bluuebunny/crossref_metadata_2025 huggingface dataset:
The molecular electronic device and the biochip computer: present status.
Authors: Haddon R C , Lamola A A
Published: May 2006

The idea that a single molecule might function as a self-contained electronic device has been of interest for some time. However, a fully integrated version--the biochip or the biocomputer, in which both production and assembly of molecular electronic components is achieved through biotechnology-is a relatively new concept that is currently attracting attention both within the scientific community and among the general public. In the present article we draw together some of the approaches being considered for the construction of such devices and delineate the revolutionary nature of the current proposals for molecular electronic devices (MEDs) and biochip computers (BCCs). With the silicon semiconductor conductor industry already in place and in view of the continuing successes of the lithographic process it seems appropriate to ask why the highly speculative MED or BCC has engendered such interest. In some respects the answer is paradigmatic as much as it is real. It is perhaps best stated as the promise of the realm of the molecular. Thus it is envisioned that devices will be constructed by assembly of individual molecular electronic components into arrays, thereby engineering from small upward rather than large downward as do current lithographic techniques. An important corollary of the construction technique is that the functional elements of such an array would be individual molecules rather than macroscopic ensembles. These two aspects of the MED/BCC--assembly of molecular arrays and individually accessible functional molecular units--are truly revolutionary. Both require scientific breakthroughs and the necessary principles, quite apart from the technology, remain essentially unknown. It is concluded that the advent of the MED/BCC still lies well before us. The twin criteria of utilization of individual molecules as functional elements and the assembly of such elements remains as elusive as ever. Biology engineers structures on the molecular scale but biomolecules do not seem to be imbued with useful electronic properties. Molecular beam epitaxy and thin-film techniques produce electronic devices but they "engineer down" and are currently unable to generate individual molecular units. The potential of the MED/BCC field is matched only by the obstacles that must be surmounted for its realization.
Deletion of 43 amino acids in the NH2-terminal half of the large tumor antigen of simian virus 40 results in a non-karyophilic protein capable of transforming established cells.
Authors: Fischer-Fantuzzi L , Vesco C
Published: May 2006

We have characterized a simian virus 40 (SV40) mutant, derived from the viral DNA insertion present in simian cell transformants, which carries a deletion affecting the NH2-terminal region of the SV40 large tumor antigen. This mutant protein is 6% smaller than normal, has lost the typical nuclear localization of the SV40 large tumor antigen, and accumulates in the cytoplasm. The deletion begins at nucleotide position 4490 of the SV40 DNA and ends in-frame at nucleotide position 4362. The missing 43 amino acids begin with proline-110 and end with serine-152 of the predicted sequence; they include a cluster of basic residues, presumably important for the viral origin-DNA binding, and most of the phosphorylation sites present in the NH2-terminal half of the molecule. The protein can still be phosphorylated considerably in vivo. This mutant viral genome is replication-defective but has conserved the competence to transform established cells, such as NIH/3T3 cells. Transfection of cloned mutant DNA into such cells resulted in the production of full transformants. Full transformants were not produced in similar transfections carried out in primary rat embryo fibroblasts, although some primary transfectants expressing the non-karyophilic large tumor antigen might be considered minimally transformed.
Molecular cloning of the human cholecystokinin gene by use of a synthetic probe containing deoxyinosine.
Authors: Takahashi Y , Kato K , Hayashizaki Y , Wakabayashi T , Ohtsuka E , Matsuki S , Ikehara M , Matsubara K
Published: May 2006

A synthetic DNA based on the known amino acid sequence of the brain/gut peptide cholecystokinin (CCK) was synthesized. This DNA contained deoxyinosines at ambiguous codon positions and was used as a probe to isolate the CCK gene directly from a human genomic library. Nucleotide sequence analysis of the isolated gene revealed that human preprocholecystokinin consists of 115 amino acid residues, with 11 amino acids in common with the human gastrin precursor, another member of the gastrin-CCK family, and that the coding region is separated by a single, long intron. CCK appears to be encoded by a single-copy gene in the haploid human genome, as revealed by genomic Southern hybridization analysis, suggesting that the same gene is expressed both in gut and brain.
L1 sequences in HeLa extrachromosomal circular DNA: evidence for circularization by homologous recombination.
Authors: Jones R S , Potter S S
Published: May 2006

Subcloned probes of the L1 family of repetitive elements were used to isolate L1-carrying clones from a plasmid library of HeLa cell extrachromosomal circular DNA. One clone was analyzed in detail by restriction mapping, cross-hybridization to L1 probes, and base sequence analysis. In addition to approximately the 3' half of a full-sized L1 element, this clone carried 390 base pairs of non-L1 sequence that is single copy in the HeLa genome. A HeLa genomic clone of this unique chromosomal region was isolated and the sequence organization of the circle clone was compared with the linear chromosomal region from which it was ultimately derived. We discuss possible mechanisms of circular DNA formation and propose homologous intrachromosomal recombination between 9-base-pair direct repeats to be most likely in this case.
Influenza viral (A/WSN/33) hemagglutinin is expressed and glycosylated in the yeast Saccharomyces cerevisiae.
Authors: Jabbar M A , Sivasubramanian N , Nayak D P
Published: May 2006

