bluuebunny/crossref_metadata_2025 huggingface dataset:
Penicillin binding proteins: role in initiation of murein synthesis in Escherichia coli.
Authors: de la Rosa E J , de Pedro M A , Vázquez D
Published: May 2006

The consequences of the specific inhibition of penicillin binding proteins (Pbps) by beta-lactam antibiotics immediately before resumption of active growth in Escherichia coli suggest that inhibition of murein biosynthesis does not prevent the earlier steps of the initiation of cell growth in mass. The activity of Pbp 2 is apparently critical for the initiation of murein biosynthesis. Provided that Pbp 2 remains active, the other Pbps (1a, 1b, 3, 4, 5, and 6) can be inhibited without any noticeable effect on the initial rate of incorporation of new precursors into macromolecular peptidoglycan. These precursors are, in addition, inserted with a high degree of cross-linkage.
Ia-transfected L-cell fibroblasts present a lysozyme peptide but not the native protein to lysozyme-specific T cells.
Authors: Shastri N , Malissen B , Hood L
Published: May 2006

We studied the antigen-presenting capacity of mouse L fibroblasts transfected with genes encoding Ia polypeptides of the major histocompatibility complex (MHC). These cells function as efficient antigen-presenting cells (APC) in stimulating peptide antigen-specific MHC-restricted proliferation of long-term T-cell lines, thus establishing the capacity of Ia-expressing L-cell transfectants to present antigens to apparently normal T cells. However, in contrast to splenic APC, L-cell transfectants fail to present native hen egg-white lysozyme to the same T cells. Since this result is similar to that obtained with physiologic APC pretreated to prevent antigen degradation, it suggests that L-cell transfectants, without such pretreatments, may be compromised in their ability to process native lysozyme. However, since such transfectant cells have been shown to present other complex polypeptides such as keyhole limpet hemocyanin, a random copolymer of glutamic acid, alanine, and tyrosine, and influenza virus neuraminidase, this observation suggests that protein antigens differ in the stringency of processing requirements.
Complete cDNA sequence of the fourth component of murine complement.
Authors: Sepich D S , Noonan D J , Ogata R T
Published: May 2006

We have used the method of Okayama and Berg to construct cDNA clones that span the entire length of the fourth component of murine complement (C4) from mouse strain B10.WR. The cDNA sequence spans 5372 nucleotides and encodes a pre-pro-C4 molecule 1738 amino acids in length; it includes 56 and 99 untranslated bases at the 5' and 3' ends, respectively, of the C4 mRNA. The deduced pre-pro-C4 molecule includes a 19 amino acid signal peptide and two highly basic interchain regions that presumably are excised during maturation of the protein. The nucleotide sequence shows 79% identity with the human C4 cDNA sequence over the length of the protein-coding region. A comparison of the B10.WR C4 sequence with those of C4 and sex-limited protein (Slp) from mouse strain FM (i) suggests that the difference in hemolytic activity between the C4 proteins from B10.WR and FM is due to structural changes distant from both the C1 cleavage site and the internal thioester site and (ii) raises the possibility that the C4 gene from B10.WR has undergone genetic exchange with its adjoining Slp genes.
Contrast and assimilation in the perception of brightness.
Authors: Shapley R , Reid R C
Published: May 2006

The rapid estimation of the brightness of objects is one of the nervous system's major visual tasks. Exactly how the eye and brain perform this basic task is still not understood. Two mechanisms that contribute to human perception of the brightness of objects have been identified previously: (i) the visual response to physical contrast and (ii) assimilation. Use of a unique visual display device allowed us to measure the relative importance of these two mechanisms. The present results reveal that assimilation is about half as effective as physical contrast in determining the apparent brightness of objects. These results imply that previous theories of vision--for instance, the retinex theory--will have to be revised; the importance of physical contrast must be weighted more strongly.
Acyl-acyl carrier protein as a source of fatty acids for bacterial bioluminescence
Authors: Byers David M. , Meighen Edward A.
Published: May 2006

Pulse-chase experiments with [
3
H]tetradecanoic acid and ATP showed that the bioluminescence-related 32-kDa acyltransferase from
Vibrio harveyi
can specifically catalyze the deacylation of a
3
H-labeled 18-kDa protein observed in extracts of this bacterium. The 18-kDa protein has been partially purified and its physical and chemical properties strongly indicate that it is fatty acyl-acyl carrier protein (acyl-ACP). Both this
V. harveyi
[
3
H]acylprotein and [
3
H]palmitoyl-ACP from
Escherichia coli
were substrates
in vitro
for either the
V. harveyi
32-kDa acyltransferase or the analogous enzyme (“34K”) from
Photobacterium phosphoreum
. TLC analysis indicated that the hexane-soluble product of the reaction is fatty acid. Phosphate ions and, to a lesser extent, organic alcohols stimulated the rate of acyl-protein cleavage. No significant cleavage of either
E. coli
or
V. harveyi
tetradecanoyl-ACP was observed in extracts of these bacteria unless the 32-kDa or 34K acyltransferase was present. Since these enzymes are believed to be responsible for the supply of fatty acids for reduction to form the aldehyde substrate of luciferase, the above results suggest that long-chain acyl-ACP is the source of fatty acids for bioluminescence.
The propeptide of rat bone gamma-carboxyglutamic acid protein shares homology with other vitamin K-dependent protein precursors.
Authors: Pan L C , Price P A
Published: May 2006

