bluuebunny/crossref_metadata_2025 huggingface dataset:
Blue-light receptor in a white mutant of Physarum polycephalum mediates inhibition of spherulation and regulation of glucose metabolism.
Authors: Schreckenbach T , Walckhoff B , Verfuerth C
Published: May 2006

Blue light induces sporulation of Physarum polycephalum macroplasmodia and reversibly inhibits spherulation (sclerotization) of microplasmodia. Illuminated microplasmodia have an abnormal appearance. The photobiological responses of the plasmodia appear to be unaffected by the absence of yellow pigment in the white mutant strain used. Illumination of microplasmodial suspensions with blue light (lambda max approximately 465 nm) results also in an early effect on glucose metabolism: glucose consumption is reversibly inhibited. By using radioactive glucose it was shown that the main products formed are a water-insoluble glucan and the disaccharide trehalose. Inhibition of glucose consumption in the light results in decreased production of these two compounds. Illumination of microplasmodial suspensions also causes a reversible effect on the pH of the medium which is interpreted as a decreased production of a yet unidentified acid from glucose. The action spectrum of the light-induced pH response shows maxima near 390, 465, and 485 nm. It resembles the absorption spectrum of a flavoprotein and confirms the existence of a blue-light receptor in P. polycephalum microplasmodia.
Switch from hapten-specific immunoglobulin M to immunoglobulin D secretion in a hybrid mouse cell line.
Authors: Neuberger M S , Rajewsky K
Published: May 2006

From a hybrid mouse cell line (B1-8) that secreted an IgM, lambda 1 anti-(4-hydroxy-3-nitrophenyl)acetyl antibody but that had no detectable surface IgM, selection for a variant with lambda 1 chains on the surface resulted in the isolation of a line that had switched from mu to delta expression. The surface and secreted Igs of this line were typed as IgD with two monoclonal antibodies, and the parental IgM and variant IgD molecules carried the same variable regions as judged by hapten-binding and idiotypic analysis. The surface and secreted delta chains of the IgD variant have apparent molecular weights of 64,000 and 61,000, respectively. However, the unglycosylated secreted delta polypeptide chain has a molecular weight of only 44,000. The secreted IgD exists predominantly in the delta 2 lambda A2 form, does not contain J protein, is relatively stable in serum, and does not fix complement.
Regulatory role for hepatic low density lipoprotein receptors in vivo in the dog.
Authors: Kovanen P T , Bilheimer D W , Goldstein J L , Jaramillo J J , Brown M S
Published: May 2006

Liver membranes from young beagle dogs were found to possess binding sites that resemble the low density lipoprotein (LDL) receptors originally described in cultured human fibroblasts. Treatment of the dogs with colestipol (a bile acid sequestrant) and mevinolin (a cholesterol synthesis inhibitor) produced a 3-fold increase in LDL binding activity. This increase correlated with a 2-fold increase in the fractional catabolic rate for intravenously administered human or canine 125I-labeled LDL, suggesting that the increased hepatic receptors were responsible for the enhanced clearance of LDL from plasma. The hepatic lipoprotein receptors of control and drug-treated dogs resembled human fibroblast LDL receptors in that they bound apoprotein E-containing lipoproteins, such as very low density lipoproteins and a subfraction of high density lipoproteins (HDL1), with 10-fold higher affinity than the apoprotein B-containing lipoprotein LDL; failed to bind canine HDL2 and human HDL3, which are devoid of apoproteins B and E; failed to bind methylated LDL; required calcium; and were destroyed by Pronase. Treatment of dogs with mevinolin not only increased the fractional catabolic rate for LDL but also reduced the synthetic rate for the lipoprotein. The current data suggest that the liver of dogs contains functional LDL receptors that are susceptible to metabolic regulation and that a drug-induced increase in the activity of these receptors can contribute to a lowering of plasma levels of LDL-cholesterol.
Molecular organization in micelles and vesicles
Authors: Dill Ken A. , Flory Paul J.
Published: May 2006

The configurations of the hydrocarbon chains in micelles are severely constrained by the space-filling requirements of the chain segments, by the continuity of the chains, and by the micellar geometry. A statistical theory that takes full account of these constraints is developed by using a lattice model. The chain ends are deduced to be nonuniformly distributed, with maximal incidence approximately midway between the center of the micelle and the outer surface. Whereas the chain disorder near the outside of the hydrophobic core may approach that of a liquid, crowding of the chains near the core center imposes a degree of order approaching that in a crystal. These results are at variance with the prevailing view that the micellar interior resembles a “liquid hydrocarbon droplet.” Also discussed are the effects of curvature on the chain configurations in monolayers and bilayers. It is found, for example, that the “disorder gradients” in inner and outer half-bilayers of small vesicles should be substantially different. Implications of these results are discussed.
Reaction site of carboxanilides and of thenoyltrifluoroacetone in complex II.
Authors: Ramsay R R , Ackrell B A , Coles C J , Singer T P , White G A , Thorn G D
Published: May 2006