Recombinant plasmids were constructed in which genes coding for either the entire or the signal-minus (amino acid residues 2-17 deleted) hemagglutinin (HA) of WSN influenza virus were placed under the control of the alcohol dehydrogenase I gene promoter of Saccharomyces cerevisiae. Both recombinant plasmids were shown to direct the synthesis of HA-specific polypeptides that were detected by immunoprecipitation with antiviral antibodies. The complete HA produced in yeast had an approximate Mr of 70,000 and was glycosylated, as determined by the endoglycosidase H sensitivity, and was bound to membrane. Therefore, the complete HA polypeptide possessing the signal sequence probably traversed the yeast secretory pathways. Signal-minus HA, on the other hand, had a lower molecular weight and was nonglycosylated. The specific binding of yeast HA with antiviral antibodies could be competitively inhibited by influenza viral HA, demonstrating that the HA produced in yeast contained antigenic determinants of the native viral HA.
DNA sequences at the ends of the genome of bacteriophage Mu essential for transposition.
Authors: Groenen M A , Timmers E , van de Putte P
Published: May 2006

We have determined the minimal DNA sequences at the ends of the genome of bacteriophage Mu that are required for its transposition. A mini-Mu was constructed on a multicopy plasmid that enabled the manipulation of the DNA sequences at its ends without affecting the genes essential for transposition. The genes A and B, which were cloned outside the ends of the mini-Mu on the same plasmid, were both needed for optimal transposition. In our experimental system the predominant end products of the transposition are cointegrates both in the presence and in the absence of B. Two regions ending approximately 25 and 160 bp from the left end and one ending approximately 50 bp from the right end appear to be essential for optimal transposition. Overlapping with these regions, a 22-base-pair sequence was recognized with the consensus Y-G-T-T-C-A-Y-T-N-N-A-A-R-Y-R-C-G-A-A-A-A, where Y and R represent any pyrimidine and purine, respectively. At the left end these sequences occur as direct repeats; at the right end this sequence is inverted with respect to those at the left end.
Evidence for linear response for the induction of mutations in human cells by x-ray exposures below 10 rads.
Authors: Grosovsky A J , Little J B
Published: May 2006

The induction of 6-thioguanine resistance (6TGR) was studied in continuous human lymphoblast cultures exposed to daily x-ray doses of 1, 2.5, 5, or 10 rads (1 rad = 0.01 gray) for periods up to 1 month. Samples were taken every 5 days for determinations of induced mutation frequency. Cells receiving daily doses of 1-10 rads showed a mutation frequency of 0.069 X 10(-6) 6TGR cell per rad, virtually identical to the value of 0.062 X 10(-6) 6TGR cell per rad determined for lymphoblasts receiving the same total dose of radiation in a single acute exposure. The effects of small daily fractions were additive, suggesting that doses as small as 1 rad are mutagenic in human lymphoblasts. Similar results were observed when resistance to trifluorothymidine, indicative of thymidine kinase deficiency, was used as a mutational marker. When 6TGR frequency was plotted against days of irradiation, a positive linear slope was observed for all dose groups. The values of these slopes were plotted against x-ray dose in rads/day to construct a dose-response relationship for 1-10 rads. A linear increase in mutation frequency was observed over this dose range, with no apparent threshold. The slope of this linear increase was 0.060 X 10(-6) 6TGR cell per rad. These results suggest that, for human lymphoblasts, the mutagenic risk of low doses of x-rays can be accurately estimated by linear extrapolation from high-dose effects.
The gene coding for carbamoyl-phosphate synthetase I was formed by fusion of an ancestral glutaminase gene and a synthetase gene.
Authors: Nyunoya H , Broglie K E , Lusty C J
Published: May 2006

A near full-length cDNA copy of rat carbamoyl-phosphate synthetase I (EC 6.3.4.16) mRNA has been cloned. The cDNA insert in the recombinant plasmid pHN234 is 5.3 kilobases long. Analysis of the sequence coding for carbamoyl-phosphate synthetase I indicates that the gene has arisen from a fusion of two ancestral genes: one homologous to Escherichia coli carA, coding for a glutaminase subunit, and the second homologous to the carB gene that codes for the synthetase subunit. A short amino acid sequence previously proposed to be part of the active site involved in glutamine amide nitrogen transfer in the E. coli and yeast carbamoyl-phosphate synthetases (EC 6.3.5.5) is also present in the rat enzyme. In the mammalian enzyme, however, the glutaminase domain lacks a cysteine residue previously shown to interact with glutamine. The cysteine is replaced by a serine residue. This substitution could, in part, account for the inability of mammalian carbamoyl-phosphate synthetase I to catalyze the hydrolysis of glutamine to glutamic acid and ammonia.
The replication initiator protein of plasmid pSC101 is a transcriptional repressor of its own cistron.
Authors: Vocke C , Bastia D
Published: May 2006

The plasmid-encoded replication initiator protein of pSC101 specifically repressed initiation of transcription of its own cistron from its natural promoter. Addition of the purified initiator had little or no visible effect on transcription initiated from a heterologous promoter. DNase protection experiments revealed that the RNA polymerase recognition sequence was overlapped by the initiator protein recognition sequences, which are vicinal to the replication origin. Using the labeled promoter sequence, we have performed competitive DNase protection experiments in two ways: by adding RNA polymerase and initiator protein simultaneously or by sequentially adding first RNA polymerase and then initiator protein to the DNase reaction mixture. The RNA polymerase protection pattern was recessive to that of the initiator regardless of whether the two proteins were added simultaneously or sequentially. This observation suggests that the mechanism of autoregulation is due to competition of the two proteins for the sequences in and around the promoter region. Furthermore, the sequential addition experiments raise the possibility of displacement of RNA polymerase from the promoter by the initiator protein.
Active site of triosephosphate isomerase: in vitro mutagenesis and characterization of an altered enzyme.
Authors: Straus D , Raines R , Kawashima E , Knowles J R , Gilbert W
Published: May 2006

