bluuebunny/crossref_metadata_2025 huggingface dataset:
An estimate of the number of G regulator proteins activated per excited rhodopsin in living Limulus ventral photoreceptors.
Authors: Kirkwood A , Weiner D , Lisman J E
Published: May 2006

Previous work by others on Limulus photoreceptors has shown that application of a variety of guanine nucleotide-binding regulatory protein (G protein) activators produces discrete waves of depolarization similar to those generated by single photos, but smaller in size. We investigated whether these events might originate at a site other than the G protein. Initiation of the events did not depend on the state of the visual pigment, suggesting that the events do not originate at the pigment level. The events could be blocked by the G-protein blocker guanosine 5'-[beta-thio]diphosphate (GDP[betaS]) and thus support the conclusion that these discrete events are due to the activation of G protein itself. Quantitative measurements indicate that the average size of these events is approximately 8 times smaller than that evoked by single photons under the same conditions. Given certain reasonable assumptions, these results imply that the gain of the first stage of transduction in vivo is approximately 8, a value considerably lower than that measured in vitro in vertebrate rods (gain, 100-500). Furthermore, independent evidence for a low first-stage gain in Limulus is derived from the observation that GDP[betaS] barely affects the size of the response to single photons, but greatly reduces the probability that a photon evokes a response. These results can be explained if rhodopsin normally activates only a few G proteins.
Human basic fibroblast growth factor gene encodes four polypeptides: three initiate translation from non-AUG codons.
Authors: Florkiewicz R Z , Sommer A
Published: May 2006

Human basic fibroblast growth factor (bFGF) is an angiogenic polypeptide mitogen present in a wide variety of mesoderm- and neuroectoderm-derived tissues. bFGF cDNA and genomic clones predict a 17.8-kDa (155-amino acid) gene product based on the presence of a single putative translational initiator ATG codon. However, a bFGF protein isolated from human placenta contains two additional amino acids NH2-terminal to the predicted initiator methionine. We report here that the human cell line SK-HEP-1 coexpresses four molecular forms (17.8, 22.5, 23.1, and 24.2 kDa) of bFGF. The 17.8-kDa bFGF protein is translationally initiated at the previously predicted methionine (AUG) codon, whereas the 22.5-, 23.1-, and 24.2-kDa proteins initiate at unusual non-AUG codons. The higher molecular weight forms are colinear NH2-terminal extensions of the 18-kDa bFGF.
Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin.
Authors: Peck K , Stryer L , Glazer A N , Mathies R A
Published: May 2006

A theory for single-molecule fluorescence detection is developed and then used to analyze data from subpicomolar solutions of B-phycoerythrin (PE). The distribution of detected counts is the convolution of a Poissonian continuous background with bursts arising from the passage of individual fluorophores through the focused laser beam. The autocorrelation function reveals single-molecule events and provides a criterion for optimizing experimental parameters. The transit time of fluorescent molecules through the 120-fl imaged volume was 800 microseconds. The optimal laser power (32 mW at 514.5 nm) gave an incident intensity of 1.8 x 10(23) photons.cm-2.s-1, corresponding to a mean time of 1.1 ns between absorptions. The mean incremental count rate was 1.5 per 100 microseconds for PE monomers and 3.0 for PE dimers above a background count rate of 1.0. The distribution of counts and the autocorrelation function for 200 fM monomer and 100 fM dimer demonstrate that single-molecule detection was achieved. At this concentration, the mean occupancy was 0.014 monomer molecules in the probed volume. A hard-wired version of this detection system was used to measure the concentration of PE down to 1 fM. This single-molecule counter is 3 orders of magnitude more sensitive than conventional fluorescence detection systems.
Functional expression of plant acetolactate synthase genes in
Escherichia coli
Authors: Smith Julie K. , Schloss John V. , Mazur Barbara J.
Published: May 2006

Acetolactate synthase (ALS; EC 4.1.3.18) is the first common enzyme in the biosynthetic pathways leading to leucine, isoleucine, and valine. It is the target enzyme for three classes of structurally unrelated herbicides, the sulfonylureas, the imidazolinones, and the triazolopyrimidines. A cloned ALS gene from the small cruciferous plant
Arabidopsis thaliana
has been fused to bacterial transcription/translation signals and the resulting plasmid has been used to transform
Escherichia coli
. The cloned plant gene, which includes sequences encoding the chloroplast transit peptide, is functionally expressed in the bacteria. It is able to complement genetically a strain of
E. coli
that lacks endogenous ALS activity. An ALS gene cloned from a line of
Arabidopsis
previously shown to be resistant to sulfonylurea herbicides has been similarly expressed in
E. coli
. The herbicide-resistance phenotype is expressed in the bacteria, as assayed by both enzyme activity and the ability to grow in the presence of herbicides. This system has been useful for purifying substantial amounts of the plant enzyme, for studying the sequence parameters involved in subcellular protein localization, and for characterizing the interactions that occur between ALS and its various inhibitors.
Concerted hexose transport curb by tunicamycin is rendered irreversible by glucose or allose in medium containing L-glutamine.
Authors: Ullrey D B , Kalckar H M
Published: May 2006