Oxathiin carboxanilides are systemic fungicides that inhibit the oxidation of succinate by interrupting electron transport between succinate dehydrogenase [succinate:(acceptor) oxidoreductase, EC 1.3.99.1] and coenzyme Q. Kinetic and electron paramagnetic resonance studies have established that the specific binding site of carboxanilides and of thenoyltrifluoroacetone responsible for the inhibition is the same. Although the binding of carboxanilides to membrane preparations of the dehydrogenase is very tight (Ki = 0.01-0.1 microM), it is noncovalent. Identification of the membrane component(s) to which specific binding occurs has therefore required the introduction of a photoaffinity label onto the carboxanilide molecule. By using [G-3H]3'-azido-5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxanilide, it was found, in accord with earlier data with other carboxanilides, that unresolved complex II specifically binds about 0.6 mol of the inhibitor per mol of succinate dehydrogenase in equilibrium dialysis experiments. The resolved components of the complex, succinate dehydrogenase and the two binding peptides CII-3 and CII-4, failed to bind the inhibitor; however, when these were recombined with reconstitution of coenzyme Q reductase activity, the initial binding titer was restored. Azidocarboxanilide-inhibited complex II was irradiated to generate covalent linkages with the binding site, and the components of the complex were separated on polyacrylamide gel. Most of the specifically bound inhibitor was found in the low molecular weight binding peptides and phospholipids.
Enzymatic release of 7-methylguanine from methylated DNA by rodent liver extracts.
Authors: Margison G P , Pegg A E
Published: May 2006

Rat and hamster liver extracts were found to contain DNA glycosylases capable of removing 3-methyladenine and 7-methylguanine from methylated DNA. The activity of 7-methylguanine-DNA glycosylase was greater tin hamster than in rat liver extracts. This finding is consistent with previous reports that the half-life of 7-methylguanine in DNA after treatment with the carcinogen dimethylnitrosamine is longer in rats than in hamsters. These enzymes may, therefore, play an important role in the removal of abnormal alkylation products from mammalian cell DNA. Rodent liver extracts also contained a DNA glycosylase able to remove from alkylated DNA the imidazole-ring-opened form of 7-methylguanine which is produced by treatment with alkali. Although this product may occur in vivo after treatment with alkylating agents to only a very small extent, the enzyme may be needed to minimize its potentially harmful biological effects.
Antibodies to rabbit progesterone receptor: crossreaction with human receptor.
Authors: Logeat F , Hai M T , Milgrom E
Published: May 2006

Progesterone receptor from rabbit uterine cytosol was purified to a specific activity of approximately 2 nmol of bound hormone per mg of protein. A goat was immunized with this preparation and, after two injections of 0.7-0.8 nmol, yielded antireceptor antibodies. The antiserum reacted with both cytosolic and nuclear rabbit progesterone receptor and also with progesterone receptor from other rabbit tissues (vagina and pituitary). A crossreaction was observed with progesterone receptors from other mammalian, especially human, tissues (cytosolic receptor from rat and guinea pig uterus, cytosolic receptor from human breast cancer, and nuclear receptor from human endometrium). On the contrary, there was no interaction with a nonmammalian receptor (chicken oviduct progesterone receptor). The antibodies did not crossreact with other rabbit steroid receptors (uterine estradiol receptor and liver glucocorticoid receptor) or with nonreceptor progesterone-binding proteins (transcortin from plasma and uteroglobin from uterine fluid).
Nucleotide sequence of the thymidine kinase gene of herpes simplex virus type 1.
Authors: Wagner M J , Sharp J A , Summers W C
Published: May 2006