We have replaced the glutamic acid-165 at the active site of chicken triosephosphate isomerase with an aspartic acid residue using site-directed mutagenesis. Expression of the mutant protein in a strain of Escherichia coli that lacks the bacterial isomerase results in a complementation phenotype that is intermediate between strains that have no isomerase and strains that produce either the wild-type chicken enzyme or the native E. coli isomerase. The value of kcat for the purified mutant enzyme when glyceraldehyde 3-phosphate is the substrate is 1/1500th that of the wild-type enzyme, and the Km is decreased by a factor of 3.6. With dihydroxyacetone phosphate as substrate, the kcat value is 1/240th that of the wild-type enzyme, and Km is 2 times higher. The value of Ki for a competitive inhibitor, phosphoglycolate, is the same for the mutant and wild-type enzymes, at 2 X 10(-5) M. By treating the enzyme-catalyzed isomerization as a simple three step process and assuming that substrate binding is diffusion limited, it is evident that the mutation of glutamic acid-165 to aspartic acid principally affects the free energy of the transition state(s) for the catalytic reaction itself.
Functional activation of the long terminal repeat of human T-cell leukemia virus type I by a trans-acting factor.
Authors: Fujisawa J , Seiki M , Kiyokawa T , Yoshida M
Published: May 2006

Promoter function for gene expression of the long terminal repeat (LTR) of human T-cell leukemia virus type I (HTLV-I) was studied by constructing plasmids containing the LTR sequence. The gene encoding chloramphenicol acetyltransferase (CATase) was linked to an HTLV-I LTR sequence (pLTR-CAT) by replacing the simian virus 40 promoter in plasmid pSV2-CAT with the LTR sequence. The transient CATase activities of cells transfected with the plasmids were compared. The results are summarized as follows: The HTLV LTR was active even in an epithelial cell line, with efficiency similar to that of the simian virus 40 promoter. pLTR-CAT expressed high CATase activity, 40-200 times that expressed by pSV2-CAT, in HTLV-I-infected T-cell lines, such as the human cell lines MT-2 and HUT-102, or in HTLV-I-infected rat cell lines. This enhanced activity of the LTR seems to be associated with HTLV gene expression, since only low activity of pLTR-CAT was observed in the HTLV-infected cell line MT-1, in which only a small percent of cells express viral antigens. In HTLV-infected rat cell lines, the pX-encoded protein p40x was the only viral protein detected. Thus, we suggest that p40x is the factor associated directly or indirectly with the enhanced activity of the LTR.
Identification and functional characterization of leukotriene B4 20-hydroxylase of human polymorphonuclear leukocytes.
Authors: Soberman R J , Harper T W , Murphy R C , Austen K F
Published: May 2006

A single reaction product was formed during the incubation of 1.5 microM (5S,12R)-dihydroxy-6,14-cis-8,10-trans-[3H]icosatetraenoic acid (leukotriene B4, LTB4) for 30 min at 37 degrees C in 10 mM potassium phosphate buffer (pH 7.5) with 100 microM NADPH and the 150,000 X g supernatant of sonicated human polymorphonuclear leukocytes (PMN). The reaction product exhibited the same mobility on reversed-phase HPLC (RP-HPLC) and TLC as standard 20-hydroxy-LTB4 (20-OH-LTB4). When the omega-oxidation product of [3H]LTB4 was eluted from a Sep-Pak, resolved by RP-HPLC, and analyzed by GC/MS, its structure was determined to be solely 20-OH-LTB4. The Km of the 20-hydroxylase for [3H]LTB4 at its optimal pH of 7.5 was 0.22 +/- 0.08 microM (mean +/- SD, n = 4) and the Vmax was 48 +/- 11 pmol/min X mg of protein (mean +/- SD, n = 4). When the concentration of [3H]LTB4 was fixed at 1.5 microM, the Km for NADPH was 1.01 +/- 0.59 microM (mean +/- SD, n = 3). The location in the 150,000 X g supernatant of the LTB4 20-hydroxylase distinguishes it from the cytochrome P-450 system of liver, lung, and kidney microsomes and from the NADPH oxidase-cytochrome b-245 system of the human PMN. The LTB4 20-hydroxylase is either a unique cytochrome P-450 or other monooxygenase.
Bone marrow engraftment efficiency is enhanced by competitive inhibition of the hepatic asialoglycoprotein receptor.
Authors: Samlowski W E , Daynes R A
Published: May 2006