The hexose transport in a hamster fibroblast mutant (DS7), unable to use glucose for generation of energy, is nevertheless subject to a marked down-regulation ("curb") after prolonged incubation of monolayer cultures with glucose; fructose is unable to exert any curb. D-Allose, an all-cis hexose, mediates a vigorous curb of the transport system. Moreover, prolonged coincubation of glucose or allose with tunicamycin (TM) brings about an additional effect that is not an inhibition of the transport system, which we shall call the "concerted" transport curb. This type of concerted transport curb requires L-glutamine in the maintenance medium; moreover, addition of cycloheximide prevents the development of this TM effect. Apparently, cellular protein synthesis or protein turnover or both are required for the development of the TM-concerted transport curb. The concerted transport curb can be reversed in sugar-free or in fructose-containing medium, even upon readdition of TM. In contrast, the sole readdition of glucose or D-allose renders the concerted curb irreversible. This raises the question of whether the cells under the condition of the concerted curb somehow have internalized the TM into the membrane.
Activation of human CDC2 protein as a histone H1 kinase is associated with complex formation with the p62 subunit.
Authors: Brizuela L , Draetta G , Beach D
Published: May 2006

p34 kinase, the product of the CDC2 gene, is a cell-cycle regulated protein kinase that is most active during mitosis. In HeLa cells, p34 kinase has previously been shown to exist in both a low- and a high-molecular-mass form, the latter of which is only found in cells in the G2/M phase of the cell cycle and contains a 62-kDa subunit. Here we show that although each form of the kinase phosphorylates casein in vitro, only the high-molecular-mass form uses histone H1 as substrate. The high-molecular-mass form of p34 kinase from nocodazole-treated HeLa cells was purified 6700-fold. The apparent molecular mass of the mitotic CDC2-encoded protein kinase complex was 220 kDa. The purified enzyme phosphorylated not only its endogenous 62-kDa subunit but also phosphorylated histone H1 with a Km of 3 microM and used ATP 40 times more efficiently than GTP (Km 54 microM and 2 mM, respectively). The enzyme activity was unaffected by cAMP, calcium/calmodulin, or by the heat-stable inhibitor of cAMP-dependent protein kinase. These characteristics are typical of growth-associated histone H1 kinase from different organisms. These results suggest that CDC2 protein may be activated as an M-phase-specific protein kinase in part by its association with the p62 subunit.
Metabolism of ethanol and carcinogens by glutathione transferases.
Authors: Bora P S , Spilburg C A , Lange L G
Published: May 2006

Nonoxidative alcohol metabolism to form fatty acid ethyl esters contributes to alcohol-related end-organ damage, and these products are formed by two synthase enzymes. We recently purified the major (pI 4.9) synthase from human myocardium. The N-terminal sequence (A P Y T V V Y F P V R G R X K A L R M L X A D) is greater than 73% identical with that of a neutral (pI 6.7) detoxification enzyme, glutathione transferase P from rat hepatocellular carcinoma (P P Y T I V Y F P V R G R C E A T R M L L A D). Moreover, both the major human fatty acid ethyl ester synthase and bovine liver glutathione transferase catalyze the formation of fatty acid ethyl esters (Vmax 105 and 98 nmol per hr per mg, respectively). In addition, both enzymes catalyze the formation of glutathione-xenobiotic conjugates (Vmax 67 and 335 mol per hr per mol of enzyme, respectively). Physiological concentrations of glutathione increase the rate of formation of fatty acid ethyl esters up to 5-fold, and the glutathione transferase substrate 1-chloro-2,4-dinitrobenzene is a potent inhibitor of human myocardial fatty acid ethyl ester synthase. Thus, the identification of the major form of human myocardial fatty acid ethyl ester synthase as an acidic glutathione transferase links alcohol and xenobiotic metabolism and may relate the enhancement of tumorigenesis by alcohol abuse with carcinogen-conjugation reactions.
Steady-state electrophoresis: a technique for measuring physical properties of macro-ions.
Authors: Godfrey J E
Published: May 2006

The basic theory of a newly discovered physical technique, steady-state electrophoresis, is described followed by analyses of several representative macro-ion systems with the technique. This method measures apparent moments of molar diminished charge (defined below) of solvated macro-ions from an analysis of a concentration gradient of the macro-ions stabilized against a semipermeable membrane by the opposing forces of diffusion and an external electric field. The experimental results confirm that the theory adequately describes the behavior of the macro-ion systems at electrophoretic steady state and suggest applications for the technique in addition to measuring macro-ion-small ion interactions.
Chromosomal inversion patterning and population differentiation in a young insular species, Drosophila silvestris.
Authors: Craddock E M , Carson H L
Published: May 2006

The recently evolved Hawaiian species Drosophila silvestris has a subdivided population structure and shows great spatial heterogeneity in chromosome inversion distributions and frequencies within its extremely limited geographic range. Pattern analysis of the 11 chromosomal polymorphisms in the context of the recently discovered morphological and behavioral divergence within the species has elucidated the history of the chromosomal differentiation. We identify four chronological groups of inversions and their probable sites of origin. Spread of the derived "3-row" bristle morphotype on the Hilo side of the Island of Hawaii has been accompanied by the acquisition of six new inversion polymorphisms. Three phylogenetically old inversions show correlations with altitude, and there are multiple cross-correlations between inversions on the same and different chromosomes, reflecting complex interaction systems. Quantification of the genetic population structure of D. silvestris by hierarchical F statistics reveals a dramatic level of genetic differentiation for an evolutionarily new species of such restricted range. This level exceeds that of older, continental Drosophila species. There is, however, minimal concordance between the chromosomal variation and the morphological-behavioral discontinuity, a consequence of the extensive cytological variation within each morphotype. Such a fragmented gene pool favors the rapid evolution and continued divergence of this insular species.
Structure of simian immunodeficiency virus regulatory genes.
Authors: Colombini S , Arya S K , Reitz M S , Jagodzinski L , Beaver B , Wong-Staal F
Published: May 2006

