bluuebunny/crossref_metadata_2025 huggingface dataset:
D1-type dopamine receptors inhibit growth cone motility in cultured retina neurons: evidence that neurotransmitters act as morphogenic growth regulators in the developing central nervous system.
Authors: Lankford K L , DeMello F G , Klein W L
Published: May 2006

Precedent exists for the early development and subsequent down-regulation of neurotransmitter receptor systems in the vertebrate central nervous system, but the function of such embryonic receptors has not been established. Here we show that stimulation of early-developing dopamine receptors in avian retina cells greatly inhibits the motility of neuronal growth cones. Neurons from embryonic chicken retinas were cultured in low-density monolayers, and their growth cones were observed with phase-contrast or video-enhanced-contrast-differential-interference-contrast (VEC-DIC) microscopy. Approximately 25% of the neurons responded to micromolar dopamine with a rapid reduction in filopodial activity followed by a flattening of growth cones and retraction of neurites. The response occurred at all ages examined (embryonic day-8 retinal neurons cultured on polylysine-coated coverslips for 1-7 days), although neurite retraction was greatest in younger cultures. Effects of dopamine on growth cone function could be reversed by haloperidol or (+)-SCH 23390, whereas forskolin elicited a response similar to dopamine; these data show the response was receptor-mediated, acting through a D1-type system, and are consistent with the use of cAMP as a second messenger. The experiments provide strong support for the hypothesis that neurotransmitters, besides mediating transynaptic signaling in the adult, may have a role in neuronal differentiation as growth regulators.
Gap-modulation infrared spectroscopy of high transition temperature superconductors
Authors: Little William A. , Collman James P.
Published: May 2006

Conventional methods of determining the coupling factor α
2
(ω)
F
(ω) for the newly discovered high transition temperature (
T
c
) cuprate superconductors by using tunneling and infrared measurements have thus far failed to show the cause of the very high
T
c
of these compounds. This is due in part to difficulties in sample preparation for tunneling studies and to difficulties in obtaining good data at relatively high tunneling voltages. Also, in IR (infrared) measurements, small differences in absorptivity between the normal and superconducting state can be masked by changes in the phonon occupation at high and low temperatures. Here we propose a technique for determing the coupling constant, which should be less dependent on the surface quality of the sample than with tunneling and should allow measurements at higher energies with greater precision than do tunneling or simple IR observations. This should make possible a definitive determination of any possible exciton contribution to this coupling term, which would appear at energies well above the range where conventional IR or tunneling measurements are effective.
Identification of a region in the S1 subunit of pertussis toxin that is required for enzymatic activity and that contributes to the formation of a neutralizing antigenic determinant.
Authors: Cieplak W , Burnette W N , Mar V L , Kaljot K T , Morris C F , Chen K K , Sato H , Keith J M
Published: May 2006

The S1 subunit of pertussis toxin possesses two regions (homology boxes), each spanning 8 residues, that are nearly identical in sequence to similarly located regions in the enzymatically active A fragments of two other ADP-ribosylating toxins: cholera toxin and Escherichia coli heat-labile toxin. This observation suggests a functional role for one or both of these regions in enzymatic activity. We have examined the role of one of these regions, located near the amino terminus of the S1 subunit, by using a high-level recombinant expression system and progressive truncation of the gene sequence encoding the amino terminus of the molecule. A series of six truncated, recombinant proteins were produced at high levels in E. coli and examined for their enzymatic and antigenic properties. The three molecules that lacked most or all of the homology box delimited by amino acid residues 8 and 15 lacked detectable enzymatic activity. All of the three molecules in which the box was retained exhibited detectable activity. Only those recombinant molecules that possessed the homology box reacted with a neutralizing and passively protective monoclonal anti-S1 antibody. These findings identify the region of homology located near the amino terminus of S1 as an apparent enzymatic subsite and a potentially important antigenic determinant.
Model for how type I restriction enzymes select cleavage sites in DNA.
Authors: Studier F W , Bandyopadhyay P K
Published: May 2006

Under appropriate conditions, digestion of phage T7 DNA by the type I restriction enzyme EcoK produces an orderly progression of discrete DNA fragments. All details of the fragmentation pattern can be explained on the basis of the known properties of type I enzymes, together with two further assumptions: (i) in the ATP-stimulated translocation reaction, the enzyme bound at the recognition sequence translocates DNA toward itself from both directions simultaneously; and (ii) when translocation causes neighboring enzymes to meet, they cut the DNA between them. The kinetics of digestion at 37 degrees C indicates that the rate of translocation of DNA from each side of a bound enzyme is about 200 base pairs per second, and the cuts are completed within 15-25 sec of the time neighboring enzymes meet. The resulting DNA fragments each contain a single recognition site with an enzyme (or subunit) remaining bound to it. At high enzyme concentrations, such fragments can be further degraded, apparently by cooperation between the specifically bound and excess enzymes. This model is consistent with a substantial body of previous work on the nuclease activity of EcoB and EcoK, and it explains in a simple way how cleavage sites are selected.
Active human immunodeficiency virus protease is required for viral infectivity.
Authors: Kohl N E , Emini E A , Schleif W A , Davis L J , Heimbach J C , Dixon R A , Scolnick E M , Sigal I S
Published: May 2006