We have determined the complete nucleotide sequence of the thymidine kinase (ATP:thymidine 5' phosphotransferase, EC 2.7.1.21) gene of herpes simplex virus type 1 strain CL101 from a plasmid clone of viral DNA derived by Enquist et al. [Enquist, L. W., Vande Woude, G. F., Wagner, M., Smiley, J. R. & Summers, W. C. (1979) Gene 7, 335-342]. A cDNA copy of the 5' end of thymidine kinase mRNA was also analyzed to locate the transcribed sequences. The transcribed portion of the gene is approximately 1300 nucleotides in length and appears to contain no intervening sequences. There is an untranslated region of 107 nucleotides at the 5' end of the mRNA followed by an open reading frame of 1128 nucleotides. The gene is thus capable of coding for a protein of 376 amino acids. Sequences similar to those thought to be involved in control of transcription and translation of a variety of eukaryotic and viral genes such as a "Hogness box" and A-A-T-A-A-A polyadenylylation signals are also present in the herpesvirus thymidine kinase gene.
Induction of the Z conformation in poly(dG-dC).poly(dG-dC) by binding of N-2-acetylaminofluorene to guanine residues.
Authors: Santella R M , Grunberger D , Weinstein I B , Rich A
Published: May 2006

Poly(dG-dC).poly(dG-dC) and poly(dG).poly(dC) were modified by treatment with N-acetoxy-N-2-acetylaminofluorene, and their conformations were examined by circular dichroism and susceptibility to nuclease S1 digestion. A sample of poly(dG-dC).poly(dG-dC) modified to an extent of 28% with acetylaminofluorene (AAF) at the C(8) position of the deoxyguanosine residues showed a circular dichroism spectrum that had the characteristics of the Z conformation seen in unmodified poly(dG-dC).poly(dG-dC) at high ethanol or salt concentrations. A sample of poly(dG-dC).poly(dG-dC) modified only 3% by AAF showed a spectrum characteristic of the B form of DNA. However, it was converted to the Z form at ethanol concentrations lower than required to convert unmodified poly(dG-dC).poly(dG-dC) from the B to the Z form. Poly(dG).poly(dC), which does not undergo the B-to-Z transition at high ethanol concentrations, did not show any large conformational changes with high AAF modification. Susceptibility to digestion with nuclease S1 also suggested differences in the conformations of the two modified polynucleotides. Poly(dG-dC).poly(dG-dC) modified by AAF to an extent of 28% was almost completely resistant to nuclease S1 digestion. However, both poly(dG).poly(dC) and DNA modified to similar levels by AAF were highly susceptible to nuclease S1 digestion. Two different conformations for AAF-modified deoxyguanosine are proposed, depending on whether its position is in alternating purine-pyrimidine sequences or in random-sequence DNA.
Partial purification and characterization of human gamma (immune) interferon.
Authors: Yip Y K , Pang R H , Urban C , Vilcek J
Published: May 2006

Human gamma (immune) interferon (IFN-gamma) was produced in lymphocyte cultures stimulated with a phorbol ester (12-O-tetradecanoylphorbol 13-acetate) and purified phytohemagglutinin. Physicochemical analysis showed that human IFN-gamma is a glycoprotein with an isoelectric point around 8.6 and an apparent molecular weight of 58,000 +/- 3000. A purification process for IFN-gamma was developed consisting of sequential chromatographic separations on controlled-pore glass, concanavalin A-Sepharose, and Bio-Gel P-200. This purification process resulted in an increase in specific activity from about 10(4) (crude culture fluid) to an estimated 10(7) units per mg of protein with a cumulative recovery of about 40% of the IFN activity.
Immune labeling of the D and E regions of human fibrinogen by electron microscopy.
Authors: Norton P A , Slayter H S
Published: May 2006

Human fibrinogen was digested with trypsin to yield core fragments D and E, and antibodies were made against the isolated fragments. The Fab' fragments derived from these antibodies were mixed with native fibrinogen, resulting in the formation of soluble immune complexes. These were rotary shadowed with platinum or negatively contrasted with uranyl acetate and examined by electron microscopy. Fab' from anti-D immunoglobulin was found to be attached to the outer nodules of fibrinogen with a frequency of 79% prior to affinity purification and 91% afterward. Fab' from anti-E immunoglobulin attached to the central nodule with a frequency of 78% prior to affinity purification and 82% afterward. The evidence clearly identifies fragment D produced by plasmin or trypsin digestion of fibrinogen with the outer nodules and the single fragment E, with the central nodule.
Isolation, culture, and immunocytochemical characterization of epididymal epithelial cells from pubertal and adult rats.
Authors: Kierszenbaum A L , Lea O , Petrusz P , French F S , Tres L L
Published: May 2006