The molecular cloning of bone gamma-carboxyglutamic acid (Gla) protein (BGP; osteocalcin) was accomplished by constructing a phage lambda gt11 cDNA library from the rat osteosarcoma cell line ROS 17/2 and screening this library with antibodies raised against BGP from rat bone. By sequencing several cloned cDNAs, we have established a 489-base-pair sequence that predicts a mature BGP of 50 amino acid residues with an NH2-terminal extension of 49 residues. The leader peptide consists of a hydrophobic signal peptide followed by a basic propeptide of 26 or 27 residues that is cleaved after an Arg-Arg dipeptide prior to secretion from the cell. Mature rat BGP is extremely homologous to BGPs from other species except for its COOH-terminal sequence. A stretch of 9 residues proximal to the NH2 terminus of secreted BGP is strikingly similar to the corresponding regions in known propeptides of the gamma-carboxyglutamic acid-containing blood coagulation factors. We suggest that this common structural feature may be involved in the posttranslational targeting of these polypeptides for vitamin K-dependent gamma-carboxylation.
Interaction of endothelial cell growth factor with heparin: characterization by receptor and antibody recognition.
Authors: Schreiber A B , Kenney J , Kowalski W J , Friesel R , Mehlman T , Maciag T
Published: May 2006

Endothelial cell growth factor (ECGF) binds specifically in vitro to membrane receptors present on the surface of several cell types, including murine and human endothelial cells and fibroblasts. Monoclonal antibodies prepared against ECGF that inhibit the mitogenic activity of the growth factor prevent receptor occupancy by the ligand. Heparin interacts structurally with ECGF [Maciag, T., Mehlman, T., Friesel, R. & Schreiber, A. B. (1984) Science 225, 932-935], potentiates the mitogenic activity of the polypeptide, restores the biological activity to inactivate ECGF, enhances the affinity of the ligand to cell surface receptors, and modifies antibody recognition of ECGF. These data suggest that the association between heparin and ECGF induces a conformational change in the polypeptide that increases or stabilizes the biological activity of the mitogen.
Recycling of epidermal growth factor in a human pancreatic carcinoma cell line.
Authors: Korc M , Magun B E
Published: May 2006

PANC-1 human pancreatic carcinoma cells readily bound and internalized 125I-labeled epidermal growth factor (EGF). Bound 125I-labeled EGF was then partially processed to a number of high molecular weight acidic species. Percoll gradient centrifugation of cell homogenates indicated that the majority of 125I activity localized to several intracellular vesicular compartments. Both intact EGF and its processed species were subsequently released into the incubation medium. A major portion of the released radioactivity was capable of rebinding to the cell. Only a small amount of bound 125I-labeled EGF was degraded to low molecular weight products, and this degradation was completely blocked by methylamine. This lysosomotropic compound did not arrest either the generation or the extrusion of the major high molecular weight species of processed EGF (pI 4.2). These findings suggest that in PANC-1 cells, bound EGF undergoes only limited processing. Both intact EGF and its major processed species bypass the cellular degradative pathways, are slowly released from the cell, and then rebind to the cell.
Variable numbers of pepsinogen genes are located in the centromeric region of human chromosome 11 and determine the high-frequency electrophoretic polymorphism.
Authors: Taggart R T , Mohandas T K , Shows T B , Bell G I
Published: May 2006

A panel of 26 mouse-human somatic cell hybrids containing different human chromosome complements was analyzed with a cloned human pepsinogen cDNA probe to determine the chromosomal location and the number of genes encoding these proteins. A complex containing variable numbers of pepsinogen genes was localized to the centromeric region of human chromosome 11 (p11----q13). Examination of somatic cell hybrids containing single copies of chromosome 11 and the corresponding human parental cell lines revealed a restriction fragment length polymorphism determined by pepsinogen haplotypes that contained two or three genes, respectively. Concurrent studies of DNA from individuals exhibiting the most common pepsinogen electrophoretic phenotypes with exon-specific probes demonstrated that the absence of one gene among the different restriction fragment patterns correlated with the absence of one specific isozymogen (Pg 5). Thus, our studies demonstrate that this genetic polymorphism involving intensity variation of individual pepsinogen isozymogens results from chromosome haplotypes that contain different numbers of genes. The regional localization of this polymorphic gene complex will facilitate detailed linkage analysis of human chromosome 11.
Interleukin 2 promotes growth and cytolytic activity in human T3+4-8- thymocytes.
Authors: de la Hera A , Toribio M L , Marquez C , Martinez C
Published: May 2006

Human thymocytes bearing T3 but neither T4 nor T8 antigens (T3+4-8- cells) were obtained after negative selection of thymocytes, either fresh or cultured in medium containing recombinant interleukin 2 (IL-2), by treatment with Na1/34, OKT4A and B9.4 monoclonal antibodies (which recognize T6, T4, and T8 antigens, respectively) and complement. Quantitative flow cytometry showed a 98% pure population of T3+4-8- lymphocytes, which included proliferating cells. The growth and maturation requirements of these thymocytes were characterized and related to the T3-receptor complex and IL-2 pathways, thought to be used by mature lymphocytes. The results show that addition of recombinant IL-2 promotes, in a dose-dependent way, proliferation and acquisition of effector functions by cultured T3+4-8- thymocytes, the growth being inhibitable by monoclonal antibody 33B73 (anti-Tac). Furthermore, cytolytic activity of T3+4-8- cells induced by recombinant IL-2 is specifically blocked by monoclonal antibody OKT3, showing that it operates via the T3-receptor complex and does not require either T4 or T8 molecules. The finding of in vitro responsiveness to recombinant IL-2 in T3+4-8- thymocytes suggests a role of IL-2 in the growth and maturation of cells committed to the T-cell lineage, during intrathymic differentiation, prior to expression of T4 and T8 molecules.
Monoclonal antiidiotypic antibodies related to a murine oncofetal bladder tumor antigen induce specific cell-mediated tumor immunity.
Authors: Lee V K , Harriott T G , Kuchroo V K , Halliday W J , Hellström I , Hellström K E
Published: May 2006