Three full-length cDNA clones were obtained from cells infected with the simian immunodeficiency virus (SIV) isolated from captive macaques (SIVMAC). Nucleotide sequence analyses suggested that these represented mRNA for the SIV MAC genes tat, rev (formerly, art/trs), and nef (formerly, 3'orf). The putative tat-specific clone was active in trans-activation of the SIV MAC long terminal repeat in COS-1 and Jurkat cells. In contrast, the human immunodeficiency virus 1 long terminal repeat was significantly trans-activated only in the COS-1 cells. This suggests that trans-activation by the SIV tat gene is modulated by cell-specific factors. The structure of all of the clones suggested an mRNA splicing pattern more complex than that described for human immunodeficiency virus 1.
Rat glucokinase gene: structure and regulation by insulin.
Authors: Magnuson M A , Andreone T L , Printz R L , Koch S , Granner D K
Published: May 2006

The glucokinase gene is 15.5-kilobases long, appears to be present as a single copy, and contains 10 exons that range in size from 96 to 977 base pairs. The transcription start site was located 127 nucleotides upstream from the translation initiation codon. The 5' flanking DNA contains several regions similar to defined promoter elements. These include a probable "TATA box," an Sp1 binding site, and several elements related to liver-specific gene expression. In addition, we determined that transcription of the glucokinase gene increased at least 20-fold when diabetic rats were treated with insulin for 2 hr.
Purification of a yeast TATA box-binding protein that exhibits human transcription factor IID activity.
Authors: Horikoshi M , Wang C K , Fujii H , Cromlish J A , Weil P A , Roeder R G
Published: May 2006

By a series of conventional chromatographic procedures we have purified from whole-cell extracts of Saccharomyces cerevisiae yeast transcription factor IID (TFIID), which functionally substitutes for human TFIID in a complementation assay comprised of the adenovirus type 2 major late promoter and HeLa cell-derived RNA polymerase II, transcription factors IIA, IIB, and IIE. Similar to its human counterpart, yeast TFIID also exhibited specific binding to the adenovirus type 2 major late promoter TATA element, as shown by both DNase I footprinting and gel mobility shift assays. NaDodSO4/PAGE analyses showed that a 27-kDa polypeptide coeluted with TFIID complementing activity through each chromatographic step. In agreement with this result and also suggesting that the native protein is a monomer, gel-filtration experiments indicated a molecular mass of 28 kDa for TFIID under nondenaturing conditions. That the 27-kDa polypeptide represented TFIID was further demonstrated by the ability of an HPLC-purified protein to bind specifically after renaturation to the adenovirus type 2 major late promoter TATA sequence.
Expression in bacteria of functional inhibitory subunit of retinal rod cGMP phosphodiesterase.
Authors: Brown R L , Stryer L
Published: May 2006

The cGMP phosphodiesterase of vertebrate retinal rod outer segments plays a key role in visual transduction. A functionally active form of the inhibitory gamma subunit of the phosphodiesterase, which keeps the enzyme inactive in the dark, has been obtained in high yield from a synthetic gene expressed in Escherichia coli. A DNA sequence encoding the 87-residue bovine gamma subunit was chemically synthesized and assembled from 10 oligonucleotides. The synthetic gene was cloned into an expression vector that uses the promoter PL of lambda phage. E. coli was transformed with this vector, which encodes a fusion protein consisting of the first 31 residues of the lambda cII protein, a 7-residue joining sequence that is specifically cleaved at its C-terminal end by clotting protease factor Xa, and the 87-residue gamma subunit. The fusion protein was solubilized in 6 M urea and purified by ion-exchange chromatography on a CM-Sephadex column. The typical yield was 1 mg of fusion protein per liter of bacterial culture, which corresponds to the amount of gamma in about 2500 bovine retinas. Proteolytic cleavage of the fusion protein by factor Xa released a synthetic gamma with the same amino acid sequence as that of native gamma. Both fusion protein and synthetic gamma inhibited trypsin-activated phosphodiesterase with high affinity (Kd less than 100 pM). Likewise, both were as effective as native gamma in inhibiting transducin-activated phosphodiesterase in rod outer segment membranes. This inhibition was reversed by the activation of additional transducin. Thus, the N terminus of gamma is not intimately involved in interactions with either the catalytic subunits of the phosphodiesterase or the activated form of transducin. In contrast, a C-terminal deletion mutant terminating at residue 74 of gamma stimulated rather than inhibited the trypsin-activated enzyme. Thus, the C-terminal region of gamma is critical for inhibition of the phosphodiesterase.
On the dynamics of a forced reaction-diffusion model for biological pattern formation.
Authors: Tsonis A A , Elsner J B , Tsonis P A
Published: May 2006