Retroviral proteins are synthesized as polyprotein precursors that undergo proteolytic cleavages to yield the mature viral proteins. The role of the human immunodeficiency virus (HIV) protease in the viral replication cycle was examined by use of a site-directed mutation in the protease gene. The HIV protease gene product was expressed in Escherichia coli and observed to cleave HIV gag p55 to gag p24 and gag p17 in vitro. Substitution of aspartic acid residue 25 (Asp-25) of this protein with an asparagine residue did not affect the expression of the protein, but it eliminated detectable in vitro proteolytic activity against HIV gag p55. A mutant HIV provirus was constructed that contained the Asn-25 mutation within the protease gene. SW480 human colon carcinoma cells transfected with the Asn-25 mutant proviral DNA produced virions that contained gag p55 but not gag p24, whereas virions from cells transfected with the wild-type DNA contained both gag p55 and gag p24. The mutant virions were not able to infect MT-4 lymphoid cells. In contrast, these cells were highly sensitive to infection by the wild-type virions. These results demonstrate that the HIV protease is an essential viral enzyme and, consequently, an attractive target for anti-HIV drugs.
Anti-tumor effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate.
Authors: Senter P D , Saulnier M G , Schreiber G J , Hirschberg D L , Brown J P , Hellström I , Hellström K E
Published: May 2006

Two anti-tumor monoclonal antibodies, L6 (anticarcinoma) and 1F5 (anti-B lymphoma), were covalently linked to alkaline phosphatase (AP), forming conjugates that could bind to the surface of antigen-positive tumor cells. The conjugates were capable of converting a relatively noncytotoxic prodrug, etoposide phosphate (EP), into etoposide--a drug with significant antitumor activity. In vitro studies with a human colon carcinoma cell line, H3347, demonstrated that while EP was less toxic than etoposide by a factor of greater than 100, it was equally toxic when the cells were pretreated with L6-AP, a conjugate that bound to the surface of H3347 cells. The L6-AP conjugate localized in H3347 tumor xenografts in nude mice and histological evaluation indicated that the targeted enzyme (AP) was distributed throughout the tumor mass. A strong antitumor response was observed in H3347-bearing mice that were treated with L6-AP followed 18-24 hr later by EP. This response, which included the rejection of established tumors, was superior to that of EP (P less than 0.005) or etoposide (P less than 0.001) given alone. The IF5-AP conjugate did not bind to H3347 cells and did not enhance the toxicity of EP on these cells in vitro. In addition, IF5-AP did not localize to H3347 tumors in nude mice and did not demonstrate enhanced antitumor activity in combination with the prodrug.
Recombinant human insulin-like growth factor I stimulates growth and has distinct effects on organ size in hypophysectomized rats.
Authors: Guler H P , Zapf J , Scheiwiller E , Froesch E R
Published: May 2006

Recombinant human insulin-like growth factor I (rhIGF-I) was infused subcutaneously into hypophysectomized rats for as long as 18 days. Three hundred micrograms (39 nmol) of rhIGF-I per day and 200 milliunits (4.5 nmol) of human growth hormone (hGH) per day increased body weight, tibial epiphyseal width, longitudinal bone growth, and trabecular bone formation similarly. Weight gains of the kidneys and spleen, however, were greater with rhIGF-I than with hGH, whereas the weight of the epididymal fat pads was reduced with rhIGF-I. The weight of the thymus was increased by rhIGF-I treatment. Thus, IGF-I administered over a prolonged period of time mimics GH effects in hypophysectomized rats. Quantitative differences between rhIGF-I and hGH treatment with respect to organ weights may be related to different forms of circulating IGF-I or may be due to independent effects of GH and IGF-I. The results support the somatomedin hypothesis, but they also stress the role of GH as a modulator of IGF-I action.
Identification of carbohydrate structures that are possible receptors for Neisseria gonorrhoeae.
Authors: Stromberg N , Deal C , Nyberg G , Normark S , So M , Karlsson K A
Published: May 2006

Different strains and isogenic variants of Neisseria gonorrhoeae were assayed for their ability to bind glycolipids extracted from various sources. Among a large number of reference glycolipids, binding was observed only to lactosylceramide [Gal(beta 1-4)Glc(beta 1-1)Cer], isoglobotriaosylceramide [Gal(alpha 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer], gangliotriaosylceramide [GalNAc(beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer], and gangliotetraosylceramide [Gal(beta 1-3)GalNAc(beta 1-4)Gal(beta 1-4)Glc(beta 1-1)Cer]. The latter two glycolipids bound gonococci with the highest affinity. Lactosylceramide and gangliotriaosylceramide were found in glycolipid preparations from ME180 cells, an epithelial cell line derived from a human cervical carcinoma, and thus are possible receptors for gonococci. The gonococcal surface component that bound the above glycolipids is a protein distinct from pilin and protein II.
Bypass and termination at apurinic sites during replication of single-stranded DNA in vitro: a model for apurinic site mutagenesis.
Authors: Hevroni D , Livneh Z
Published: May 2006