Rat monoclonal antibody 6.10 recognizes a 175-kDa protein expressed in all BALB/c mouse transitional cell bladder carcinomas tested, in epithelial cells of the mouse embryo, and in a few epithelial cells of adult mice. The antibody was used as an immunogen to generate two mouse monoclonal antibodies, 21D9 and 43A10, which bind to idiotopes on antibody 6.10 associated with the binding site for the 175-kDa antigen. The antiidiotypic antibodies induced bladder tumor-specific, cell-mediated immunity when injected into syngeneic mice, as shown by delayed-type hypersensitivity reactions in vivo and leukocyte adherence inhibition reactions in vitro. Tumor specificity was demonstrated by employing as controls a chemically induced BALB/c fibrosarcoma, MCA-1511 (MCA, 3-methylcholanthrene), and its corresponding antiidiotypic antibody, 5.96. Lymphocytes from mice sensitized with antibody 21D9 or 5.96 specifically recognized antigens in extracts of BALB/c bladder carcinoma BTCC-1660 (BTCC, bladder transitional cell carcinoma) and sarcoma MCA-1511, respectively, as shown by leukocyte adherence inhibition reactivity. This reactivity was selectively abrogated by prior treatment of the sensitized cells with the appropriate antiidiotypic antibodies and complement. An antigen recognized in vitro by antibody 21D9-sensitized lymphocytes could be separated from BTCC-1660 extract by immunoabsorption with antibody 6.10 and elution with acidic buffer. Our findings indicate that the oncofetal antigen defined by antibody 6.10 is recognized by the immune system of syngeneic mice and suggest that antiidiotypic antibodies related to certain oncofetal antigens can be used to immunize against syngeneic tumors.
Angiotensin II receptor binding sites in brain microvessels.
Authors: Speth R C , Harik S I
Published: May 2006

We assessed the specific binding of 125I-labeled angiotensin II (125I-Ang II) to particulate fractions of the cerebral cortex and cerebellum and to microvessels obtained by bulk isolation from these two brain regions in the dog. 125I-Ang II binds to cerebral and cerebellar microvessels in a specific, saturable, and reversible manner and with high affinity (dissociation constant about 1 nM). Maximal binding of 125I-Ang II to brain microvessels was about 2-fold higher than the maximal binding to particulate fractions of the cerebellum and more than 15-fold higher than that of the cerebral cortex. No significant differences were noted between cerebral and cerebellar microvessels in their specific binding of Ang II. Furthermore, our finding that analogues of Ang II displace specific 125I-Ang II binding to brain microvessels in a rank order that correlates with their pharmacological activities confers biological relevance on the ligand-binding studies. These results strongly suggest that specific Ang II receptor binding sites are present in brain microvessels. Such Ang II receptors may have an important role in regulating the microcirculation of the brain.
The "beta-like-globin" gene domain in human erythroid cells.
Authors: Tuan D , Solomon W , Li Q , London I M
Published: May 2006

We have mapped the distribution of the major and minor DNase I-hypersensitive sites in the human "beta-like-globin" gene domain. The minor DNase I-hypersensitive sites map close to the 5' end of each of the beta-like-globin genes. Their presence is specifically associated with the transcription of the immediate downstream beta-like-globin genes. The major DNase I-hypersensitive sites map in what appear to be the 5' and 3' boundary areas of the human beta-like-globin gene domain, a region estimated to span at least 90 kilobases of DNA. These major sites are present in various erythroid cells, which express predominantly either the embryonic, the fetal, or the adult beta-like-globin genes, and seem to be involved in defining the active beta-like-globin genes domain in cells of erythroid lineage. The four major DNase I-hypersensitive sites in the 5' boundary area, when correlated with sequencing data, are shown to be located in DNA regions containing enhancer core-like sequences and alternating purine and pyrimidine bases.
Isolation of the human insulin-like growth factor genes: insulin-like growth factor II and insulin genes are contiguous.
Authors: Bell G I , Gerhard D S , Fong N M , Sanchez-Pescador R , Rall L B
Published: May 2006

Overlapping recombinant clones that encompass the insulin-like growth factor (IGF) I and II genes have been isolated from a human genomic DNA library. Each gene is present once per haploid genome; the IGF-I gene spans greater than 35 kilobase pairs (kbp) and the IGF-II gene is at least 15 kbp. The exon-intron organization of these genes is similar, each having four exons, which is one more than the related insulin gene. Comparison of the restriction endonuclease cleavage maps of the IGF-II and insulin genes, including their flanking regions and hybridization with an IGF-II cDNA probe, revealed that they are adjacent to one another. The IGF-II and insulin genes have the same polarity and are separated by 12.6 kbp of intergenic DNA that includes a dispersed middle repetitive Alu sequence. The order of the genes is 5'-insulin-IGF-II-3'.
Identification of new gene products coded from X regions of human T-cell leukemia viruses.
Authors: Shimotohno K , Miwa M , Slamon D J , Chen I S , Hoshino H , Takano M , Fujino M , Sugimura T
Published: May 2006