Ideas from the theory of dynamical systems are applied in biological pattern formation. By considering a simple reaction-diffusion model subjected to an external excitation, we find that the system can give rise to a great variety of periodic, quasiperiodic, and chaotic evolutions.
Recombinant interleukin 2 therapy in severe combined immunodeficiency disease.
Authors: Pahwa R , Chatila T , Pahwa S , Paradise C , Day N K , Geha R , Schwartz S A , Slade H , Oyaizu N , Good R A
Published: May 2006

Severe combined immunodeficiency disease (SCID) is a congenital disorder of severe B- and T-lymphocyte dysfunction in which several pathogenic mechanisms have been identified. The present study describes a female child with SCID who had a primary defect in the ability of T cells to secrete interleukin 2 (IL-2). B- and T-cell numbers were normal, but their functions were severely deficient. Mitogen and antigen-driven lymphoproliferative responses were diminished but were correctable in vitro with recombinant IL-2 (rIL-2). The patient's phytohemagglutinin-stimulated lymphocytes expressed IL-2 receptors normally. Despite the presence of the gene for IL-2, the patient's cells were grossly deficient in messenger RNA for IL-2 and endogenous IL-2 production. Pokeweed mitogen-driven B-cell differentiation was decreased and was not corrected by the addition of normal T cells to the B cells. Two attempts at immune reconstitution by haploidentical bone marrow transplantation failed. Therapy with rIL-2 (30,000 units/kg, given daily i.v.) resulted in marked clinical improvement as well in improved T-cell functions. The child, now 3 yr old, has been on rIL-2 therapy for 2 yr and receives rIL-2 (30,000 units/kg) three times weekly at home. This case study points to a new direction in the treatment of such disorders with rIL-2.
Inhibin and activin modulate the release of gonadotropin-releasing hormone, human chorionic gonadotropin, and progesterone from cultured human placental cells.
Authors: Petraglia F , Vaughan J , Vale W
Published: May 2006

Although it is clear that human chorionic gonadotropin (hCG) and progesterone play fundamental roles in pregnancy, the regulation of placental production of these hormones remains to be defined. Recent evidence suggests that the human placenta expresses proteins related to inhibin (alpha beta subunits) or activin (beta beta subunits). Inhibin and activin (follicle-stimulating hormone-releasing protein) possess opposing activities in several biological systems including pituitary follicle-stimulating hormone (follitropin) secretion, erythroid differentiation, and gonadal sex-steroid production. The actions of purified inhibin and activin on hormonogenesis by primary cultures of human placental cells were studied. The addition of activin increased gonadotropin-releasing hormone (GnRH) and progesterone production and potentiated the GnRH-induced release of hCG. Inhibin by itself did not modify placental immunoreactive GnRH, hCG, and progesterone secretion but reversed the activin-induced changes. Neither inhibin nor activin influenced the release of human placental lactogen. Furthermore, transforming growth factor beta, structurally related to inhibin/activin, did not significantly influence hormone release from cultured placental cells. These results support the hypothesis that inhibin and activin may play a role in regulating the release of GnRH, hCG, and progesterone from placenta and implicate inhibin-related proteins in the endocrine physiology of human pregnancy.
RETRACTED: Activation of protein kinase C by elevation of glucose concentration: proposal for a mechanism in the development of diabetic vascular complications.
Authors: Lee T S , Saltsman K A , Ohashi H , King G L
Published: May 2006

Hyperglycemia is believed to be the major cause of diabetic vascular complications involving both microvessels and arteries as in the retina, renal glomeruli, and aorta. It is unclear by which mechanism hyperglycemia is altering the metabolism and functions of vascular cells, although changes in nonenzymatic protein glycosylation and increases in cellular sorbitol levels have been postulated to be involved. Previously, we have reported that the elevation of extracellular glucose levels with cultured bovine retinal capillary endothelial cells causes an increase in protein kinase C (PKC) activity of the membranous pool with a parallel decrease in the cytosol without alteration of its total activity. Now we demonstrate that the mechanism for the activation of PKC is due to an enhanced de novo synthesis of diacylglycerol as indicated by a 2-fold increase of [14C]diacylglycerol labeling from [14C]glucose. The elevated diacylglycerol de novo synthesis is secondarily due to increased formation of precursors derived from glucose metabolism; this formation is enhanced by hyperglycemia as substantiated by elevated [3H]glucose conversion into water. This effect of hyperglycemia on PKC is also observed in cultured aortic smooth muscle and endothelial cells and the retina and kidney of diabetic rats, but not in the brain. Since PKC in vascular cells has been shown to modulate hormone receptor turnover, neovascularization in vitro, and cell growth, we propose that this mechanism of enhancing the membranous PKC activities by hyperglycemia plays an important role in the development of diabetic vascular complications.
Extra-renal transcription of the renin genes in multiple tissues of mice and rats.
Authors: Ekker M , Tronik D , Rougeon F
Published: May 2006

Expression of the mouse renin genes (Ren-1 and Ren-2) and of the unique rat renin gene was determined in several extra-renal tissues of mice and rats by primer-directed enzymatic amplification of cDNAs. In addition to the adrenal glands, testis, and ovaries, renin transcripts are detected in the liver, whole brain, and hypothalamus and, at lower levels, in spleen, thymus, lung, and prostate. Expression of the rat renin gene correlates with that of the mouse Ren-1 gene with the notable exception of the submaxillary gland where renin transcripts are found only in mice. The levels of renin transcripts in the liver of females from both species are higher than in males. In mice, the relative levels of Ren-1 and Ren-2 transcripts vary widely in different tissues. These results support the hypothesis of a local renin-angiotensin system in multiple extra-renal sites and imply the existence of complex mechanisms of regulation of the renin gene, previously thought to be expressed in a tissue-specific manner.
Visual responses in adult cat visual cortex depend on N-methyl-D-aspartate receptors.
Authors: Miller K D , Chapman B , Stryker M P
Published: May 2006