Mutations produced in Escherichia coli by apurinic sites are believed to arise via SOS-assisted translesion replication. Analysis of replication products synthesized on depurinated single-stranded DNA by DNA polymerase III holoenzyme revealed that apurinic sites frequently blocked in vitro replication. Bypass frequency of an apurinic site was estimated to be 10-15%. Direct evidence for replicative bypass was obtained in a complete single-stranded----replicative form replication system containing DNA polymerase III holoenzyme, single-stranded DNA binding protein, DNA polymerase I, and DNa ligase, by demonstrating the sensitivity of fully replicated products to the apurinic endonuclease activity of E. coli exonuclease III. Termination at apurinic sites, like termination at pyrimidine photodimers, involved dissociation of the polymerase from the blocked termini, followed by initiations at available primer templates. When no regular primer templates were available, the polymerase underwent repeated cycles of dissociation and rebinding at the blocked termini and, while bound, carried out multiple polymerization-excision reactions opposite the apurinic sites, leading to turnover of dNTPs into dNMPs. From the in vitro turnover rates, we could predict with striking accuracy the specificity of apurinic site mutagenesis, as determined in vivo in depurinated single-stranded DNA from an M13-lac hybrid phage. This finding is consistent with the view that DNA polymerase III holoenzyme carries out the mutagenic "misinsertion" step during apurinic site mutagenesis in vivo and that the specificity of the process is determined primarily by the polymerase. SOS-induced proteins such as UmuD/C might act as processivity-like factors to stabilize the polymerase-DNA complex, thus increasing the efficiency of the next stage of past-lesion polymerization required to complete the bypass reaction.
Interferon-gamma-induced transcriptional activation is mediated by protein kinase C.
Authors: Fan X D , Goldberg M , Bloom B R
Published: May 2006

Interferon-gamma (IFN-gamma) regulates a variety of biological functions and is the principal lymphokine known to activate macrophages. In studies of the molecular mechanisms by which these cells are regulated by IFN-gamma, the transcriptional activation of an IFN-gamma-inducible gene, gamma.1, in human macrophage-like cell lines was examined. Transcription of this gene is rapidly induced by 0.1-1 unit of IFN-gamma. In addition, gamma.1 transcription is efficiently induced by phorbol 12-myristate 13-acetate, which is known to activate protein kinase C (PKC). Both stimulators of gamma.1 transcription induce the translocation of PKC from the cytosol of a membrane fraction. Two selective inhibitors of PKC, H7 and sphingosine, suppressed not only the induction of gamma.1 mRNA but transcription of HLA-DR by IFN-gamma as well. These findings establish that PKC plays a significant role in the signal transduction pathway leading to transcriptional activation of some IFN-gamma-regulated genes of cells of the mononuclear phagocyte lineage.
Structure of a fish (rainbow trout) growth hormone gene and its evolutionary implications.
Authors: Agellon L B , Davies S L , Chen T T , Powers D A
Published: May 2006

We have isolated and sequenced a clone from a rainbow trout (Salmo gairdneri) genomic library that carries a gene encoding a fish growth hormone (GH). This gene spans a region of approximately equal to 4 kilobases, nearly twice that of mammalian GH genes. The trout GH gene is comprised of six exons, in contrast with five exons in mammals. The additional intron in the fish gene interrupts translated regions that are analogous to the last exon of its mammalian counterpart. In addition, the alleged internally repeating sequence in mammalian GH, prolactin (Prl), or placental lactogen (PL) is not observed in the predicted polypeptide sequence of fish GH. Direct repeats that flank exons I, III, and V of the mammalian GH, Prl, and PL genes are absent in the fish GH gene. These findings indicate that the rainbow trout GH gene structure does not support the current hypothesis that internally repeated regions in GH, Prl, and PL arose from a small primordial gene.
Therapeutic agents with dramatic antiretroviral activity and little toxicity at effective doses: aromatic polycyclic diones hypericin and pseudohypericin.
Authors: Meruelo D , Lavie G , Lavie D
Published: May 2006

Two aromatic polycyclic diones hypericin and pseudohypericin have potent antiretroviral activity; these substances occur in plants of the Hypericum family. Both compounds are highly effective in preventing viral-induced manifestations that follow infections with a variety of retroviruses in vivo and in vitro. Pseudohypericin and hypericin probably interfere with viral infection and/or spread by direct inactivation of the virus or by preventing virus shedding, budding, or assembly at the cell membrane. These compounds have no apparent activity against the transcription, translation, or transport of viral proteins to the cell membrane and also no direct effect on the polymerase. This property distinguishes their mode of action from that of the major antiretro-virus group of nucleoside analogues. Hypericin and pseudohypericin have low in vitro cytotoxic activity at concentrations sufficient to produce dramatic antiviral effects in murine tissue culture model systems that use radiation leukemia and Friend viruses. Administration of these compounds to mice at the low doses sufficient to prevent retroviral-induced disease appears devoid of undesirable side effects. This lack of toxicity at therapeutic doses extends to humans, as these compounds have been tested in patients as antidepressants with apparent salutary effects. Our observations to date suggest that pseudohypericin and hypericin could become therapeutic tools against retroviral-induced diseases such as acquired immunodeficiency syndrome (AIDS).
Glucocorticoid stimulates expression of corticotropin-releasing hormone gene in human placenta.
Authors: Robinson B G , Emanuel R L , Frim D M , Majzoub J A
Published: May 2006