Antibodies were raised against oligopeptides deduced from the nucleotide sequence in the conserved region located between env and the 3' long terminal repeat in human T-cell leukemia virus type I (HTLV-I) and type II (HTLV-II) to detect a protein coded from this region in virus-infected cells. Two of these antibodies precipitated a protein of 41 kilo-daltons in HTLV-I-infected cell lines and a protein of 38 kilo-daltons in HTLV-II-infected cells. The protein in HTLV-I-infected cells was precipitated by plasma from patients with adult T-cell leukemia but not by plasma from a normal adult. These results indicate that these proteins were translated from new coding regions (X) present in HTLV-I and HTLV-II.
Prediction of homology and divergence in the secondary structure of polypeptides.
Authors: Pongor S , Szalay A A
Published: May 2006

A quantitative procedure is described for the comparison of secondary structure of homologous proteins. Standard predictive methods are used to generate probability profiles from pairs of homologous amino acid sequences; correlation coefficients (R) are then computed between each pair of amino acids for alpha-helix (R alpha), extended structure (R beta), turn (R(t)), and coil (R(c)). R values are >0.2 for correctly aligned homologous sequences. Unrelated or incorrectly aligned sequences give R values near zero. Lack of correlation for a segment of otherwise well-correlated sequences is used to identify structural divergence, which is then evaluated graphically by using difference profiles. A combination of these techniques correctly predicts secondary structural differences between melittin or beta-endorphin and their respective synthetic analogs. The method is potentially useful to describe evolutionary changes in protein secondary structure as well as in the design of peptide analogs.
Combining solid-state and solution-state 31P NMR to study in vivo phosphorus metabolism.
Authors: Cholli A L , Yamane T , Jelinski L W
Published: May 2006

Otherwise unavailable information concerning the distribution of phosphorylated compounds in biological systems is obtained by a combined solid-state/solution-state NMR approach, illustrated here for oocytes from Rana pipiens. General methodology is developed, and further extensions are proposed. The following conclusions pertain to the specific system under examination. (i) Nucleoside phosphates can be observed by magic-angle sample spinning of the lyophilized material. (ii) The solid-state NMR technique of dipolar decoupling provides no additional resolution of the phospholipid and phosphoprotein components of the yolk. However, cellular death produces sufficient pH changes to cause the phospholipid and protein phosphate peaks to become resolvable. The concentration of nucleoside phosphates also decreases. (iii) The phospholipid and phosphoprotein components are shown by computer simulation to be present in a ratio of 40:60, respectively. (iv) The amounts of inorganic phosphate, nucleoside phosphates, and sugar phosphates are determined by solution-state NMR observation of the perchloric acid extract of the oocytes.
Phase-change kinetics for a microtubule with two free ends.
Authors: Hill T L
Published: May 2006

The two-phase macroscopic kinetic model of the end of a microtubule is extended to microtubules in solution, with two free ends. The theoretical treatment of this system is complicated by the possibility of microtubules shortening all the way to disappearance. Another possibility, if a microtubule is shortening from one end only (and has a GTP cap on the other end), is that completed shortening will leave a residual cap from which growth can then take place at both ends. Two approximations are introduced.
Muscarinic cholinergic receptor in the human heart evidenced under physiological conditions by positron emission tomography.
Authors: Syrota A , Comar D , Paillotin G , Davy J M , Aumont M C , Stulzaft O , Maziere B
Published: May 2006

The muscarinic receptor was studied in vivo in the human heart by a noninvasive method, positron emission tomography (PET). The study showed that the binding sites of 11C-labeled methiodide quinuclidinyl benzilate [( 11C]-MQNB), a muscarinic antagonist, were mainly distributed in the ventricular septum (98 pmol/cm3 of heart) and in the left ventricular wall (89 pmol/cm3), while the atria were not visualized. A few minutes after a bolus intravenous injection, the concentration of [11C]MQNB in blood fell to a negligible level (less than 100th of the concentration measured in the ventricular septum). When injected at high specific radioactivity, the concentration of [11C]MQNB in the septum rapidly increased and then remained constant with time. This result was explained by rebinding of the ligand to receptors. It was the major difference observed between the kinetics of binding of [11C]MQNB to receptor sites after intravenous injection in vivo and that of [3H]MQNB to heart homogenates in vitro. The MQNB concentrations in the ventricular septum of different individuals were found to be highest when the heart rate at the time of injection was slow. This result suggests that the antagonist binding site is related to a low-affinity conformational state of the receptor under predominant vagal stimulation. Thus, positron emission tomography might be the ideal method to study the physiologically active form of the muscarinic acetylcholine receptor in man.
Phorbol ester effects on neurotransmission: interaction with neurotransmitters and calcium in smooth muscle.
Authors: Baraban J M , Gould R J , Peroutka S J , Snyder S H
Published: May 2006

Stimulation of the phosphatidylinositol cycle by neurotransmitters generates diacylglycerol, an activator of protein kinase C, which may regulate some forms of neurotransmission. Phorbol esters, potent inflammatory and tumor-promoting compounds, also activate protein kinase C. We demonstrate potent and selective effects of phorbol esters on smooth muscle, indicating a role for protein kinase C in neurotransmission. In rat vas deferens and dog basilar artery, phorbol esters synergize with calcium to mimic the contractile effects of neurotransmitters that act through the phosphatidylinositol cycle. In guinea pig ileum and rat uterus, phorbol esters block contractions produced by these neurotransmitters.
Complete coding sequence of rat tyrosine hydroxylase mRNA.
Authors: Grima B , Lamouroux A , Blanot F , Biguet N F , Mallet J
Published: May 2006