We have investigated the role of the N-methyl-D-aspartate (NMDA) receptor, a subtype of glutamate receptor, in the responses of cells in adult cat visual cortex. After intracortical infusion of the NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (DL-APV) for one day, iontophoretic responses to NMDA, to kainate, and to quisqualate revealed a receptor blockade specific to NMDA receptors and extending several millimeters from the cannula. In this region, neuronal responses to visual stimulation were profoundly suppressed, in a manner strongly correlated with the degree of NMDA receptor blockade. Neither NMDA receptor blockade nor activity suppression was caused by the inactive stereoisomer L-APV. Hence, we conclude that NMDA receptors make a major contribution to normal excitatory transmission in adult visual cortex.
Physical association of the 2,6-diamino-4-hydroxy-5N-formamidopyrimidine-DNA glycosylase of Escherichia coli and an activity nicking DNA at apurinic/apyrimidinic sites.
Authors: O'Connor T R , Laval J
Published: May 2006

The 2,6-diamino-4-hydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase of Escherichia coli, which is coded for by the fpg gene, excises purine bases with ring-opened imidazoles. In addition to the DNA glycosylase activity, we report that the Fapy-DNA glycosylase of E. coli has an associated activity, resistant to EDTA, that nicks DNA at apurinic/apyrimidinic (AP) sites. The levels of Fapy-DNA glycosylase and AP-nicking activity were parallel in crude lysates of E. coli HB101 harboring different plasmids constructed from the fpg gene. The fpg gene is different from the xth, nth, and nfo genes of E. coli, whose gene products also cleave DNA at AP sites. The Fapy-DNA glycosylase was purified to electrophoretic homogeneity. During this purification, the Fapy-DNA glycosylase copurified with an AP-nicking activity using chromatographic separations based on ion-exchange, molecular weight exclusion, and hydrophobicity. The cleavage at AP sites by the Fapy-DNA glycosylase left a 5'-phosphomonoester nucleotide at one terminus. In addition, DNA containing reduced AP sites was not nicked by the Fapy-DNA glycosylase. These data suggest that the mechanism of cleavage involved beta elimination. Therefore, this activity of the Fapy-DNA glycosylase nicking DNA at AP sites should be referred to as an AP lyase. The 3' terminus did not prime nick-translation by E. coli DNA polymerase I. However, the 3' terminus becomes a substrate for nick-translation if first allowed to react with calf intestine phosphatase or the E. coli exonuclease III. These data suggest that the repair of the Fapy lesion at least to some extent results in the formation of both 5'- and 3'-phosphomonoester nucleotides and the release of the deoxyribose.
Is there an excitonic interaction or antenna system in bacteriorhodopsin?
Authors: El-Sayed M. A. , Lin C. T. , Mason W. R.
Published: May 2006

The presence of a biphasic circular dichroism (CD) observed in the visible absorption spectrum of retinal in bacteriorhodopsin (bR) has been believed for many years to be due to excitonic-type interaction within the trimeric structure of the retinal in the protein membrane. In the present work, we present data and discuss previous observations that strongly suggest the absence of such an excitonic interaction. The magnetic CD spectrum of the trimer is found to be similar to that of the monomer and shows no sign of absorption to the doubly degenerate state predicted to be present from the exciton theory. This, together with the previous observations on the CD spectra of the photocycle intermediates of bR as well as the linear polarization studies of the fluorescence and the daughter absorption, sheds doubt on the presence of exciton interaction and thus suggests the presence of neither an antenna system nor a viable special reaction center in bR. Possible explanation for the observed biphasic nature of the CD spectrum is given in terms of having more than one type of bR with different protein conformations around the retinals in the trimer giving each similar absorption maximum but opposite signs for its rotary dispersion power.
Identification of a vitamin D-responsive protein on the surface of human osteosarcoma cells.
Authors: Shull S , Tracy R P , Mann K G
Published: May 2006

Monoclonal antibodies were elicited to membrane constituents of the osteoblastic human osteosarcoma cell line Saos-2. Two types of antibody reactivities were characterized: one group of antibodies identified fibroblastic and osteoblastic cultured cells, whereas the other group was specific for the parent cell line, Saos-2. Primary endothelial cells and hepatoma cells were not recognized by either group of antibodies. Through indirect immunofluorescent microscopy, the Saos-2-specific antigen was demonstrated to reside on the surface of these osteosarcoma cells. Metabolic radiolabeling of cultured Saos-2 cells and subsequent immunoprecipitation, electrophoretic separation, and autoradiography revealed this protein to have a Mr of 80,000. Similar experiments in the presence of hormones showed that the expression of this cell surface protein was influenced in an opposing fashion by the bone-regulating hormones parathyroid hormone and vitamin D. Vitamin D stimulated expression by 300%, whereas parathyroid hormone depressed expression by 50%. Thus, Saos-2 human osteoblastic cells demonstrate hormonal regulation through an apparently specific membrane protein.
Isolation of peroxisome-deficient mutants of Saccharomyces cerevisiae.
Authors: Erdmann R , Veenhuis M , Mertens D , Kunau W H
Published: May 2006