Primary cultures of purified human cytotrophoblasts have been used to examine the expression of the corticotropin-releasing hormone (CRH) gene in placenta. We report here that glucocorticoids stimulate placental CRH synthesis and secretion in primary cultures of human placenta. This stimulation is in contrast to the glucocorticoid suppression of CRH expression in hypothalamus. The positive regulation of CRH by glucocorticoids suggests that the rise in CRH preceding parturition could result from the previously described rise in fetal glucocorticoids. Furthermore, this increase in placental CRH could stimulate, via adrenocorticotropic hormone, a further rise in fetal glucocorticoids, completing a positive feedback loop that would be terminated by delivery.
Lovastatin, an inhibitor of cholesterol synthesis, induces hydroxymethylglutaryl-coenzyme A reductase directly on membranes of expanded smooth endoplasmic reticulum in rat hepatocytes.
Authors: Singer I I , Scott S , Kazazis D M , Huff J W
Published: May 2006

Lovastatin is a potent competitive inhibitor of the rate-limiting enzyme of cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (NADPH) [HMG-CoA reductase; (S)-mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]. We determined the subcellular distribution of HMG-CoA reductase at high resolution by means of immunoelectron microscopy on ultrathin frozen liver sections of rats treated with lovastatin and cholestyramine. High concentrations of reductase were located on the outer (cytoplasmic) surfaces of smooth endoplasmic reticulum (SER) membranes induced in hepatocytes by acute drug administration. The enzyme was specifically localized over the whorled SER membranes and was absent from nonwhorled SER, rough endoplasmic reticulum, and peroxisomes. Intense HMG-CoA reductase labeling was only observed in hepatocytes containing high levels of HMG-CoA reductase activity; no staining was detected in untreated livers. These observations show that HMG-CoA reductase is induced as an integral component of the SER membranes that form in rat hepatocytes subsequent to lovastatin treatment and suggest that the formation of SER whorls in rat hepatocytes is due to mechanism-based effects of lovastatin.
Selective destruction of protein function by chromophore-assisted laser inactivation.
Authors: Jay D G
Published: May 2006

Chromophore-assisted laser inactivation of protein function has been achieved. After a protein binds a specific ligand or antibody conjugated with malachite green (C.I. 42,000), it is selectively inactivated by laser irradiation at a wavelength of light absorbed by the dye but not significantly absorbed by cellular components. Ligand-bound proteins in solution and on the surfaces of cells can be denatured without other proteins in the same samples being affected. Chromophore-assisted laser inactivation can be used to study cell surface phenomena by inactivating the functions of single proteins on living cells, a molecular extension of cellular laser ablation. It has an advantage over genetics and the use of specific inhibitors in that the protein function of a single cell within the organism can be inactivated by focusing the laser beam.
Differential expression of the homeobox gene Hox-1.3 in F9 embryonal carcinoma cells.
Authors: Murphy S P , Garbern J , Odenwald W F , Lazzarini R A , Linney E
Published: May 2006

The Hox-1.3 gene is located on mouse chromosome 6 and has been previously shown to be expressed in mouse embryos and adults. In this study, we have examined the steady-state levels of the Hox-1.3 transcripts in undifferentiated and differentiated F9 embryonal carcinoma cells. We find that there is a rapid increase of Hox-1.3 transcripts after differentiation induction of F9 cells. The level of the major 1.85-kilobase (kb) transcript peaks at 16-24 hr after differentiation induction of F9 cells. By using primer extension techniques the 5' ends of the major 1.85-kb transcript have been mapped to two sites in induced F9 cells. Cellular fractionation of RNA and transfer blot gel analysis has localized one minor transcript to the nucleus, whereas the major transcript and two additional minor transcripts appear in the nucleus and the cytoplasm of induced F9 cells. The results of nuclear run-off experiments with uninduced and induced F9 cell nuclei indicate that there is a substantial increase in the rate of Hox-1.3 transcription upon induction of F9 cells with retinoic acid.
Induction of protective immunity against Schistosoma mansoni by vaccination with schistosome paramyosin (Sm97), a nonsurface parasite antigen.
Authors: Pearce E J , James S L , Hieny S , Lanar D E , Sher A
Published: May 2006

Paramyosin (Sm97), a 97-kDa myofibrillar protein identified by the unusually monospecific antibody response induced by intradermal vaccination of mice with a complex soluble worm antigen preparation (SWAP) of adult Schistosoma mansoni administered with bacillus Calmette-Guérin (BCG), was purified and tested for its capacity to protect mice against challenge infection. When administered intradermally with BCG at total doses of only 4-40 micrograms per mouse, both the native molecule and a recombinant expression product containing approximately 50% of the whole protein were found to confer significant resistance (26-33%) against challenge infection, while 2 mg of unfractionated SWAP was required to induce similar levels of protection. In addition, paramyosin was shown to stimulate T lymphocytes from vaccinated mice to produce lymphokines [e.g., gamma interferon (IFN-gamma)] that activate macrophages to kill schistosomula. Neither schistosome myosin nor a heterologous paramyosin from a different invertebrate genus were protective, indicating a requirement for specific epitopes in the immunization. That the protection induced by paramyosin involves a T-cell-mediated mechanism was supported by the failure of anti-paramyosin antibodies to passively transfer significant resistance to infection to recipient mice. Lymphocytes from mice vaccinated with paramyosin were found to produce IFN-gamma in response to living schistosomula, suggesting that during challenge infection of vaccinated hosts, paramyosin (a nonsurface antigen) may elicit a protective T-cell response as a consequence of its release from migrating parasite larvae. Paramyosin-depleted SWAP was also found to be protective as well as stimulatory for T lymphocytes from SWAP-vaccinated mice, indicating that other antigens in this preparation may have immunoprophylactic potential. In summary, these results (i) suggest that the induction of T-cell-dependent cell-mediated immunity against soluble nonsurface antigens may be an effective strategy for immunization against multicellular parasites and (ii) in the case of schistosomes, identify paramyosin as a candidate vaccine immunogen in this category.
The short arm of chromosome 11 is a "hot spot" for hypermethylation in human neoplasia.
Authors: de Bustros A , Nelkin B D , Silverman A , Ehrlich G , Poiesz B , Baylin S B
Published: May 2006