Several clones specific for tyrosine hydroxylase [tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] have been identified from a rat PC12 library by using the previously characterized clone pTH-1. The most complete of these, pTH-51, is 1758 base pairs long and covers most of the length of the mRNA, including the entire coding and 3' untranslated region. The polypeptide has an estimated molecular weight of 55,903 and some of its characteristic features are discussed.
Effect of DNA polymerase I and DNA helicase II on the turnover rate of UvrABC excision nuclease.
Authors: Husain I , Van Houten B , Thomas D C , Abdel-Monem M , Sancar A
Published: May 2006

UvrABC excision nuclease (UvrA, UvrB, and UvrC proteins) of Escherichia coli removes nucleotide mono- and diadducts from DNA in the form of oligonucleotides 12 or 13 bases long. We find that the purified enzyme dissociates from DNA very slowly, if at all, in the absence of other proteins implicated in excision repair. Addition of DNA polymerase I and helicase II (UvrD protein) to the reaction mixture stimulates the turnover rate of the excision nuclease to a level comparable to that observed in vivo.
Human apolipoprotein B cDNA clone isolation and demonstration that liver apolipoprotein B mRNA is 22 kilobases in length.
Authors: Huang L S , Bock S C , Feinstein S I , Breslow J L
Published: May 2006

An expression library made in plasmids pUC8 and pUC9 with mRNA derived from the human hepatoma cell line HepG2 was screened with a rabbit antiserum to human low density lipoprotein (LDL). Approximately 12,000 clones were screened and five positives were identified. The cDNA inserts were all 1500-1600 base pairs in length. The insert from one clone, pB8, was isolated, labeled by nicktranslation, and found to cross-hybridize strongly with the other four cDNA clones. The pB8 clone produces a fusion protein of approximately equal to 37.5 kDa that reacts in electrophoretic transfer blot analysis with rabbit anti-human LDL. This reactivity can be abolished by pretreatment of the antiserum with purified human LDL, p = 1.025 - 1.050 g/ml. A pB8-derived probe was used to demonstrate that apolipoprotein B (apo B) mRNA is present in HepG2 cells and liver extracts but not in HeLa cells or extracts from small intestine, heart, aorta, spleen, brain, skeletal muscle, lung, kidney, or ovary. RNA transfer blot analysis revealed that HepG2 cell apo B mRNA was approximately equal to 22 kilobases in length. These cDNA clones should allow the isolation of the apo B gene and ultimately the elucidation of the primary structure of this protein.
Microtubule distribution in cultured cells and intact tissues: improved immunolabeling resolution through the use of reversible embedment cytochemistry.
Authors: Gorbsky G , Borisy G G
Published: May 2006

To investigate the detailed distributions of microtubules in cultured cells and intact tissues we developed a reversible embedment method for antibody labeling of sectioned material. Fixed tissues were infiltrated with fully polymerized polymethylmethacrylate dissolved in an organic solvent. Evaporation of the solvent left the tissue embedded in hard plastic. After sectioning by conventional methods, the plastic was extracted and sections were processed for indirect immunofluorescence to label microtubules. Clear images of microtubules were observed in sections of cultured epithelial cells, intact chick intestinal epithelium, and dividing sea urchin eggs. Microtubules in the differentiated epithelium of the chick intestine generally paralleled the long axis of the cells and did not focus on a microtubule-organizing center. Mitotic cells of the intestinal epithelium appeared similar to the mitotic cells of epithelial lines in culture. In sections of dividing sea urchin eggs detailed images of spindle and astral fibers were revealed. Immunoelectron microscopic labeling for tubulin was performed on sections of Pt K1 cells using secondary antibodies adsorbed to 20-nm gold particles. Semi-thick sections viewed by high-voltage electron microscopy showed both the overall distribution of microtubules and their detailed interactions with other cellular organelles. Mitochondria were often aligned along labeled microtubules. Reversible embedment cytochemistry should provide a general method for high resolution labeling of cells and tissues with affinity probes.
Multiple forms of tubulin in the cilia and cytoplasm of Tetrahymena thermophila.
Authors: Suprenant K A , Hays E , LeCluyse E , Dentler W L
Published: May 2006

Most higher eukaryotic tubulins are separated into alpha- and beta-tubulin when electrophoresed in NaDodSO4- denaturing gels, while many lower eukaryotic tubulins are poorly resolved under these conditions, which include a stacking gel (pH 6.80) and a separating gel (pH 8.80). By lowering the pH of the separating gel to 8.25, we have found that tubulin isolated from the protozoan Tetrahymena thermophila is resolved by one-dimensional polyacrylamide gel electrophoresis into two alpha-tubulins and one beta-tubulin. Moreover, at least five alpha- and two beta-tubulin isotypes are identified in Tetrahymena by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis. Three of these alpha isotypes and one beta isotype are found specifically in ciliary microtubules, while the other two isotypes are found only in the cytoplasmic tubulin pool that was isolated and induced to self-assemble into microtubules in vitro. Peptide mapping by limited proteolytic digestion indicates that the tubulins are closely related. Possible mechanisms for the generation and selection of these tubulin isotypes are discussed.
Peptidergic modulation of patterned motor activity in identified neurons of
Helisoma
Authors: Murphy A. D. , Lukowiak Ken , Stell W. K.
Published: May 2006