A method for the isolation and culture of epididymal epithelial cells obtained from pubertal and old adult rats is described. This method permits the establishment of primary cultures of these cells in monolayers from aggregates isolated from whole epididymides and major epididymal anatomical segments (caput, corpus, and cauda) after trypsin and collagenase digestions. A large number of cultured epididymal cells retain a differentiated function as demonstrated by the immunocytochemical and radioimmunoassay finding of acidic epididymal glycoprotein, a spermatozoa-coating protein secreted by the principal cells of rat epididymis. The proliferative potential of cultured epididymal cells obtained from pubertal and old adult donors can be documented by [3H]thymidine labeling and mitotic indices without significant loss of gene expression for acidic epididymal glycoprotein. Results of this study demonstrate that epididymal epithelial cells, consisting of a predominant population of principal cells, can be isolated, cultured, and maintained for up to 3 months.
Modulation of fertilization in vitro by peptides released during hamster sperm-zona pellucida interaction.
Authors: Hartmann J F , Hutchison C F
Published: May 2006

The penetration of the zona-pellucida by hamster sperm in vitro (assay drops) was inhibited by aliquots of ultrafiltrates obtained from another set of drops (experimental drops) in which capacitated sperm had interacted with the surface of the zona pellucida. This inhibition was inversely related to the concentration of sperm in the assay drop, and it was noted that the inhibitory response declined when the gametes in the assay drop were incubated together beyond the standard 90-min period. Thus, inhibition was not irreversible. A number of criteria suggest that the inhibitory activity resides in at least some of the peptides (S1 factors) released at four different times after contact between the surfaces of the sperm and zona pellucida. Like the S1 factors, the substances responsible for the inhibitory activity of the supernatants, collected at 2 and 50 min after gamete combination, traversed ultrafilters with Mr cutoffs of 5000 and 2000, respectively, and were inactivated by subtilisin. In addition, the quantity of inhibitory material recovered in the ultrafiltrates from experimental drops containing various numbers of eggs was well correlated with the amount of 2- and 50-min S1 factors from similar experimental drops. These results suggest that the S1 factors are peptide modulators of the penetration process and it is speculated that one of their functions may be to prevent or at least to discourage polyspermic penetration of the hamster zona pellucida.
Metabolic stimulation by light in a pigmented freshwater invertebrate
Authors: Byron Earl R.
Published: May 2006

The pigmentation of terrestrial invertebrates has been shown to function in the optimization of solar warming, yet pigmented aquatic invertebrates have never been examined for the ability to utilize solar illumination in a similar manner. The degree of carotenoid pigmentation in calanoid copepods is easily quantified, and comparisons between populations suggest that variation in copepod pigmentation is related to variation in water temperature, with the most darkly pigmented copepods occurring in the coldest lakes. Darkly pigmented copepods show significantly higher metabolic rates in the light than in the dark, whereas lightly pigmented copepods do not. The metabolic experiments provide evidence that dark coloration is adaptive to cold-water copepods because it facilitates significant internal warming of tissues by sunlight. This hypothesis may be used to explain the pigmentation of many other cold-water invertebrates.
Does OKT3 monoclonal antibody react with an antigen-recognition structure on human T cells?
Authors: Chang T W , Kung P C , Gingras S P , Goldstein G
Published: May 2006

OKT3 monoclonal antibody to human T cells inhibits the target cell lysis mediated by allogeneic cytotoxic T cells and the generation of these effector cells in mixed lymphocyte culture. This marked inhibition of cell-mediated lysis is not found with other monoclonal antibodies also reactive with cell surface antigens of human T cells (OKT1, OKT4, OKT5, OKT6, OKT8, and OKT11). OKT3 antibody is mitogenic and this effect appears to require receptor activation in that it occurs at low concentrations (10(-12) M range) of OKT3 antibody, requires intact OKT3 IgG, and is inhibited by a factor(s) in human plasma. By contrast, the inhibition of allogeneic cell-mediated lysis by OKT3 antibody appears to be due to steric hindrance in that it requires higher concentrations of OKT3 antibody (10(-8) M range), Fab fragments retain approximately 10% activity, and inhibition is demonstrable in the presence of human plasma. These findings are consistent with the suggestion that OKT3 antibody reacts with the human T-cell antigen-recognition structure.
Isolation of lymphocyte membrane complement receptor type two (the C3d receptor) and preparation of receptor-specific antibody.
Authors: Lambris J D , Dobson N J , Ross G D
Published: May 2006