Two mutants of Saccharomyces cerevisiae affected in peroxisomal assembly (pas mutants) have been isolated and characterized. Each strain contains a single mutation that results in (i) the inability to grow on oleic acid, (ii) accumulation of peroxisomal matrix enzymes in the cytosol, and (iii) absence of detectable peroxisomes at the ultrastructural level. These lesions (pas1-1 and pas2) are shown to be nonallelic and recessive. Crossing of pas1-1 and pas2 strains resulted in diploid cells that had regained the ability to grow on oleic acid as sole carbon source and to form peroxisomes. These pas mutants may provide useful tools for future studies on the molecular mechanisms involved in peroxisomal assembly.
Dysfunction of chromosomal loop attachment sites: illegitimate recombination linked to matrix association regions and topoisomerase II.
Authors: Sperry A O , Blasquez V C , Garrard W T
Published: May 2006

A family of A + T-rich sequences termed MARs ("matrix association regions") mediate chromosomal loop attachment. Here we demonstrate that several MARs both specifically bind and contain multiple sites of cleavage by topoisomerase II, a major protein of the mitotic chromosomal scaffold. Interestingly, "hotspots" of enzyme cutting occur within the MAR of the mouse immunoglobulin kappa-chain gene at the breakpoint of a previously described chromosomal translocation. Since topoisomerase II can mediate illegitimate recombination in prokaryotes, we explored further the possibility that MARs might be targets for this process in eukaryotes. We found that a MAR had been deleted from one of the two rabbit immunoglobulin kappa-chain genes and that MARs reside next to a long interspersed repetitive element within the recombination junction of a human ring chromosome 21. These results, taken together with other accounts of nonhomologous recombination, lead to the proposal that a dysfunction of MARs is illegitimate recombination.
Murine complement component C4 and sex-limited protein: identification of amino acid residues essential for C4 function.
Authors: Ogata R T , Cooper N R , Bradt B M , Mathias P , Picchi M A
Published: May 2006

Murine sex-limited protein (Slp) is an isotype of murine complement component C4 that shares 95% sequence identity with C4 as well as the intramolecular thioester necessary for C4 function but has no complement activity. Slp is nonfunctional at least in part because it is not cleaved by the activated form of complement protease C1s (C1s), which proteolytically activates C4 in the classical complement pathway. Slp is also distinct from C4 in that its expression in some mouse strains is under testosterone control. In the present studies, we used site-directed mutagenesis of C4 and expression of the mutant proteins in cultured cells to identify the amino acid substitutions in Slp that are responsible for resistance to C1s cleavage. We focused on sequence changes immediately downstream of the cleavage site in C4 because the arginine at that site is conserved in Slp, but the downstream sequences diverge substantially, with six differences in the first 7 residues followed by a 3-residue deletion in Slp. We found that a C4 mutant carrying only the 3-residue deletion is not cleaved by C1s and has essentially no hemolytic activity, whereas a mutant carrying only the six replacement changes is cleaved by C1s and has normal hemolytic activity. Both mutants have intact thioesters. A third mutant in which two acidic residues in the segment deleted in Slp were replaced by aliphatic residues is also cleaved by C1s, has an intact thioester group, and has normal hemolytic activity. These results indicate that the downstream mutations are responsible for the resistance of Slp to C1s cleavage and suggest that the length rather than the specific sequence of this segment is critical in determining susceptibility to the protease.
IgE secretion by Epstein-Barr virus-infected purified human B lymphocytes is stimulated by interleukin 4 and suppressed by interferon gamma.
Authors: Thyphronitis G , Tsokos G C , June C H , Levine A D , Finkelman F D
Published: May 2006

The cytokine interleukin 4 (IL-4) has been shown to induce lipopolysaccharide-activated murine B cells to differentiate into IgE-secreting cells and to stimulate IgE secretion by cultured human peripheral blood lymphoid cells. It is unclear, however, whether this effect of IL-4 on human peripheral blood lymphoid cells is a direct effect on the B cell because IL-4 can stimulate T cells and monocytes as well as B cells and does not induce purified human B cells to secrete immunoglobulin. To investigate this issue we studied the ability of IL-4 to induce IgE secretion by purified human B cells (93-96% CD20+, less than 1% CD3+) that were cultured with Epstein-Barr virus (EBV). Although B cells cultured with IL-4 alone did not secrete Ig and B cells cultured with EBV alone secreted IgM, IgG, and IgA but less than 150 pg of IgE per ml, the combination of EBV and IL-4 induced an IgE response that ranged from 11.4 to 40.3 ng/ml of culture supernatant after 26 days of culture. While IL-4 also enhanced IgM, IgG, and IgA secretion, as well as proliferation by EBV-infected B cells, these effects were less pronounced, occurred earlier during culture, and required a lower concentration of IL-4 than did the stimulation of IgE secretion. Furthermore, interferon gamma at 10 units per ml was found to inhibit IL-4/EBV-induced IgE secretion without inhibiting the other stimulatory effects of IL-4. We conclude that (i) IL-4 and interferon gamma can act directly on polyclonally activated human B cells to respectively stimulate and suppress IgE secretion and (ii) IL-4, in addition to its specific effect on IgE secretion, has a general stimulatory effect on the growth and differentiation of EBV-infected human B cells.
Transmembrane signaling by a chimera of the Escherichia coli aspartate receptor and the human insulin receptor.
Authors: Moe G R , Bollag G E , Koshland D E
Published: May 2006