The neuroactive peptides SCP
B
(small cardioactive peptide B) and FMRFamide (Phe-Met-Arg-Phe-NH
2
), both originally isolated from molluscs, have potent modulatory effects upon the production of patterned motor activity in identified neurons (e.g., B5 and B19) in the buccal ganglia of the snail
Helisoma
. Such patterned motor activity has previously been shown to underlie feeding behavior. Micromolar concentrations of SCP
B
initiate patterned motor activity in quiescent ganglia and increase the rate of activity in ganglia that are spontaneously active. Micromolar concentrations of FMRFamide inhibit patterned motor activity in
Helisoma
buccal ganglia, and 10 μM FMRFamide completely suppresses such activity. In addition, there are both anti-SCP
B
-and anti-FMRFamide-immunoreactive neurons in
Helisoma
buccal ganglia. Our results suggest that peptides may play a prominent role in the regulation of feeding behavior in
Helisoma
.
Electron microscopic observation of domain movement in reconstituted erythrocyte membranes.
Authors: He N B , Hui S W
Published: May 2006

The movement of labeled concanavalin A (Con A) receptors in reconstituted human erythrocyte membranes was observed directly in an electron microscope, using an environmental stage that kept the sample fully hydrated at all experimental temperatures. Human erythrocyte membrane ghosts were spread on the air/water interface in a Langmuir trough. The surface monolayer film contained most native proteins and lipids of the erythrocyte membrane. The Con A receptors in the film were labeled with Con A-conjugated, 25-nm-diameter gold microspheres. Unsupported bilayer membranes were reconstituted by dipping a 1000-mesh grid through the labeled surface film. The reconstituted membrane samples were observed under low beam current and photographed by timed exposures with sensitive x-ray films. The total radiation per exposure was kept below the damage threshold of 5 X 10(-4) coulomb/cm2. The Con A-gold labels were observed to move in unison within local areas (domains) of the reconstituted membrane. The size of the domains and the velocity of the labels were measured as functions of temperature. The typical domain size was 10 micron2 and the typical velocity of the labels was 7 nm/sec. The minimum domain size and velocity were found at 17 degrees-28 degrees C. Reduction of the amount of cholesterol in the precursor erythrocyte membrane caused the domain velocity at 7 degrees C to decrease and the domain size to increase; the opposite effect was observed with cholesterol enrichment. The results indicate that the components of the erythrocyte membrane tended to form moving domains and that the motion was related to lipid phase separation in the bilayer.
Nasal absorption of insulin: enhancement by hydrophobic bile salts.
Authors: Gordon G S , Moses A C , Silver R D , Flier J S , Carey M C
Published: May 2006

We demonstrate that therapeutically useful amounts of insulin are absorbed by the nasal mucosa of human beings when administered as a nasal spray with the common bile salts. By employing a series of bile salts with subtle differences in the number, position, and orientation of their nuclear hydroxyl functions and alterations in side chain conjugation, we show that adjuvant potency for nasal insulin absorption correlates positively with increasing hydrophobicity of the bile salts' steroid nucleus. As inferred from studies employing various concentrations of unconjugated deoxycholate and a constant dose of insulin, insulin absorption begins at the aqueous critical micellar concentration of the bile salt and becomes maximal when micelle formation is well established. These and other data are consistent with the complementary hypotheses that bile salts act as absorption adjuvants by producing high juxtamembrane concentrations of insulin monomers via solubilization in mixed bile salt micelles and forming reverse micelles within nasal membranes, through which insulin monomers can diffuse through polar channels from the nares into the blood stream.
Expression at the cell surface of biologically active fusion and hemagglutinin/neuraminidase proteins of the paramyxovirus simian virus 5 from cloned cDNA.
Authors: Paterson R G , Hiebert S W , Lamb R A
Published: May 2006

cDNAs encoding the mRNAs for the fusion protein (F) and the hemagglutinin/neuraminidase protein (HN) of the paramyxovirus simian virus 5 have been inserted into a eukaryotic expression vector under the control of the simian virus 40 late promoter. The F and HN proteins synthesized in recombinant infected cells are indistinguishable in terms of electrophoretic mobility and glycosylation from the proteins synthesized in simian virus 5-infected cells. In addition, the expressed F and HN proteins have been shown to be anchored in the plasma membrane in a biologically active form by indirect live cell immunofluorescence, the F-mediated formation of syncytia, and the ability of HN to cause the hemadsorption of erythrocytes to the infected cell surface.
Expression of hepatitis B surface antigen with a recombinant adenovirus.
Authors: Davis A R , Kostek B , Mason B B , Hsiao C L , Morin J , Dheer S K , Hung P P
Published: May 2006

Early region 1 of the adenovirus type 5 genome was replaced with a DNA sequence containing the gene coding for the hepatitis B surface antigen (HBsAg) flanked by the major late promoter from adenovirus 2 and processing and polyadenylylation signals from simian virus 40. In one type of hybrid virus only the adenovirus 2 major late promoter, including just 33 base pairs of the adenovirus type 2 tripartite leader, preceded the coding region of the HBsAg gene. In another, this region was preceded by both the adenovirus major late promoter and almost the entire tripartite leader. The structure of the substituted sequence in each of the recombinant viral DNAs was identical to that in the plasmids used to construct the viruses. Approximately equivalent amounts of HBsAg-specific mRNA were produced late in infection with each recombinant virus. Although HBsAg production was detected late in infection of the hybrid virus not containing the full tripartite leader sequence, its level was 1/70th of that obtained with the hybrid virus containing this sequence. One likely interpretation is that the presence of the tripartite leader at the 5' end of this mRNA is critical for the synthesis of HBsAg polypeptide in the late stage of infection. HBsAg produced upon infection with the hybrid adenoviruses was glycosylated and secreted into the culture medium as particles that were essentially indistinguishable from the 22-nm particles found in human serum.
Mobilization of specific maternal RNA species into polysomes after fertilization in Xenopus laevis.
Authors: Dworkin M B , Shrutkowski A , Dworkin-Rastl E
Published: May 2006