A glycoprotein binding complement component C3d was isolated from media used for culture of Raji human lymphoblastoid cells. Analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and gas/liquid chromatography indicated that the C3d-binding glycoprotein consisted of a single polypeptide chain with extensive intrachain disulfide bonds, a molecular weight of 72,000, and several different bound carbohydrates. Several lines of evidence indicated that this medium-derived C3d-binding protein originated from membrane complement receptor type two (CR2, the C3d receptor), presumably shed during membrane turnover. The C3d-binding protein bound to sheep erythrocytes coated with C3d (EC3d) but not to sheep erythrocytes coated with C3b (EC3b). Antisera, prepared by immunization with the purified C3d-binding glycoprotein, inhibited lymphocyte rosette formation with EC3d but not with EC3b. Analysis by sodium dodecyl sulfate gel electrophoresis of the radiolabeled and solubilized lymphocyte antigens reactive with the anti-C3d-binding protein sera revealed a single-chain cell-surface protein of molecular weight 72,000 that was apparently identical to the isolated C3d-binding protein. Parallel assay of lymphocytes for Cr2 by direct immunofluorescence with F(ab')2 anti-C3d-binding protein (anti-CR2) and rosette formation with EC3d indicated that both assays had the same specificity and nearly the same sensitivity. With both systems CR2 expression was limited to B cells, and was undetectable on T cells, monocytes, or neutrophils.
Characterization and distribution of nucleic acid sequences of a novel type C retrovirus isolated from neoplastic human T lymphocytes.
Authors: Reitz M S , Poiesz B J , Ruscetti F W , Gallo R C
Published: May 2006

A type C retrovirus (designated HTLV) recently isolated from a cell line derived from a lymph node and later from peripheral blood of a person with cutaneous T-cell lymphoma (mycosis fungoides) was characterized by nucleic acid hybridization experiments. HTLV [3H]cDNA hybridized 90% to its own 70S RNA with kinetics consistent with the genetic complexity of other retroviruses, but it did not hybridize substantially to RNA or proviral DNA from any animal retroviruses (types B, C, and D), including those from nonhuman primates. Conversely, [3H]cDNA from other retroviruses did not hybridize to RNA or DNA of the human T-cell line producing HTLV. HTLV proviral sequences were present (two to three copies per haploid genome) in DNA of these cells, and homologous sequences were present in the cell cytoplasmic RNA (0.3% viral sequences by weight). HTLV-related nucleic acid sequences were not found in DNA from various other human tissues. The results indicate that HTLV is a new class of type C virus that is not an endogenous (genetically transmitted) retrovirus in man.
Spectrophotometry of H II regions in the spiral galaxy M101
Authors: Sedwick K. E. , Aller L. H.
Published: May 2006

Spectral line intensity data are presented for ionized hydrogen regions in the giant spiral galaxy M101. The influence of interstellar extinction is assessed and electron temperatures of the gas clouds are derived.
Identification of endogenous binding proteins for the lectin discoidin-I in Dictyostelium discoideum.
Authors: Breuer W , Siu C H
Published: May 2006

Recent biochemical and genetic evidence has shown that the endogenous lectin discoidin-I is involved in intercellular adhesion during development of the cellular slime mold Dictyostelium discoideum. We have prepared discoidin-I affinity columns and used them to isolate the lectin receptors. By using the cell surface radioiodination method, 11 discoidin-I binding proteins were identified in wild-type NC4 cells by gel electrophoresis, with apparent molecular weights of 95,000, 85,000, 78,000, 72,000, 60,000, 33,000, 31,000, 28,000, 25,000, 21,000, and 15,000. Only three (gp33, gp31, and gp28) were under developmental regulation. The amount of gp31 increased 8- to 10-fold during aggregation, and it was the predominant discoidin-I binding protein synthesized at the aggregation stage. Discoidin-I binding proteins derived from aggregation stage cells were potent inhibitors of discoidin-I in a hemagglutination assay in vitro. The same preparation was found to promote aggregation of cells bearing discoidin-I on the surface, suggesting a multivalent interaction.
31P NMR studies of intracellular pH and phosphate metabolism during cell division cycle of Saccharomyces cerevisiae.
Authors: Gillies R J , Ugurbil K , den Hollander J A , Shulman R G
Published: May 2006

We have analyzed changes in intracellular pH and phosphate metabolism during the cell cycle of Saccharomyces cerevisiae (NCYC 239) by using high-resolution 31P NMR spectroscopy. High-density yeast cultures (2 x 10(8) cells per ml) were arrested prior to "start" by sequential glucose deprivation, after which they synchronously replicated DNA and divided after a final glucose feeding. Oxygenation of arrested cultures in the absence of glucose led to increased levels of sugar phosphates and ATP and an increase in intracellular pH. However, these conditions did not initiate cell cycle progression, indicating that energization is not used as an intracellular signal for initiation of the cell division cycle and that the cells need exogenous carbon sources for growth. Glucose refeeding initiated an alkaline intracellular pH transient only in the synchronous cultures, showing that increased intracellular pH accompanies the traversal of start. Changes in phosphate flow and utilization also were observed in the synchronous cultures. In particular, there was increased consumption of external phosphate during DNA synthesis. When external phosphate levels were low, the cells consumed their internal polyphosphate stores. This shows that, under these conditions, polyphosphate acts as a phosphate supply.
Capacity for alternating sites cooperativity in catalysis by succinyl-coenzyme A synthetase.
Authors: Wolodko W T , O'Connor M D , Bridger W A
Published: May 2006