Inactivation of normally expressed genes may play a role in the formation and/or progression of human cancers. Methylation of cytosine in DNA could potentially participate in such alterations of gene expression. Abnormalities in DNA methylation are a consistent feature of human neoplasms, and we now show that these include not only previously recognized widespread genomic hypomethylation, but also regional increases in gene methylation. A hot spot for abnormal methylation of C + G-rich areas has been detected on the short arm of chromosome 11 in an area known to harbor tumor suppressor genes. This change occurs consistently in common forms of human cancer and appears early during the transformation of cells with viruses including members of the human T-cell leukemia (HTLV) family. Furthermore, in one chromosome 11 gene examined, calcitonin, the increased methylation in somatic tumor cells coincides with the presence of an "inactive" chromatin pattern in the transcriptional regulatory area. The increased regional DNA methylation demonstrated may then participate in or mark chromosomal changes associated with gene inactivation events that are central to the genesis and/or progression of human cancers.
A-type potassium channels expressed from Shaker locus cDNA.
Authors: Iverson L E , Tanouye M A , Lester H A , Davidson N , Rudy B
Published: May 2006

A-type K+ currents are expressed in Xenopus oocytes injected with in vitro-synthesized transcripts from cDNAs for the Drosophila Shaker (Sh) locus. A single Sh gene product, possibly as a multimer, is sufficient for formation of functional A channels. Various Sh RNAs express A currents with distinct kinetic properties. An analysis of structure-function relationships shows that the conserved central region of Sh polypeptides determines ionic selectivity and overall channel behavior, whereas the divergent amino and carboxyl termini can modify channel kinetics. Alternative splicing of Sh gene transcripts may provide one mechanism for the generation of K+ channel diversity.
Conformational analysis of 9β,19-cyclopropyl sterols: Detection of the pseudoplanar conformer by nuclear Overhauser effects and its functional implications
Authors: Nes W. David , Benson Mabry , Lundin Robert E. , Le Phu H.
Published: May 2006

Nuclear Overhauser difference spectroscopy and variable temperature studies of the 9β,19-cyclopropyl sterols 24,25-dehydropollinastanol (4,4-desmethyl-5α-cycloart-24-en-3β-ol) and cyclolaudenol [(24
S
)-24-methyl-5α-cycloart-25(27)-en-3β-ol] have shown the solution conformation of the B/C rings to be twist-chair/twist-boat rather than boat/chair as suggested in the literature. This is very similar to the known crystal structure conformation of 9β,19-cyclopropyl sterols. The effect of these conformations on the molecular shape is highly significant: the first conformation orients into a pseudoplanar or flat shape analogous to lanosterol, whereas the latter conformation exhibits a bent shape. The results are interpreted to imply that, for conformational reasons, cyclopropyl sterols can be expected to maintain the pseudoplanar shape in membrane bilayers.
Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.
Authors: Doehmer J , Dogra S , Friedberg T , Monier S , Adesnik M , Glatt H , Oesch F
Published: May 2006

V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltransferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B1, which is activated by this enzyme.
Deprotonation of lipid-depleted bacteriorhodopsin.
Authors: Jang D J , el-Sayed M A
Published: May 2006

The removal of 75% of the lipid from bacteriorhodopsin caused the following: (i) decreased efficiency and rate of deprotonation of the protonated Schiff base (as monitored by absorption of the M412 intermediate); (ii) increased efficiency of deprotonation of deionized samples; (iii) a decrease by 1 unit in the pH at which deprotonation ceases; (iv) increased intensity of Eu3+ emission in Eu3+-regenerated deionized delipidated samples; (v) increased exposure of the Eu3+ sites to water; and (vi) elimination of the dependence of the deprotonation efficiency on the metal cation concentration. These results are discussed in terms of changes in the protein conformation upon delipidation, which in turn control the deprotonation mechanism.
The role of lipids in Plasmodium falciparum invasion of erythrocytes: a coordinated biochemical and microscopic analysis.
Authors: Mikkelsen R B , Kamber M , Wadwa K S , Lin P S , Schmidt-Ullrich R
Published: May 2006