Unfertilized eggs of many species contain large amounts of maternal mRNA that are used to support protein synthesis during the first few hours of development, before the onset of embryonic transcription. We have examined the accumulation of nonpolysomal maternal RNAs in polysomes after fertilization in Xenopus laevis by measuring the distributions of specific sequences in nonpolysomal and polysomal fractions. In an arbitrary selection of 18 maternal sequences that are largely nonpolysomal in the full-grown oocyte, 13 became enriched in polysomes by the 16-cell cleavage stage. One sequence accumulated only 50% in polysomes at this time, while four sequences became polysomal later than the 16-cell stage. Several RNA sequences decreased in titer during early embryogenesis and were rare during organogenesis. Sequences that are mobilized rapidly and efficiently into polysomes shortly after fertilization and whose cellular concentrations are highest in embryos before organogenesis may provide genetic information for developmental functions restricted to very early embryogenesis. These experiments serve to identify such sequences in Xenopus.
Genes from plasmid pKM101 in Haemophilus influenzae: separation of functions of mucA and mucB.
Authors: Balganesh M , Setlow J K
Published: May 2006

Haemophilus influenzae, normally not mutable by UV, became UV mutable with a recombinant plasmid insertion. A 7.8-kilobase-pair (kbp) fragment of the plasmid pKM101 containing the mucA and mucB genes was ligated to the shuttle vector pDM2, and a Rec- strain of H. influenzae was transformed with the ligated mixture. All of the transformants, unlike the parent Rec- strain, were resistant to UV, could carry out postreplication repair and Weigle reactivation, showed greatly increased spontaneous mutation, and contained a plasmid carrying an insert of only 1.2 rather than 7.8 kbp. This plasmid in a umuC mutant strain of Escherichia coli complemented a pKM101 derivative lacking mucA function but with an intact mucB gene, although there was no complementation with a mucA+ mucB- plasmid, suggesting that the newly constructed plasmid coded for the mucA protein; this is in accord with the restriction analysis and hybridization between the plasmid and a probe containing all of the mucA gene but only a small fraction of mucB. When one of the H. influenzae Rec- transformants lost the plasmid, the resistance to UV was retained but the high spontaneous mutation and UV mutability were not. The fact that there was hybridization between the chromosome of the "cured" strain and a probe containing both muc genes but none when almost no mucB was present suggested that at least part of the mucB gene had been integrated into the Rec- chromosome. Five different postreplication repair-proficient strains became UV mutable and had high spontaneous mutation rates caused by the putative mucA plasmid, indicating that these strains already possessed a chromosomal equivalent of the mucB gene.
Ontogeny of electrically excitable cells in cultured olfactory epithelium.
Authors: Schubert D , Stallcup W , LaCorbiere M , Kidokoro Y , Orgel L
Published: May 2006

A primary system has been developed in which it is possible to study the production of electrically excitable neuron-like cells from a precursor population of olfactory epithelial cells. Rat nasal epithelium was dissociated and placed in culture. The initial surviving cells are flat and ciliated and contain glial fibrillary acidic protein (GFAP). After 3-5 days electrically excitable cells appear that contain neuron-specific enolase but not GFAP. These round cells originate by means of the differentiation of the GFAP-positive flat cell to a round cell, followed by the division of the round cell. Therefore, neuron-like cells can be derived from cells that synthesize GFAP.
Identification of the porcine intestinal 1,25-dihydroxyvitamin D3 receptor on sodium dodecyl sulfate/polyacrylamide gels by renaturation and immunoblotting.
Authors: Dame M C , Pierce E A , DeLuca H F
Published: May 2006

Identification of the porcine 1,25-dihydroxyvitamin D3 receptor protein on NaDodSO4/polyacrylamide slab gels was accomplished by two separate techniques: (i) assay of the specific binding activity of tritiated 1,25-dihydroxyvitamin D3 to protein eluted from NaDodSO4/polyacrylamide gels and renatured and (ii) immunoblotting of the partially purified receptor using two anti-receptor monoclonal antibodies. The porcine receptor preparation used in these studies was isolated from a crude nuclear extract of intestinal mucosa followed by chromatography on DNA-cellulose, ammonium sulfate precipitation, gel filtration HPLC, and DEAE-Sepharose chromatography. These receptor fractions were then electrophoresed on NaDodSO4/polyacrylamide gels. The receptor was eluted from the gel, renatured, and assayed for its ability to bind tritiated 1,25-dihydroxyvitamin D3. The renatured receptor appears as a single peak of specific tritiated 1,25-dihydroxyvitamin D3 binding activity. This binding activity corresponds to a band on a silver-stained gel that correlates with the receptor peak eluted from the DEAE-Sepharose column. It also corresponds to the highest molecular weight species identified on an immunoblot with anti-receptor monoclonal antibodies. The 1,25-dihydroxyvitamin D3 receptor protein has a molecular weight of 55,000 as deduced from its migration on NaDodSO4/polyacrylamide gels.
Lysine residue 121 in the proposed ATP-binding site of the v-mos protein is required for transformation.
Authors: Hannink M , Donoghue D J
Published: May 2006