Since many receptors apparently contain only one or two membrane-spanning segments, their transmembrane topology should be similar. This feature suggests that these receptors share common mechanisms of transmembrane signaling. To test the degree of conservation of signaling properties, a chimeric receptor containing the ligand-binding extracellular domain of the Escherichia coli aspartate chemoreceptor and the cytosolic portion of the human insulin receptor was constructed. This chimeric receptor is active as a tyrosine kinase, and aspartate stimulates its activity. Some interesting differences are noted in the target proteins phosphorylated by the chimera compared to the wild-type insulin receptor. These results indicate that features of the signaling mechanisms used by these diverse receptors are conserved, but that interesting changes in the protein properties are caused by differences in the neighboring domains.
Altered regulation of β-amylase activity in mutants of
Arabidopsis
with lesions in starch metabolism
Authors: Caspar Timothy , Lin Tsan-Piao , Monroe Jonathan , Bernhard Werner , Spilatro Steven , Preiss Jack , Somerville Chris
Published: May 2006

Three classes of mutants of
Arabidopsis thaliana
(L.) Heynhold with alterations in starch metabolism were found to have higher levels of leaf amylase activity than the wild type when grown in a 12-hr photoperiod. This effect was dependent upon the developmental stage of the plants and was largely suppressed during growth in continuous light. The various amylolytic activities in crude extracts were separated by electrophoresis in nondenaturing polyacrylamide gels and visualized by activity staining. The increased amylase activity in the mutants was due to an up to 40-fold increase in the activity of an extrachloroplast β-amylase (EC 3.2.1.2). These observations indicate the existence of a regulatory mechanism that controls the amount of β-amylase activity in response to fluctuations in photosynthetic carbohydrate metabolism. It is paradoxical that β-amylase appears to be a highly regulated enzyme, but as yet no physiologically relevant function can be assigned to this enzyme due to the absence of starch in the cytoplasmic compartment of leaf cells.
Tumor-elicited polymorphonuclear cells, in contrast to "normal" circulating polymorphonuclear cells, stimulate invasive and metastatic potentials of rat mammary adenocarcinoma cells.
Authors: Welch D R , Schissel D J , Howrey R P , Aeed P A
Published: May 2006

Circulating polymorphonuclear cell (PMN) levels rise in proportion to the metastatic potential of the tumor in 13762NF mammary adenocarcinoma tumor-bearing rats. These tumor-elicited PMNs (tcPMNs) secrete high levels of the basement-membrane-degrading enzymes, type IV collagenase and heparanase, suggesting that metastatic tumor cells stimulate neutrophilia so that the tcPMNs might assist tumor cell extravasation during metastasis. To test this hypothesis, purified proteose peptone-elicited PMNs from peritoneal exudate, circulating normal PMNs, and tcPMNs were evaluated for their effects on in vitro invasive and in vivo metastatic potentials of syngeneic 13762NF mammary adenocarcinoma tumor cells. tcPMNs caused a dose-dependent increase in invasion through a reconstituted basement membrane barrier in an in vitro invasion assay. At PMN:tumor cell ratios of 30:1, invasion potential significantly (P less than 0.05) rose to 26-fold, 40-fold, and 37-fold for poorly metastatic MTLn2 cells, highly metastatic MTLn3 cells, and moderately metastatic MTF7 cells, respectively. In contrast, purified proteose peptone-elicited PMNs and circulating normal PMNs did not significantly alter invasive potential. Intravenous coinjections of purified proteose peptone-elicited PMNs did not change the number of experimental lung metastases, but tcPMNs at ratios to 50:1 significantly raised the mean number of metastases 23-fold for MTLn2, 3- to 4-fold for MTLn3, and 1.6- to 1.8-fold for MTF7. These results demonstrate that tcPMNs contribute to the metastatic propensity of mammary adenocarcinoma clones by increasing efficiency of invasion through basement membrane.
Tn5supF, a 264-base-pair transposon derived from Tn5 for insertion mutagenesis and sequencing DNAs cloned in phage lambda.
Authors: Phadnis S H , Huang H V , Berg D E
Published: May 2006

We constructed a derivative of transposon Tn5 called Tn5supF for insertion mutagenesis and sequencing DNAs cloned in phage lambda. This element carries a supF amber-suppressor tRNA gene. Its insertion into lambda can be selected by plaque formation by using nonsuppressing (sup0) Escherichia coli for amber mutant lambda phage and sup0 dnaB-amber E. coli for nonamber lambda phage. Tn5supF is just 264 base pairs long. It transposes efficiently and inserts quasi-randomly into DNA targets. The unique sequences near its termini can be used as primer binding sites for dideoxynucleotide DNA sequencing, thus permitting the direct sequencing of DNAs cloned in phage lambda without subcloning.
Reordering of nine exons is necessary to form a functional actin gene in Oxytricha nova.
Authors: Greslin A F , Prescott D M , Oka Y , Loukin S H , Chappell J C
Published: May 2006