Succinyl-coenzyme A synthetase [succinate:CoA ligase (ADP-forming), EC 6.2.1.5] of Escherichia coli in an alpha 2 beta 2 tetramer. A histidyl residue in the alpha subunit is phosphorylated as a catalytic intermediate. It has been suggested [Bild, G. S., Janson, C. & Boyer, P. D. (1980) J. Biol. Chem. 255, 8109--8115] that the mechanism of action of this enzyme involves intersubunit cooperativity in which attachment of substrates at one of the two active sites promotes catalytic events at the other. This scheme would require that the two active sites, although otherwise equivalent, should act alternately. We have prepared a hybrid enzyme species that contains one 35S-labeled alpha subunit (dephosphorylated), one nonradioactive alpha subunit (phosphorylated), and two beta subunits per tetrameric molecule. With the aid of a selective chromatographic procedure for the isolation of peptides that contain phosphohistidyl residues, we have shown that each of the alpha subunits undergoes phosphorylation when the hybrid enzyme is exposed briefly to substrates. This result demonstrates that the two active sites are capable of alternate activity and lends support to the concept of alternating sites cooperativity. The half-of-the-sites phosphorylation that occurs with this enzyme is not a consequence of permanent asymmetry or other lack of equivalence of the two alpha subunits.
Procedure for production of hybrid genes and proteins and its use in assessing significance of amino acid differences in homologous tryptophan synthetase alpha polypeptides.
Authors: Schneider W P , Nichols B P , Yanofsky C
Published: May 2006

Hybrid tryptophan synthetase alpha and beta polypeptides were produced by genetic recombination between the trpB--trpA regions of Escherichia coli and Salmonella typhimurium contained on compatible, multicopy plasmids. Intragenic recombination was decreased but still evident in recA cells. Genetic exchange occurred at many sites within trpA, but every recombinant gene produced a functional alpha polypeptide despite many amino acid differences from one or the other of the parental polypeptides. The five hybrid tryptophan synthetase alpha subunits examined resembled the parental polypeptides in catalytic function but differed in thermostability. The stability differences suggest that, as amino acid changes occurred in these proteins during the course of evolution, subsequent changes were limited to those that would allow retention of a desired protein conformation.
Released protease-nexin regulates cellular binding, internalization, and degradation of serine proteases.
Authors: Low D A , Baker J B , Koonce W C , Cunningham D D
Published: May 2006

Protease-nexin (PN), a component released by normal human fibroblasts into culture medium, forms covalent linkages with thrombin (Th) and the urinary plasminogen activator urokinase, apparently with their catalytic site serines. The present studies explored the function of PN by examining the interaction of protease-PN complexes with human fibroblasts and the consequences of this interaction. Th-PN and urokinase-PN complexes bind to cells via the PN portion of the complexes. The binding is selectively inhibited by heparin. Because PN has a heparin-binding site, this indicates that protease-PN complexes might bind to a cellular heparin-like site. After binding, the complexes are internalized. By inhibiting endocytosis with phenylarsine oxide, which does not affect cellular binding of Th-PN complexes, we showed that complexes must be internalized before they are degraded. Kinetic analysis of internalization and degradation of Th-PN showed that complexes are internalized more rapidly than they dissociate from the cell surface; by 120 min of incubation at 37 degrees C most cell-bound Th-PN complexes are degraded to amino acids. The results are summarized in a model showing how PN mediates the cellular binding, internalization, and degradation of serine proteases through formation of protease-PN complexes. This series of events may be involved in the regulation of serine protease activity at the cell surface and in the extracellular environment.
Polyamines are necessary for the survival of human small-cell lung carcinoma in culture.
Authors: Luk G D , Goodwin G , Marton L J , Baylin S B
Published: May 2006