The role of lipids in Plasmodium falciparum invasion of erythrocytes was investigated by biochemical and fluorescent microscopic analysis. Metabolic incorporation of radioactive oleate or palmitate and fractionation of radiolabeled phospholipids by thin-layer chromatography revealed no difference in the major phospholipid classes of schizonts and early ring forms after merozoite invasion. Fluorescent anthroyloxy derivatives of oleate and palmitate were also metabolically incorporated into parasite phospholipids. By microscopic analysis, the fluorescent phospholipids were seen localized in the plasma membrane and, within the merozoite, concentrated near the apical end. During invasion fluorescent phospholipid appeared to be injected from the apical end of the merozoite into the host membrane, both within and outside the parasite-host membrane junctions. After invasion fluorescent lipid was only found in the parasite plasma membrane and/or parasitophorous vacuole membrane. Parallel experiments with a fluorescent cholesterol derivative, incorporated into parasite membranes by exchange, revealed neither heterogeneous distribution of label within the parasite nor evidence for cholesterol transfer from merozoite to host cell membrane. Results suggest that during invasion no major covalent alteration of parasite lipids, such as lysophospholipid formation, occurs. However, invasion and formation of the parasitophorous vacuolar membrane apparently involves insertion of parasite phospholipids into the host membrane.
RED1: a yeast gene required for the segregation of chromosomes during the reductional division of meiosis.
Authors: Rockmill B , Roeder G S
Published: May 2006

A mutation at the RED1 locus was identified in a search for sporulation-proficient, meiotic-lethal yeast mutants. The few viable spores produced in the red1-1 mutant are highly aneuploid, suggesting that the spore lethality results from a high frequency of chromosome nondisjunction. Disomic spores produced by the red1-1 mutant contain nonsister chromatids and the red1-1 spore inviability phenotype is alleviated in red1-1 spo13 double mutants; these results indicate that nondisjunction occurs at the first meiotic division. The red1-1 mutant is recombination-proficient. The RED1 gene was cloned by complementation of the meiotic lethal phenotype; strains carrying a disruption of the gene are mitotically viable. We propose that the RED1 gene product is involved in meiosis I chromosome disjunction, perhaps by maintaining the connections between homologous chromosomes through metaphase I.
Structure and action of buccalin: a modulatory neuropeptide localized to an identified small cardioactive peptide-containing cholinergic motor neuron of Aplysia californica.
Authors: Cropper E C , Miller M W , Tenenbaum R , Kolks M A , Kupfermann I , Weiss K R
Published: May 2006

A model system that consists of a muscle utilized in biting, the accessory radula closer (ARC), and the two cholinergic motor neurons innervating this muscle, neurons B15 and B16, has been used to study the expression of food-induced arousal in the marine mollusk Aplysia. The ARC muscle receives modulatory input from an extrinsic source, the serotonergic metacerebral cells, which partially accounts for the progressive increase in the strength of biting seen in aroused animals. Another source of modulation may arise from the ARC motor neurons themselves, which synthesize neuropeptides that can potentiate ARC contractions. Neuron B15 synthesizes the two homologous peptides, small cardioactive peptides A and B, whereas neuron B16 synthesizes the structurally unrelated peptide myomodulin. Here we report the purification and sequencing of a neuropeptide termed buccalin and show that it is colocalized with the small cardioactive peptides to neuron B15. Buccalin is also bioactive at the ARC neuromuscular junction but, in contrast to the small cardioactive peptides, when exogenously applied, it decreases rather than increases the size of muscle contractions elicited by firing of the motor neurons. Also unlike the small cardioactive peptides, which exert postsynaptic actions, buccalin seems to act only presynaptically. It has no effect on muscle relaxation rate and decreases motor neuron-elicited excitatory junction potentials in the ARC without affecting contractions produced by direct application of acetylcholine to the muscle. Neuron B15, therefore, appears to contain three modulatory neurotransmitters, two of which may act postsynaptically on the muscle to potentiate the action of the primary neurotransmitter acetylcholine and one of which may act presynaptically on nerve terminals to inhibit acetylcholine release.
Cholinergic amacrine cells of the rabbit retina contain glutamate decarboxylase and gamma-aminobutyrate immunoreactivity.
Authors: Brecha N , Johnson D , Peichl L , Wässle H
Published: May 2006

The transmitters acetylcholine and gamma-aminobutyrate (GABA) play critical roles in the formation of receptive-field properties of retinal ganglion cells. In rabbit retina, cholinergic amacrine and displaced amacrine cells were identified by immunohistochemical staining for the enzyme choline acetyltransferase and by their avid accumulation of the fluorescent dye 4',6-diamidino-2-phenylindole. Several GABA-immunoreactive and glutamate decarboxylase-immunoreactive cell types, including a prominent population of small, round amacrine and displaced amacrine cells, were also identified. Double-label experiments demonstrated that all amacrine and displaced amacrine cells that prominently accumulate 4',6-diamidino-2-phenylindole contain GABA and glutamate decarboxylase immunoreactivity. However, not all GABA-immunoreactive cells accumulate this dye. Quantitative analysis of the ganglion cell layer of whole mount preparations of the retina showed that choline acetyltransferase-immunoreactive cells and the majority of GABA-immunoreactive cells have a small, round shape and similar cell density profiles that parallel that of displaced amacrine cells. These studies establish that cholinergic cells are a major subpopulation of GABA-immunoreactive amacrine and displaced amacrine cells. The role these cells have in the formation of ganglion cell receptive-field properties may be parsimoniously explained by an excitatory postsynaptic action mediated by acetylcholine and an inhibitory presynaptic action mediated by GABA.
Biosynthesis of a protein containing a nonprotein amino acid by Escherichia coli: L-2-aminohexanoic acid at position 21 in human epidermal growth factor.
Authors: Koide H , Yokoyama S , Kawai G , Ha J M , Oka T , Kawai S , Miyake T , Fuwa T , Miyazawa T
Published: May 2006