The transforming gene product encoded by Moloney murine sarcoma virus clone 124, p37mos, contains a lysine residue (lysine-121) that is conserved among all members of the protein kinase family. This lysine has been shown to be part of a conserved ATP-binding site in both the catalytic subunit of the cAMP-dependent protein kinase and p60v-src. We wished to determine whether this lysine is required for the transforming activity of p37mos. Two site-specific mutations were therefore constructed, which result in the substitution of an aspartic acid or arginine codon in place of the codon for lysine-121. Both mutations abolished the ability of the mos gene to transform cells. These results show that lysine-121 is required for the ability of p37mos to transform cells and provide evidence for an ATP-binding site in p37mos. Furthermore, these results suggest that the conserved lysine residue is specifically involved in the catalytic activity of protein kinases in general.
Milrinone and thyroid hormone stimulate myocardial membrane Ca2+-ATPase activity and share structural homologies.
Authors: Mylotte K M , Cody V , Davis P J , Davis F B , Blas S D , Schoenl M
Published: May 2006

We have recently shown that thyroid hormone in physiological concentrations stimulates sarcolemma-enriched rabbit-myocardial-membrane Ca2+-ATPase in vitro. In this study, milrinone [2-methyl-5-cyano-(3,4'-bipyridin)-6(1H)-one], a cardiac inotropic agent, was thyromimetic in the same system. At clinically achievable concentrations (50-500 nM), milrinone significantly stimulated membrane Ca2+-ATPase in vitro. This action was antagonized by W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], an agent that also blocks thyroid hormone action on the Ca2+-ATPase, at concentrations as low as 5 microM. Progressive additions of milrinone to membranes incubated with a fixed concentration of thyroxine (0.10 nM) or triiodothyronine resulted in a progressive obliteration of the thyroid hormone effect on Ca2+-ATPase. Amrinone [5-amino-(3,4'-bipyridin)-6(1H)-one], the parent bipyridine of milrinone, had no effect on myocardial Ca2+-ATPase activity. X-ray crystallographic analysis of milrinone and amrinone revealed structural homologies between the phenolic ring of thyroxine and the substituted ring of milrinone, whereas amrinone did not share these homologies. The mechanism(s) of the inotropic actions of thyroxine and of milrinone is not clearly understood, but these observations implicate Ca2+-ATPase, a calcium pump-associated enzyme, as one mediator of the effects on the heart of these two compounds.
TnphoA: a transposon probe for protein export signals.
Authors: Manoil C , Beckwith J
Published: May 2006

We constructed a derivative of transposon Tn5 that permits the generation of hybrid proteins composed of alkaline phosphatase (EC 3.1.3.1) lacking its signal peptide fused to amino-terminal sequences of other proteins. Such a hybrid gives alkaline phosphatase activity if the protein fused to alkaline phosphatase contributes sequences that promote export and thus compensate for the missing alkaline phosphatase signal peptide. Fusions to both a secreted periplasmic protein and a complex cytoplasmic membrane protein led to alkaline phosphatase activity. TnphoA fusions should help localize export signals within the structure of a protein, such as a transmembrane protein, as well as identify new chromosomal genes for secreted and transmembrane proteins.
A defect in cholesterol esterification in Niemann-Pick disease (type C) patients.
Authors: Pentchev P G , Comly M E , Kruth H S , Vanier M T , Wenger D A , Patel S , Brady R O
Published: May 2006

The demonstration of a defect of cholesterol esterification in a mutant strain of BALB/c mice with an attendant reduction of sphingomyelinase activity [Pentchev, P. G., Boothe, A. D., Kruth, H.S., Weintroub, H., Stivers, J. & Brady, R. O. (1984) J. Biol. Chem. 259, 5784-5791] prompted us to examine the capacity of cultured human Niemann-Pick fibroblasts to esterify exogenously derived cholesterol. Cholesterol was supplied to cell cultures in the form of native or chemically modified, positively charged low density lipoprotein or as non-lipoprotein cholesterol. Cholesterol esterification was not impaired in cell cultures derived from patients with type A or B Niemann-Pick disease. However, esterification of exogenously administered cholesterol was deficient in 20 type C Niemann-Pick cell lines that were available for testing. Fluorescence histochemical staining of unesterified cholesterol in type C cells suggested that these cells were able to internalize and lysosomally process lipoprotein cholesterol. Acyl-CoA:cholesterol acyltransferase activity did not appear deficient in type C cell extracts. The error in cholesterol esterification may provide an opportunity for probing the molecular lesion in this disorder and may afford a useful and reliable means for establishing diagnosis.
Nonconsensus branch-site sequences in the in vitro splicing of transcripts of mutant rabbit beta-globin genes.
Authors: Padgett R A , Konarska M M , Aebi M , Hornig H , Weissmann C , Sharp P A
Published: May 2006

Mutants of the rabbit beta-globin gene lacking the natural site of branch formation in the second intervening sequence have been analyzed for in vitro splicing activity. RNAs transcribed from these mutants were spliced, via lariat formation, at a reduced rate compared to wild-type RNA. The sites of branch formation were mapped by direct RNA analysis and primer-extension analysis. The sequences at the branch sites in the three mutants examined did not conform to the previously determined consensus sequence, nor were the 5' splice sites and branch sites complementary.