The efficiency of pluripotent stem cell engraftment following bone marrow transplantation is predicated upon many poorly understood factors. These include the processes by which intravenously injected stem cells circulate and localize in microenvironments that contain the stromal elements necessary to facilitate their continued proliferation. We have recently established that lymphoid cells that bind the lectin peanut agglutinin are subject to prolonged sequestration following their interaction with the hepatic asialoglycoprotein receptor. Since bone marrow stem cells are also known to bind peanut agglutinin, we hypothesized that the physiologic function of the asialoglycoprotein receptor might significantly impair their ability to localize in anatomic sites where they are able to proliferate. Competitive inhibition with asialoglycoproteins was employed to establish a temporary receptor blockade during the initial 3-4 hr after transplantation. This procedure resulted in a 5- to 10-fold increase in splenic hematopoietic colony formation. Our findings suggest that inhibition of the liver asialoglycoprotein receptor during murine bone marrow transplantation results in more efficient stem cell localization to hematopoietic-inducing microenvironments. This enhancement in engraftment efficiency was paralleled by a more rapid recovery of peripheral blood leukocyte and platelet counts, an increase in megakaryocytic colony formation, as well as increased recipient survival. Techniques designed to inhibit the liver sequestration of bone marrow stem cells may have direct applicability to human bone marrow transplantation procedures.
A chemically cleavable biotinylated nucleotide: usefulness in the recovery of protein-DNA complexes from avidin affinity columns.
Authors: Shimkus M , Levy J , Herman T
Published: May 2006

A biotinylated nucleotide analog containing a disulfide bond in the 12-atom linker joining biotin to the C-5 of the pyrimidine ring has been synthesized. This analog, Bio-SS-dUTP, is an efficient substrate for Escherichia coli DNA polymerase I. Bio-SS-dUTP supported DNA synthesis in a standard nick-translation reaction at 35%-40% the rate of an equal concentration of the normal nucleotide, TTP. DNA containing this analog was bound to an avidin-agarose affinity column and subsequently eluted after reduction of the disulfide bond by dithiothreitol. The ability to recover biotinylated DNA from an avidin affinity column under nondenaturing conditions should prove useful in the isolation of specific protein-DNA complexes. As a demonstration of this approach, Bio-SS-DNA was reconstituted with histones to form 11S monomer nucleosomes. Bio-SS-nucleosomes were shown to selectively bind to avidin-agarose. Ninety percent of the bound Bio-SS-nucleosomes were recovered from the affinity column by elution with buffer containing 50-500 mM dithiothreitol. The recovered nucleosomes were shown to be intact 11S particles as judged by velocity sedimentation in a sucrose gradient. This approach may prove to be generally useful in the isolation of protein-DNA complexes in a form suitable for further analysis of their native unperturbed structure.
Poliovirus protease does not mediate cleavage of the 220,000-Da component of the cap binding protein complex.
Authors: Lloyd R E , Etchison D , Ehrenfeld E
Published: May 2006

Poliovirus infection of HeLa cells results in a rapid shutoff of host protein synthesis but does not inhibit the translation of poliovirus mRNA. It has been suggested that this virus-induced translational control is mediated by the inactivation of a cap binding protein (CBP) complex, and it has been shown that the 220,000-Da component(s) (p220) of the CBP complex is cleaved in infected HeLa cells to form antigenically related peptides of 100,000-130,000 Da. To determine whether the known viral protease (peptide 3C) was the mediator of the cleavage of p220, we used immunoblot techniques to analyze partially purified infected HeLa cell extracts for cleavage activity. We report here that p220 cleavage activity does not copurify with viral peptide 3C or with any precursors containing 3C sequences. We also show that cleavage of p220 can be demonstrated in vitro in HeLa cell extracts under conditions where the functional activity of the poliovirus protease is inhibited by specific antibody.
Avian myelocytomatosis virus immortalizes differentiated quail chondrocytes.
Authors: Gionti E , Pontarelli G , Cancedda R
Published: May 2006

Quail embryo chondrocytes in culture display two morphological phenotypes: polygonal epithelial-like and floating cells. Both cell populations synthesize cartilage extracellular matrix proteins (type II collagen and specific proteoglycans), whereas type X collagen, which appears to be a marker of later stages of chondrocyte differentiation, is expressed only by the epithelial-like cells. Avian myelocytomatosis virus strain MC29 does not induce morphological transformation in quail embryo chondrocytes but stimulates these cells to proliferate with a progressively reduced doubling time. MC29-infected chondrocytes can be established in culture as a continuous cell line, whereas control (uninfected) cultures only survive a few months. Rapidly dividing MC29-infected chondrocytes still express type II collagen and cartilage proteoglycans but do not synthesize type X collagen.
Susceptibility of human malaria parasites to chloroquine is pH dependent.
Authors: Yayon A , Cabantchik Z I , Ginsburg H
Published: May 2006

Chloroquine (CQ) accumulates in the acidic food vacuole of intraerythrocytic malaria parasites (Plasmodium falciparum) by virtue of its weak base properties. In the present work, the extent of CQ accumulation was determined by the transvacuolar pH gradient: modification of the latter--either by changing the external pH or by adding the acidotropic agent NH4Cl--led to a corresponding change in CQ distribution between cells and medium. Changes in pH gradient provoked a change in the susceptibility of parasites to CQ: at external pH values of 8.0, 7.4, and 6.8, the IC50 values for CQ were 0.48 X 10(-7) M, 1.8 X 10(-7) M, and 3.3 X 10(-7) M, respectively. Marked resistance to CQ (IC50 = 9.8 X 10(-7) M) was conferred upon cells by exposing them simultaneously to CQ and 10 mM NH4Cl, at pH 7.4. The final concentration of CQ attained within the acidic compartment of the parasite was correlated with inhibition of parasite growth. At therapeutic drug levels, CQ accumulation caused minor changes in the food vacuole pH, whereas at higher CQ concentrations substantial alkalinization was observed. The antimalarial activity of CQ is suggested to be exerted by the interference of the high concentrations of the accumulated drug with vital functions of the food vacuole.
Intraterminal injection of synapsin I or calcium/calmodulin-dependent protein kinase II alters neurotransmitter release at the squid giant synapse.
Authors: Llinás R , McGuinness T L , Leonard C S , Sugimori M , Greengard P
Published: May 2006

