin step size: measurement of the unit displacement per ATP hydrolyzed in an in vitro assay.
Authors: Toyoshima Y Y , Kron S J , Spudich J A
Published: May 2006

Chemomechanical coupling in muscle contraction may be due to "swinging crossbridges," such that a change in the angle at which the myosin head binds to the actin filament is tightly coupled to release of products of ATP hydrolysis. This model would limit the step size, the unit displacement of actin produced by a single ATP hydrolysis, to less than twice the chord length of the myosin head. Recent measurements have found the step size to be significantly larger than this geometric limit, bringing into question any direct correspondence between the crossbridge and ATP-hydrolysis cycles. We have measured the rate of ATP hydrolysis due to actin sliding movement in an in vitro motility assay consisting of purified actin and purified myosin. We have calculated an apparent myosin step size well within the geometric limit set by the size of the myosin head. These data are consistent with tight coupling between myosin crossbridge movement and ATP hydrolysis.
Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction.
Authors: Noonan K E , Beck C , Holzmayer T A , Chin J E , Wunder J S , Andrulis I L , Gazdar A F , Willman C L , Griffith B , Von Hoff D D , Roninson I B
Published: May 2006

The resistance of tumor cells to chemotherapeutic drugs is a major obstacle to successful cancer chemotherapy. In human cells, expression of the MDR1 gene, encoding a transmembrane efflux pump (P-glycoprotein), leads to decreased intracellular accumulation and resistance to a variety of lipophilic drugs (multidrug resistance; MDR). The levels of MDR in cell lines selected in vitro have been shown to correlate with the steady-state levels of MDR1 mRNA and P-glycoprotein. In cells with a severalfold increase in cellular drug resistance, MDR1 expression levels are close to the limits of detection by conventional assays. MDR1 expression has been frequently observed in human tumors after chemotherapy and in some but not all types of clinically refractory tumors untreated with chemotherapeutic drugs. We have devised a highly sensitive, specific, and quantitative protocol for measuring the levels of MDR1 mRNA in clinical samples, based on the polymerase chain reaction. We have used this assay to measure MDR1 gene expression in MDR cell lines and greater than 300 normal tissues, tumor-derived cell lines, and clinical specimens of untreated tumors of the types in which MDR1 expression was rarely observed by standard assays. Low levels of MDR1 expression were found by polymerase chain reaction in most solid tumors and leukemias tested. The frequency of samples without detectable MDR1 expression varied among different types of tumors; MDR1-negative samples were most common among tumor types known to be relatively responsive to chemotherapy.
Posttranslational modification of class III beta-tubulin.
Authors: Lee M K , Rebhun L I , Frankfurter A
Published: May 2006

The charge heterogeneity of class III beta-tubulin (beta III) during neural development was analyzed by high-resolution isoelectric focusing/two-dimensional polyacrylamide gel electrophoresis in combination with site-specific proteolytic digestion and immunological detection. The number of beta III isoforms (charge variants) gradually increases from one in embryonic brain to seven in adult brain. All of the charge heterogeneity is due to posttranslationally modified sites located within the extreme C-terminal region of the beta III polypeptide. One beta III isoform is present in testis, the only other tissue in which this isotype is expressed. The testis beta III isoform cofocuses with the earliest-appearing embryonic brain beta III charge variant. Our results indicate that the posttranslational modifications of beta III are developmentally regulated, occur at more than one site, and are neuron-specific. The location of these modifications within the extreme C-terminal domain suggests that their function is to modulate the interaction of tubulin with microtubule-associated proteins.
An upstream region of the mouse ZP3 gene directs expression of firefly luciferase specifically to growing oocytes in transgenic mice.
Authors: Lira S A , Kinloch R A , Mortillo S , Wassarman P M
Published: May 2006

The gene encoding the mouse egg primary receptor for sperm, a zona pellucida glycoprotein called ZP3, is expressed exclusively in growing oocytes within ovaries of sexually immature and mature female mice. We have constructed a transgene in which 6.5 kilobases of ZP3 gene 5'-flanking sequence is fused to the coding region of the firefly luciferase gene, and we have generated four independent lines of transgenic mice. In these animals, the transgene is expressed exclusively in ovaries. Furthermore, within ovaries, expression is confined to growing oocytes, and luciferase activity can be detected by assaying individual, isolated oocytes. The pattern of firefly luciferase expression during oocyte growth is similar to that observed in previous studies of ZP3 expression during oogenesis in mice. Observations reported here strongly suggest that cis-acting elements present in the ZP3 gene 5'-flanking region regulate oocyte-specific and, therefore, sex-specific expression of the sperm receptor gene during mouse development. They also suggest that such elements can be used to direct expression of cloned genes specifically to oocytes of transgenic mice and to evaluate the effects of such expression on various aspects of early mammalian development.
Conservation of infectivity in purified fibrillary extracts of scrapie-infected hamster brain after sequential enzymatic digestion or polyacrylamide gel electrophoresis.
Authors: Brown P , Liberski P P , Wolff A , Gajdusek D C
Published: May 2006

Infectious extracts of scrapie-infected hamster brain enriched for scrapie-associated fibrils and scrapie amyloid protein (PrP) were partially denatured and subjected to either polyacrylamide gel electrophoresis with subsequent isolation of the PrP band or sequential enzymatic digestion with deglycosidase, phospholipase, proteinase, and several different nucleases. Infectivity measurements of these various specimens revealed a convincing association between infectivity and scrapie amyloid protein, with or without its sugar chains and disulfide bonds, and did not support the hypothesis that nucleic acid is involved in replication.