During the development of a macronucleus from a micronucleus after cell mating in hypotrichs all the genes (approximately 20,000) are excised from micronuclear chromosomes as individual small DNA molecules. Telomeres are added to the ends of each gene-sized molecule and each is amplified, mostly by approximately 1000-fold, to yield a transcriptionally active macronucleus. As a part of the study of the excision of genes from chromosomes, we have cloned six fragments of chromosomal DNA from Oxytricha nova, each containing a full copy of an actin gene, for comparison with the structure of the actin-encoding DNA molecule in the macronucleus. All six micronuclear actin clones had the same overall organization as judged by restriction mapping. Two micronuclear actin clones were sequenced. These differ from one another at a few nucleotide positions but both prescribe precisely the same actin polypeptide. Both micronuclear actin genes contain nine exons separated by eight intron-like sequences. The macronuclear gene contains these nine exons without intron-like segments. Assigning the order 1 through 9 to the nine micronuclear exons, the order in the macronucleus is 8-7-1-2-4-3-5-9-6. In the micronuclear actin gene, all nine exons possess terminal repeat sequences. These repeat sequences provide precise directions for reordering and joining of the nine exons to yield the exon order in the macronuclear gene. Polymerase chain reaction analysis of micronuclear DNA of the related species, Oxytricha trifallax, shows that the actin gene has an unorthodox arrangement in this species also.
Influence of Mycobacterium leprae and its soluble products on the cutaneous responsiveness of leprosy patients to antigen and recombinant interleukin 2.
Authors: Kaplan G , Sampaio E P , Walsh G P , Burkhardt R A , Fajardo T T , Guido L S , de Miranda Machado A , Cellona R V , Abalos R M , Sarno E N
Published: May 2006

Experiments were carried out in the skin of patients with leprosy to examine whether suppressor cell populations either exist in the skin of multibacillary lepromatous leprosy patients, can be activated with antigen, or are induced to emigrate into a cutaneous site from the circulation. For this purpose, purified protein derivative of tuberculin, a delayed-type antigen that generates a cell-mediated immune response, was introduced into the skin alone or with nonviable Mycobacterium leprae bacilli. Areas of induration and the resulting numbers and phenotypes of emigratory cells were not influenced by M. leprae and its products. Further studies examined the ability of M. leprae and its soluble products to modify the cutaneous response to intradermal injection of recombinant interleukin 2 (IL-2), a lymphokine that mimics a cell-mediated response. Neither the simultaneous injection of M. leprae and IL-2, nor the prior injection of M. leprae followed in 2 days by IL-2, nor the prior administration of IL-2 followed in 4 days by M. leprae, into the same skin site, modified the zone of induration generated by IL-2. In addition, the immunocytochemical and histopathological evaluation of biopsy specimens of skin sites showed no difference between sites injected with IL-2 and sites injected with IL-2 and M. leprae. We conclude that suppressor T cells, if they exist, do not influence the gross or microscopic responsiveness of a cell-mediated skin reaction to antigen and IL-2. IL-2 did, however, enhance the responsiveness of skin-test-positive tuberculoid patients and family contacts to M. leprae antigens by a synergistic effect on the zone of induration and local cell accumulation.
The alpha 3 beta 3 complex, the catalytic core of F1-ATPase.
Authors: Miwa K , Yoshida M
Published: May 2006

The alpha 3 beta 3 complex was reconstituted from alpha and beta subunits of the thermophilic bacterium PS3 F1-ATPase (TF1) and then isolated. It is less stable at high and low temperatures than TF1, and the complex dissociates into subunits during native polyacrylamide gel electrophoresis. The alpha 3 beta 3 complex has about 20% of the ATPase activity of TF1. Its enzymic properties are similar to those of the native TF1, exhibiting similar cooperative kinetics as a function of ATP concentration, similar substrate specificity for nucleotide triphosphates, and the presence of two peaks in its temperature-activity profile. Differing from TF1, the ATPase activity of the alpha 3 beta 3 complex is insensitive to N3- inhibition, its divalent cation specificity is less stringent, and its optimum pH shifts to the alkaline side. The addition of the gamma subunit to the alpha 3 beta 3 complex leads to the formation of the alpha 3 beta 3 gamma complex, indicating that the alpha 3 beta 3 complex is an intermediate in the process of assembly of the holoenzyme from each subunit. These results definitely show that the essential structure for eliciting the ATPase activity of F1-ATPase is trimeric alpha beta pairs and that the kinetic cooperativity of the F1-ATPase is an inherent property of this trimeric structure but is not due to the presence of single-copy subunits. In this sense, the alpha 3 beta 3 complex is the catalytic core of F1-ATPase.
Isolation and expression of the Pneumocystis carinii thymidylate synthase gene.
Authors: Edman U , Edman J C , Lundgren B , Santi D V
Published: May 2006

The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar to TS from other organisms and is most closely related to the enzyme from Saccharomyces cerevisiae with 65% identity. TS is found on a 330-kilobase-pair chromosome in P. carinii. While TS and dihydrofolate reductase reside on a single polypeptide chain in all protozoa studied to date, TS is not linked to dihydrofolate reductase in P. carinii. The TS gene shows the presence of four small intervening sequences, some of which interrupt the coding sequence in highly ordered structural regions of the protein. Heterologous expression of P. carinii TS in E. coli was accomplished by cloning the coding sequence into plasmid vectors under control of the lac and tac promoters. These constructs direct the synthesis of catalytically active enzyme to the extent of 2% of total soluble protein.