Many human small-cell lung carcinoma culture lines grow as multicellular aggregate spheroids, for which high L-dopa decarboxylase activity is a marker. During the initial cell aggregation and the exponential growth phase, there is a marked increase in ornithine decarboxylase activity and an accumulation of polyamines. alpha-Difluoromethylornithine, a specific enzyme-activated, irreversible ornithine decarboxylase inhibitor, blocks the increase in ornithine decarboxylase activity and in polyamines and inhibits human small-cell lung carcinoma cell growth. After the onset of a decreased proliferation rate, the multicellular spheroid aggregates become poorly formed, cell loss ensues, and there is a decrease in L-dopa decarboxylase activity. These findings support the hypothesis that ornithine decarboxylase and the polyamines play an essential role not only in the proliferative phase but also in the viability of human small-cell lung carcinoma cells in culture. The results suggest that alpha-difluoromethylornithine, a virtually nontoxic compound, may be potentially useful in the therapy of this human tumor.
Micropinocytic ingestion of glycosylated albumin by isolated microvessels: possible role in pathogenesis of diabetic microangiopathy.
Authors: Williams S K , Devenny J J , Bitensky M W
Published: May 2006

Microvessels isolated from rat epididymal fat exhibit differential vesicular ingestion rates for unmodified and non-enzymatically glycosylated rat albumin. While unmodified rat albumin is excluded from ingestion by endothelial micropinocytic vesicles, glycosylated albumin is avidly taken up by endocytosis. Interaction of albumin and glycosylated albumin with endothelium was studied with a double-label fluorescence assay of micropinocytosis. When glycosylated albumin was present at a concentration of 6% with respect to total albumin (the level found in "non diabetic" serum), only glycosylated albumin was ingested. At higher concentrations of glycosylated albumin (those found in diabetic serum), both albumin and glycosylated albumin are ingested. Glycosylation of endothelial membrane components results in stimulated ingestion of glycosylated albumin, persistent exclusion of unmodified albumin, and unaltered micropinocytic ingestion of native ferritin. These results indicate that nonenzymatic glycosylation of serum albumin may result in rapid vesicle-mediated extravasation of albumin. Chronic microvascular leakage of glycosylated albumin could contribute to the pathogenesis of diabetic microangiopathy.
Intercellular communication in plants: Evidence for a rapidly generated, bidirectionally transmitted wound signal
Authors: Davies Eric , Schuster Anne
Published: May 2006

Wounding (whether by excision, abrasion, or puncture) elicited rapid, massive, and enduring formation of polysomes in aged pea stems and other mature tissues. The response depended on temperature and severity of wounding but not on water uptake, and it occurred in tissues adjacent to, distant from, above, and below the site of injury. The kinetics of polysome formation were similar in tissues adjacent to or up to 150 mm distant from the point of injury. The wound-induced increases in protein-synthesizing capacity of the polysomes both
in vivo
and
in vitro
were much greater than the increases in rRNA and poly(A)
+
RNA. The results indicate that wounding evokes the almost immediate production of a wound signal that travels rapidly both acropetally and basipetally to stimulate the recruitment of preexisting ribosomes onto primarily preexisting mRNA, thence forming polysomes with greatly enhanced protein-synthesizing capacity.
Cytotoxic T lymphocyte hybridomas that mediate specific tumor-cell lysis in vitro.
Authors: Kaufmann Y , Berke G , Eshhar Z
Published: May 2006

Cytotoxic hybridomas were generated by polyethylene glycol-induced fusion of cytotoxic T lymphocytes (CTL) and BW5147 lymphoma cells. The CTL populations used for fusion were obtained from BALB/c (H-2d) mice primed with leukemia EL4 of C57BL/6 (H-2b) and restimulated either in vivo or in vitro. To circumvent possible CTL-mediated nonspecific lysis of BW5147 cells during fusion, the CTL were transiently inactivated by trypsin prior to fusion. Four cytolytically active hybridomas were obtained, cloned, and subcloned. Hybrid clones lysed all H-2b leukemic target cells tested but not lipopolysaccharide- or concavanalin A-stimulated C57BL/6 lymphoblasts or non-H2b target tumor cells. The mechanism of hybridoma-mediated killing of target cells in vitro appears to be similar to that of parental CTL, although some differences have been observed. The hybridomas appear to possess neither natural killing nor antibody-dependent cytolytic activity. Clones of hybrids propagated in culture for over 6 months without the addition of known external stimulus (i.e., independent of cell growth factor and antigen) exhibit specific lytic activity against H-2b tumor cells. Such autonomous hybridomas will provide a tool for studying the mechanism of CTL-mediated lysis and the nature of the CTL receptors.
Reactivity of Langerhans cells with hybridoma antibody.
Authors: Fithian E , Kung P , Goldstein G , Rubenfeld M , Fenoglio C , Edelson R
Published: May 2006