Synapsin I and calcium/calmodulin-dependent protein kinase II were pressure-injected into the preterminal digit of the squid giant synapse to test directly the possible regulation of neurotransmitter release by these substances. Neurotransmitter release was determined by measuring the amplitude, rate of rise, and latency of the postsynaptic potential generated in response to presynaptic depolarizing steps under voltage clamp conditions. Injection of dephosphosynapsin I decreased the amplitude and rate of rise of the postsynaptic potential, whereas injection of either phosphosynapsin I or heat-treated dephosphosynapsin I was without effect. Conversely, injection of calcium/calmodulin-dependent protein kinase II, which phosphorylates synapsin I on site II, increased the rate of rise and amplitude and decreased the latency of the postsynaptic potential. The effects of these proteins were observed without any detectable change in the initial phase of the presynaptic calcium current. A synapsin I-like protein and calcium/calmodulin-dependent protein kinase II were demonstrated by biochemical and immunochemical techniques to be present in squid nervous tissue. The data support the hypothesis that synapsin I regulates the availability of synaptic vesicles for release; we propose that calcium entry into the nerve terminal activates calcium/calmodulin-dependent protein kinase II, which phosphorylates synapsin I on site II, dissociating it from the vesicles and thereby removing a constraint in the release process.
Two distinct Ca-dependent K currents in bullfrog sympathetic ganglion cells.
Authors: Pennefather P , Lancaster B , Adams P R , Nicoll R A
Published: May 2006

Healthy bullfrog sympathetic ganglion cells often show a two-component afterhyperpolarization (AHP). Both components can be reduced or abolished by adding Ca-channel blockers or by removing external Ca. Application of a single electrode "hybrid clamp"--i.e., switching from current- to voltage-clamp at the peak of the AHP, reveals that the slow AHP component is generated by a small, slow, monotonically decaying outward current, which we call IAHP. IAHP is blocked by Ca-removal or by apamin and is a pure K current. It is slightly sensitive to muscarine and to tetraethylammonium ion but is much less so than muscarine-sensitive (IM) and fast Ca-dependent (IC) K currents. It also can be recorded in dual-electrode voltage-clamp experiments, where it is seen as a slow, small component of the outward tail current that follows brief depolarizations to 0 mV or beyond. IC is seen as an early, fast, large component of the same tail current. Both components are blocked by Ca removal, but only the IC component is blocked by low doses of tetraethylammonium ion. Thus, bullfrog ganglion cells exhibit two quite distinct Ca-dependent K currents, which differ in size, voltage-sensitivity, kinetics, and pharmacology. These two currents also play quite separate roles in shaping the action potential.
Fucose-containing glycolipids are stage- and region-specific antigens in developing embryonic brain of rodents.
Authors: Yamamoto M , Boyer A M , Schwarting G A
Published: May 2006

A monoclonal antibody (against 7A antigen), raised against a homogenate of embryonic rat forebrain, marks the distribution of region- and developmental stage-specific cell-surface antigens in the mammalian central nervous system. In the mouse, immunocytochemical staining revealed that 7A antigen is expressed almost exclusively in germinal layers of the cerebral cortex beginning as early as day 11 of gestation and becomes undetectable by birth. The typical staining is seen at embryonic days 13-15, in which only the ventricular and the subventricular zones of the cerebral cortex are intensely labeled, whereas most other areas of the brain are unlabeled. Tissue culture studies indicate that the 7A antigen is localized at the plasma membrane. Molecular species possessing the antigen are identified as neutral glycolipids that contain the X-determinant trisaccharide: (formula; see text) This glycoconjugate is expressed in embryonic brains of many mammalian species (mouse, rat, rabbit, calf, and human) and may be a cell-surface component important in normal development of the central nervous system.
Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs.
Authors: Schatten H , Schatten G , Mazia D , Balczon R , Simerly C
Published: May 2006

The forms and locations of centrosomes in mouse oocytes and in sea urchin eggs were followed through the whole course of fertilization and first cleavage by immunofluorescence microscopy. Centrosomes were identified with an autoimmune antiserum to centrosomal material. Staining of the same preparations with tubulin antibody and with the DNA dye Hoechst 33258 allowed the correlation of the forms of the centrosomes with the microtubule structures that they generate and with the stages of meiosis, syngamy, and mitosis. The results with sea urchin eggs conform to Boveri's view on the paternal origin of the functional centrosomes. Centrosomes are seen in spermatozoa and enter the egg at fertilization. Initially, the centrosomes are compact, but as the eggs enter the mitotic cycle the forms of the centrosomes go through a cycle in which they spread during interphase, apparently divide, and condense into two compact poles by metaphase. In anaphase, they spread to form flat poles. In telophase and during reconstitution of the daughter nuclei, the centrosomal material is disposed as hemispherical caps around the poleward surfaces of the nuclei. Mouse sperm lack centrosomal antigen. In the unfertilized mouse oocyte, the meiotic spindle poles are displayed as broad-beaded centrosomes. In addition, centrosomal material is detected in the cytoplasm as particles, about 16 in number, which are foci of small aster-like arrays of microtubules. The length and number of astral microtubules correlate with the size of the centrosomal foci. After sperm incorporation, as the pronuclei develop and more cytoplasmic microtubules assemble, a few of the foci associate with the peripheries of the nuclei. The number of foci multiplies during the first cell cycle. At the end of interphase, all of the centrosomal foci have concentrated on the nuclear peripheries and the cytoplasmic microtubules have disappeared. At prophase, the centrosomes are seen as two irregular clusters, marking the poles which, at metaphase and anaphase, appear as rough bands with foci, and the spindle is typically barrel-shaped. At telophase, the centrosomes are seen as arcs that lie on the nuclear peripheries after cleavage. The ordering of microtubules in all the stages reflects the shapes of the centrosomes. The findings on the sea urchin confirm the classical theory of the paternal origin of centrosomes and contrast with observations tracing the mitotic poles of the mouse egg to maternal centrosomal material. This evidence strengthens the conclusion that mouse centrosomes derive from the oocyte.
Two insulin-like growth factor I messenger RNAs are expressed in human liver.
Authors: Rotwein P
Published: May 2006