Endeavoring to develop a method to biosynthesize proteins substituted with nonprotein amino acids, we attempted the incorporation of L-2-aminohexanoic acid (Ahx) into human epidermal growth factor (hEGF). Escherichia coli YK537 strain harboring plasmid pTA1522, which has the phoA promoter-phoA signal peptide-hEGF gene, was used. Cells were cultured first in high-phosphate medium and then, for induction of the hEGF-encoding gene, transferred to low-phosphate medium containing Ahx (0.25 mg/ml). hEGF and Ahx-substituted hEGF, [Ahx21]hEGF, secreted into the periplasm were recovered. After treatment with H2O2, [Ahx21]-hEGF was clearly separated from methionine-oxidized hEGF by one-step reverse-phase HPLC. Substitution of the methionine residue of hEGF with Ahx was confirmed by the amino acid analysis of [Ahx21]hEGF. The three biological activities of [Ahx21]hEGF were the same as those of hEGF. From the successful production of [Ahx21]hEGF, a basic strategy was established for preparing proteins substituted with nonprotein amino acid (alloprotein). Induction of the phoA promoter of pho regulon and secretion of the product to the periplasm may depress heat shock-like responses and subsequent hydrolysis of the product by cytoplasmic protease.
Cloning and expression of a human kidney cDNA for an alpha 2-adrenergic receptor subtype.
Authors: Regan J W , Kobilka T S , Yang-Feng T L , Caron M G , Lefkowitz R J , Kobilka B K
Published: May 2006

An alpha 2-adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet alpha 2-adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet alpha 2-adrenergic receptor and is consistent with the structure of other members of the family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the alpha 2-adrenergic ligand [3H]rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the alpha 2B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet alpha 2-adrenergic receptor (alpha 2A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective alpha-adrenergic ligands.
Primary structure of the mouse sperm receptor polypeptide determined by genomic cloning.
Authors: Kinloch R A , Roller R J , Fimiani C M , Wassarman D A , Wassarman P M
Published: May 2006

The mouse sperm receptor, a glycoprotein called ZP3, is synthesized and secreted by growing oocytes. It is present in more than a billion copies in the unfertilized egg's extracellular coat, or zona pellucida. We have cloned and characterized a region of the mouse (CD-1) genome that spans 10 kilobases of the ZP3 locus. The genomic clones described encompass the entire ZP3 coding region, which contains eight exons. The exons were identified, mapped, and sequenced, yielding the entire primary structure of the ZP3 polypeptide chain (424 amino acids; Mr, 46,300), which includes a 22-amino acid signal sequence. In addition, sequencing of genomic clones has revealed some unusual features of ZP3 mRNA and a region just downstream of the ZP3 gene.
v-Ha-ras oncogene insertion: a model for tumor progression of human small cell lung cancer.
Authors: Mabry M , Nakagawa T , Nelkin B D , McDowell E , Gesell M , Eggleston J C , Casero R A , Baylin S B
Published: May 2006

Small cell lung cancer (SCLC) manifests a range of phenotypes in culture that may be important in understanding its relationship to non-SCLCs and to tumor progression events in patients. Most SCLC-derived cell lines, termed "classic" SCLC lines, have properties similar to SCLC tumors in patients, including high expression of neuroendocrine markers and low c-myc oncogene expression. A significant number of SCLC lines characterized as "biochemical or morphologic variant" SCLC lines have decreased levels of endocrine differentiation markers associated with increased proliferative indices, amplification of the c-myc oncogene, and growth patterns and biochemical markers more typical of non-SCLCs. To delineate further the relationships between these phenotypes and the molecular events involved, we have inserted the v-Ha-ras gene in SCLC cell lines with (biochemical variant) and without (classic) an amplified c-myc gene. These two SCLC subtypes had markedly different phenotypic responses to similar levels of expression of v-Ha-ras RNA. No biochemical or morphologic changes were observed in classic SCLC cells. In contrast, in biochemical variant SCLC cells, v-Ha-ras expression induced features typical of large cell undifferentiated lung carcinoma, including adherent monolayer growth patterns, increased cloning efficiency, increased levels of non-SCLC cell markers, ultrastructural characteristics and an acquired resistance to polyamine depletion typical of large cell carcinoma, but not SCLC, in vitro. Expression of v-Ha-ras in biochemical variant SCLC cells directly demonstrates that important transitions can occur between phenotypes of human lung cancer cells and that these may play a critical role in tumor progression events in patients. The findings provide a model system to study molecular events involved in tumor progression steps within a series of related tumor types.
Central mammalian neurons normally resistant to glutamate toxicity are made sensitive by elevated extracellular Ca2+: toxicity is blocked by the N-methyl-D-aspartate antagonist MK-801.
Authors: Hahn J S , Aizenman E , Lipton S A
Published: May 2006