Through use of a synthetic oligonucleotide probe, human insulin-like growth factor I (IGF-I) cDNA clones were isolated from a liver library. Two types of cDNAs were defined by restriction enzyme analysis and DNA sequencing. Both encode IGF-I precursors of either 195 or 153 amino acids. The two predicted protein precursors are identical from their amino terminus to a lysine residue 16 codons beyond the IGF-I sequence, and then they diverge. Both cDNAs predict additional unique carboxyl-terminal extension peptides. Since there is only one IGF-I gene in the human genome, the finding of two different cDNAs suggests that alternative RNA processing plays a role in IGF-I gene expression. The functions of the different extension peptides remain to be elucidated.
Cloning and sequence analysis of cDNA species coding for the two subunits of inhibin from bovine follicular fluid.
Authors: Forage R G , Ring J M , Brown R W , McInerney B V , Cobon G S , Gregson R P , Robertson D M , Morgan F J , Hearn M T , Findlay J K
Published: May 2006

The primary amino acid structures of the 43-kDa (A) and 15-kDa (B) subunits of the 58-kDa form of the hormone inhibin have been elucidated by cloning and analysis of cDNA species derived from bovine granulosa cell mRNA. The A subunit (Mr = 32,298) is a protein of 300 amino acids with two potential N-glycosylation sites and two potential proteolytic processing sites and has a pre-pro region of 60 amino acids. The mature B subunit (Mr = 12,977) is a protein of 116 amino acids synthesized from a separate mRNA. These data establish that a 31-kDa form of inhibin also isolated from bovine follicular fluid, with subunits of 20 kDa (Ac) and 15 kDa (B), is derived from the 58-kDa form by proteolytic processing of the A subunit.
Human prostate-specific antigen: structural and functional similarity with serine proteases.
Authors: Watt K W , Lee P J , M'Timkulu T , Chan W P , Loor R
Published: May 2006

The complete amino acid sequence of the prostate-specific antigen (PA) from human seminal plasma has been determined from analyses of the peptides generated by cyanogen bromide, hydroxylamine, endoproteinases Arg-C and Lys-C. The single polypeptide chain of PA contains 240-amino acid residues and has a calculated Mr of 26,496. An N-linked carbohydrate side chain is predicted at asparagine-45, and O-linked carbohydrate side chains are possibly attached to serine-69, threonine-70, and serine-71. The primary structure of PA shows a high degree of sequence homology with other serine proteases of the kallikrein family. The active site residues of histidine, aspartic acid, and serine comprising the charge-relay system of typical serine proteases were found in similar positions in PA (histidine-41, aspartic acid-96, and serine-192). At pH 7.8, PA hydrolyzed insulin A and B chains, recombinant interleukin 2, and--to a lesser extent--gelatin, myoglobin, ovalbumin, and fibrinogen. The cleavage sites of these proteins by PA were chemically analyzed as the alpha-carboxyl side of some hydrophobic residues, tyrosine, leucine, valine, and phenylalanine, and of basic residues histidine, lysine, and arginine. The chymotrypsin-like activity of PA exhibited with the chromogenic substrate N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide yielded a specific activity of 9.21 microM per min per mg of PA and Km and kcat values of 15.3 mM and 0.075s-1, respectively. "Trypsin-like" activity of PA was also detected with N alpha-benzoyl-DL-arginine p-nitroanilide and gave a specific activity of 1.98 microM per min per mg of PA. Protease inhibitors such as phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, L-1-tosylamido-2-phenylethyl chloromethyl ketone, aprotinin, leupeptin, soybean trypsin inhibitor as well as Zn2+ and spermidine were effective inhibitors of PA enzymatic activity.
Analysis of a sea urchin gene cluster coding for the small nuclear U7 RNA, a rare RNA species implicated in the 3' editing of histone precursor mRNAs.
Authors: De Lorenzi M , Rohrer U , Birnstiel M L
Published: May 2006

A genomic 9.3-kilobase DNA fragment of the sea urchin Psammechinus miliaris, containing a cluster of five U7-RNA genes (or pseudogenes), has been isolated and analyzed by partial DNA sequencing. The U7-RNA coding sequences differ from one another by one or two nucleotides, one of the five gene sequences being identical to those of the cDNA U73 clone prepared earlier [Strub, K., Galli, G., Busslinger, M. & Birnstiel, M. L. (1984) EMBO J. 3, 2801-2807]. The spacer sequences separating the genes have, on the whole, a low degree of homology; hence, the five genes must have arisen by an ancient duplication event. The sequences preceding the coding portion contain three highly conserved sequence motifs but no "TATA box." The 3' flanking sequences include a highly conserved AAAGNNAGA sequence that is held in common with other U-RNA genes from both sea urchins and vertebrates. Our findings confirm our classification of the U7 RNA as a genuine, if sparsely represented, member of the U-RNA family.
Distribution of ions around DNA, probed by energy transfer.
Authors: Wensel T G , Meares C F , Vlachy V , Matthew J B
Published: May 2006

Measurements of the effect of DNA on rates of bimolecular energy transfer between ions provide a direct indication of how cations cluster in regions near DNA and how anions are repelled from the same regions. Energy transfer from luminescent lanthanide ions (in the "rapid-diffusion" limit) probes collision frequencies that are dependent on the equilibrium spatial distributions of ions. The addition of 1 mM DNA (phosphate) to a 2 mM salt solution increases the overall collision frequency between monovalent cations by a factor of 6 +/- 1.5; it increases the divalent-monovalent cation collision frequency by a factor of 29 +/- 3; and it decreases the divalent cation-monovalent anion collision frequency by a factor of 0.24 +/- 0.03. Comparisons are made with the changes in collision frequencies predicted by several different theoretical descriptions of ion distributions. The closest agreement with experimental results for monovalent ions at 1 mM DNA is obtained with a static accessibility-modified discrete charge calculation, based on a detailed molecular model of B-DNA. At high DNA concentration (10 mM), the best results are obtained by numerical solutions of the Poisson-Boltzmann equation for a "soft-rod" model of DNA. Poisson-Boltzmann calculations for a "hard-rod" model greatly overestimate the effects of DNA on collision frequencies, as does a calculation based on counterion-condensation theory.
Specific binding of the mononuclear phagocyte colony-stimulating factor CSF-1 to the product of the v-fms oncogene.
Authors: Sacca R , Stanley E R , Sherr C J , Rettenmier C W
Published: May 2006

Cells transformed by the McDonough strain of feline sarcoma virus (SM-FeSV) express a v-fms-encoded glycoprotein whose expression at the cell surface correlates with the transformed phenotype. The mouse mononuclear phagocyte growth factor CSF-1 specifically binds to SM-FeSV-transformed cells at high-affinity sites indistinguishable from those detected on normal feline macrophages. A monoclonal antibody to a v-fms-encoded epitope competed for CSF-1 binding to SM-FeSV-transformed cells, and chemical crosslinking demonstrated that murine CSF-1 bound to the v-fms gene product at the cell surface. Although SM-FeSV-transformed fibroblast lines were found to secrete CSF-1, the growth of transformed cells was not affected by antibodies to the v-fms gene product or to the growth factor. Tyrosine phosphorylation of the v-fms products in membranes was observed in the absence of CSF-1 and was not enhanced by addition of the murine growth factor. The data support the hypothesis that the c-fms protooncogene product is related, and possibly identical, to the CSF-1 receptor and suggest that the v-fms-encoded kinase functions in the absence of an exogenous growth factor.
Role of cholecystokinin in corticotropin release: coexistence with vasopressin and corticotropin-releasing factor in cells of the rat hypothalamic paraventricular nucleus.
Authors: Mezey E , Reisine T D , Skirboll L , Beinfeld M , Kiss J Z
Published: May 2006

Cholecystokinin-8 (CCK8)-containing cell bodies in the parvocellular region of the rat paraventricular nucleus (PVN) contain vasopressin and corticotropin-releasing factor (CRF). The CCK8 and vasopressin in these cells can readily be visualized in adrenalectomized, but not in shamoperated animals. Furthermore, CCK8 levels as measured by RIA change in the PVN and in the median eminence in response to adrenalectomy. CCK8 has a stimulatory effect on corticotropin (ACTH) release from primary cultures of the anterior pituitary. This stimulation is additive with that produced by vasopressin; CCK8 plus vasopressin have an effect as great as CRF in stimulating ACTH release. Our results suggest that CCK8 may participate in the regulation of ACTH release under certain physiological conditions.
Low-dimensional chaos in an instance of epilepsy.
Authors: Babloyantz A , Destexhe A
Published: May 2006

Using a time series obtained from the electroencephalogram recording of a human epileptic seizure, we show the existence of a chaotic attractor, the latter being the direct consequence of the deterministic nature of brain activity. This result is compared with other attractors seen in normal human brain dynamics. A sudden jump is observed between the dimensionalities of these brain attractors (4.05 +/- 0.05 for deep sleep) and the very low dimensionality of the epileptic state (2.05 +/- 0.09). The evaluation of the autocorrelation function and of the largest Lyapunov exponent allows us to sharpen further the main features of underlying dynamics. Possible implications in biological and medical research are briefly discussed.
A revised set of values of single-bond radii derived from the observed interatomic distances in metals by correction for bond number and resonance energy
Authors: Pauling Linus , Kamb Barclay
Published: May 2006

An earlier discussion [Pauling, L. (1947)
J. Am. Chem. Soc.
69, 542] of observed bond lengths in elemental metals with correction for bond number and resonance energy led to a set of single-bond metallic radii with values usually somewhat less than the corresponding values obtained from molecules and complex ions. A theory of resonating covalent bonds has now been developed that permits calculation of the number of resonance structures per atom and of the effective resonance energy per bond. With this refined method of correcting the observed bond lengths for the effect of resonance energy, a new set of single-bond covalent radii, in better agreement with values from molecules and complex ions, has been constructed.
Hepatocellular uptake of oleate is energy dependent, sodium linked, and inhibited by an antibody to a hepatocyte plasma membrane fatty acid binding protein.
Authors: Stremmel W , Strohmeyer G , Berk P D
Published: May 2006