It is widely held that a glutamate-like toxin that resembles N-methyl-D-aspartate may be responsible for the death of nerve cells seen after severe neurological insults including stroke, seizures, and degenerative disorders, such as Huntington disease, Alzheimer disease, and the amyotrophic lateral sclerosis-parkinsonism-dementia complex found on Guam. One puzzling fact about these maladies is the differential vulnerability of specific groups of neurons peculiar to each condition. We report here that an identified population of central neurons, rat retinal ganglion cells, are resistant to the neurotoxic effects of millimolar concentrations of glutamate under otherwise normal culture conditions. Patch-clamp experiments show that this resistance is associated with a very small ionic current response to N-methyl-D-aspartate. Varying the ionic milieu by increasing the extracellular Ca2+ concentration, however, results in a striking increase in glutamate-induced cell death in this population. Under these conditions, Mg2+ or the amino acid antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo-(alpha,gamma)-cyclohepten-5 ,10-imine maleate], blockers of N-methyl-D-aspartate receptor-coupled ion channels, completely abrogate the lethal effects of glutamate. These findings strongly suggest that Ca2+ entry through N-methyl-D-aspartate-activated channels is responsible for this type of neuronal death and suggest strategies that may be clinically useful in the treatment of various neurological disorders.
Tissue-specific expression of transfected human insulin genes in pluripotent clonal rat insulinoma lines induced during passage in vivo.
Authors: Madsen O D , Andersen L C , Michelsen B , Owerbach D , Larsson L I , Lernmark A , Steiner D F
Published: May 2006

The pluripotent rat islet tumor cell line MSL-G2 expresses primarily glucagon or cholecystokinin and not insulin in vitro but changes phenotype completely after prolonged in vivo cultivation to yield small-sized hypoglycemic tumors composed almost entirely of insulin-producing beta cells. When a genomic DNA fragment containing the coding and upstream regulatory regions of the human insulin gene was stably transfected into MSL-G2 cells no measurable amounts of insulin or insulin mRNA were detected in vitro. However, successive transplantation of two transfected clones resulted in hypoglycemic tumors that efficiently coexpressed human and rat insulin as determined by human C-peptide-specific immunoreagents. These results demonstrate that cis-acting tissue-specific insulin gene enhancer elements are conserved between rat and human insulin genes. We propose that the in vivo differentiation of MSL-G2 cells and transfected subclones into insulin-producing cells reflects processes of natural beta-cell ontogeny leading to insulin gene expression.
Genomic sequencing and in vivo footprinting of an expression-specific DNase I-hypersensitive site of avian vitellogenin II promoter reveal a demethylation of a mCpG and a change in specific interactions of proteins with DNA.
Authors: Saluz H P , Feavers I M , Jiricny J , Jost J P
Published: May 2006

Genomic sequencing was used to study the in vivo methylation pattern of two CpG sites in the promoter region of the avian vitellogenin gene. The CpG at position +10 was fully methylated in DNA isolated from tissues that do not express the gene but was unmethylated in the liver of mature hens and estradiol-treated roosters. In the latter tissue, this site became demethylated and DNase I hypersensitive after estradiol treatment. A second CpG (position -52) was unmethylated in all tissues examined. In vivo genomic footprinting with dimethyl sulfate revealed different patterns of DNA protection in silent and expressed genes. In rooster liver cells, at least 10 base pairs of DNA, including the methylated CpG, were protected by protein(s). Gel-shift assays indicated that a protein factor, present in rooster liver nuclear extract, bound at this site only when it was methylated. In hen liver cells, the same unmethylated CpG lies within a protected region of approximately equal to 20 base pairs. In vitro DNase I protection and gel-shift assays indicate that this sequence is bound by a protein, which binds both double- and single-stranded DNA. For the latter substrate, this factor was shown to bind solely the noncoding (i.e., mRNA-like) strand.
Analysis of gene amplification in human tumor cell lines.
Authors: Fukumoto M , Shevrin D H , Roninson I B
Published: May 2006

Oncogene amplification has been observed in various primary tumors and tumor-derived cell lines. In several types of cancer, amplification of specific oncogenes is correlated with the stage of tumor progression. To estimate the frequency of gene amplification in other tumor types and to determine whether the ability to grow in vivo is associated with gene amplification in tumor cell lines, we have developed a modified version of the in-gel renaturation assay that detects human DNA sequences of unknown nature amplified as little as 7- to 8-fold. This assay was used to screen 16 cell lines derived from various solid tumors and leukemias. Amplified DNA sequences were detected in only one cell line, Calu-3 lung adenocarcinoma. This cell line was found to contain coamplified NGL (formerly termed neu) and ERBA1 oncogenes. However, when one of the amplification-negative cell lines, PC-3 prostatic carcinoma, was selected for in vivo growth in nude mice, amplified DNA sequences became detectable in these cells. The amplified sequences included the MYC oncogene, which showed no amplification in the parental cell line but was amplified 10- to 12-fold in the in vivo-selected cells. MYC amplification may, therefore, provide tumor cells with a selective advantage specific for in vivo growth.