Several studies suggest that a portion of hepatocellular nonesterified fatty acid uptake may be carrier mediated. To further investigate this process, initial rates (Vo) of [14C]oleate uptake into rat hepatocytes, isolated by collagenase perfusion and incubated at 37 degrees C with oleate in the presence of bovine serum albumin, were studied as a function of the concentration of unbound [14C]oleate in the medium. Vo was saturable with increasing unbound oleate concentration (Km = 8.3 X 10(-8) M; Vmax = 197 pmol per min per 5 X 10(4) hepatocytes) and was not inhibited by up to 40 microM sulfobromophthalein, taurocholate, or cholic acid. Oleate uptake was sodium dependent. Vo was significantly diminished when Li+, K+, choline, or sucrose were substituted for Na+ in the incubation medium and was reduced 46% by 1 mM ouabain. Uptake was also markedly reduced after exposure of cells to metabolic inhibitors (e.g., 2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, antimycin, KCN). To evaluate the physiologic significance of the previously isolated rat liver plasma membrane fatty acid-binding protein, the effect of an antibody directed against this protein on hepatocellular [14C]oleate uptake was examined. Preincubation of hepatocytes with the IgG fraction of this antiserum inhibited Vo of [14C]oleate by up to 65% in dose-related fashion, without altering Vo for [35S]sulfobromophthalein, [14C]taurocholate, or [3H]cholate. These data indicate that at least a portion of hepatocellular oleate uptake is energy dependent, sodium linked, and mediated by a specific liver plasma membrane-fatty acid-binding protein.
Cell-specific enhancers in the rat exocrine pancreas.
Authors: Boulet A M , Erwin C R , Rutter W J
Published: May 2006

Pancreatic amylase, chymotrypsin B, and trypsin I genes are specifically expressed in the exocrine pancreas. The 5'-flanking regions of these genes direct preferential expression of a linked reporter function (chloramphenicol acetyltransferase) in the pancreatic exocrine cell line AR4-2J. The sequences upstream of the amylase and chymotrypsin genes that are required for this cell specific activity possess the characteristics of transcriptional enhancers. We have mapped the regions responsible for enhancer activity by deletion analysis. Modification of specific nucleotide sequences within these regions can alter or eliminate enhancer function. Comparison of the 5'-flanking regions of nine genes expressed in the exocrine pancreas identifies a family of short related sequences. These sequences are located within the enhancer regions that we have mapped and may play a role in the regulation of the expression of pancreatic exocrine-specific genes.
Three-dimensional structure of proteins determined by molecular dynamics with interproton distance restraints: application to crambin.
Authors: Brünger A T , Clore G M , Gronenborn A M , Karplus M
Published: May 2006

Model calculations are performed to evaluate the utility of molecular dynamics with NMR interproton distance restraints for determining the three-dimensional structure of proteins. The system used for testing the method is the 1.5-A resolution crystal structure of crambin (a protein of 46 residues) from which a set of 240 approximate interproton distances of less than 4 A are derived. The convergence properties of the method are examined by using different dynamics protocols and starting from two initial structures; one is a completely extended beta-strand, and the other has residues 7-19 and 23-30 in the form of alpha-helices (as in the crystal structure) with the remaining residues in the form of extended beta-strands. In both cases global and local convergence to the correct final structure is achieved with rms atomic differences between the restrained dynamics structures and the crystal structure of 1.5-2.1 A for the backbone atoms and 2.1-2.8 A for all atoms; the averaged structure has backbone and all atom rms deviations of 1.3 and 1.9 A, respectively. Further, it is shown that a restrained dynamics structure with significantly larger deviations (i.e., 5.7 A for the backbone atoms) can be characterized as incorrect, independent of a knowledge of the crystal structure.
Enzymatic reactions of ascorbate and glutathione that prevent peroxide damage in soybean root nodules
Authors: Dalton David A. , Russell Sterling A. , Hanus F. J. , Pascoe Gary A. , Evans Harold J.
Published: May 2006

The critical problem of oxygen toxicity for nitrogen-fixing organisms may be related to damage caused by oxygen radicals and peroxides. An enzymatic mechanism is described for removal of peroxides in root nodules of soybean (
Glycine max
). The system utilizes ascorbate as an antioxidant and glutathione as a reductant to regenerate ascorbate. The enzymes involved are ascorbate peroxidase (ascorbate:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), dehydroascorbate reductase (glutathione:dehydroascorbate oxidoreductase, EC 1.8.5.1), and glutathione reductase (NADPH:oxidized-glutathione oxidoreductase, EC 1.6.4.2). The reactions are essentially the same as those involving scavenging of H
2
O
2
in chloroplasts. Glutathione peroxidase (glutathione:hydrogenperoxide oxidoreductase, EC 1.11.1.9) was not detected. During the course of early nodule development, ascorbate peroxidase and dehydroascorbate reductase activities and total glutathione contents of nodule extracts increased strikingly and were positively correlated with acetylene reduction rates and nodule hemoglobin contents. The evidence indicates an important role of glutathione, ascorbate, ascorbate peroxidase, dehydroascorbate reductase, and glutathione reductase as components of a peroxide-scavenging mechanism in soybean root nodules.