Reactivity of a monoclonal antibody with human Langerhans cells was demonstrated by a double-labeling immunofluorescence technique. Ia-bearing cells of the epidermis (Langerhans cells) were reactive with this antibody both in frozen sections and in cell suspensions prepared from human epidermis. This monoclonal antibody was unreactive with non-Ia-bearing epidermal cells and with peripheral blood B cells, T cells, and monocytes but did not bind to 70% of intrathymic lymphocytes. These observations further distinguish Langerhans cells from classical monocytes. Furthermore, this monoclonal antibody is a highly specific marker for the in vivo identification and in vitro isolation of Langerhans cells.
Binding of amphiphilic peptides to phospholipid/cholesterol unilamellar vesicles: a model for protein--cholesterol interaction.
Authors: Fukushima D , Yokoyama S , Kézdy F J , Kaiser E T
Published: May 2006

In earlier studies, we prepared a docosapeptide, 1, designed with minimum homology as an amphiphilic alpha-helical model of apolipoprotein A-I (apo A-I) and described its lipid-binding characteristics, surface properties, and enzyme-activating ability. Although the affinity of 1 for egg lecithin unilamellar vesicles was comparable with that for the binding of apo A-I, the affinity of 1 for mixed lecithin/cholesterol (4:1 mol/mol) vesicles was less than that of apo A-I. It appeared possible that the 3-hydroxyl group of cholesterol may have a deleterious interaction with the hydrophobic portion of the amphiphilic helix of 1 that is inserted into the vesicles. Examination of the amphiphilic alpha-helical segments of apo A-I suggested that the preferential interaction of apo A-I with the mixed vesicles might be due to the presence of polar arginine residues in the otherwise hydrophobic regions of two of the helices. Therefore, we synthesized a model docosapeptide, 2, corresponding to the sequence of 1 but containing arginine rather than leucine at position 10 in the hydrophobic region of the alpha helix to assess the role of the alcohol function of cholesterol in protein--cholesterol interactions. The results of studies on the binding of 2 to unilamellar vesicles containing lecithin only, lecithin/cholesterol, lecithin/cholesterol hemisuccinate, or lecithin/cholesterol methyl ether were consistent with the postulate that the major role of cholesterol in the binding of proteins to phospholipid surfaces is the creation of free space between the phospholipid head groups that can accommodate the amphiphilic peptide chains at the interface.
Regulation of collagen synthesis by ascorbic acid.
Authors: Murad S , Grove D , Lindberg K A , Reynolds G , Sivarajah A , Pinnell S R
Published: May 2006

After prolonged exposure to ascorbate, collagen synthesis in cultured human skin fibroblasts increased approximately 8-fold with no significant change in synthesis of noncollagen protein. This effect of ascorbate appears to be unrelated to its cofactor function in collagen hydroxylation. The collagenous protein secreted in the absence of added ascorbate was normal in hydroxylysine but was mildly deficient in hydroxyproline. In parallel experiments, lysine hydroxylase (peptidyllysine, 2-oxoglutarate:oxygen 5-oxidoreductase, EC 1.14.11.4) activity increased 3-fold in response to ascorbate administration whereas proline hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate:oxygen oxidoreductase, EC 1.14.11.2) activity decreased considerably. These results suggest that collage polypeptide synthesis, posttranslational hydroxylations, and activities of the two hydroxylases are independently regulated by ascorbate.
Posttranslational uptake and processing of in vitro synthesized ornithine transcarbamoylase precursor by isolated rat liver mitochondria.
Authors: Conboy J G , Rosenberg L E
Published: May 2006

The mitochondrial matrix enzyme ornithine transcarbamoylase (OTCase; ornithine carbamoyltransferase; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) is encoded by a nuclear gene on the X chromosome, synthesized on cytoplasmic ribosomes, and translocated across both mitochondrial membranes. Using specific immunoprecipitation, we presented evidence previously that the primary in vitro translation product of OTCase in rat liver is a polypeptide about 4000 daltons larger than the "mature" OTCase augment subunit purified from homologous mitochondria. In this report we augment the immunological identification of this cell-free translation product (pOTCase) with structural information and show, by electrophoresis of proteolysis products, that pOTCase is structurally similar to mitochondrial OTCase. Moreover, we now demonstrate that, when pOTCase is incubated posttranslationally with isolated rat liver mitochondria, it is converted to the size of mature OTCase and is sequestered within the mitochondria in such a way that it becomes resistant to externally added proteases. Such posttranslational processing is catalyzed specifically by the mitochondrial fraction of rat liver cells and is dependent both on the duration of incubation with mitochondria and on the amount of mitochondrial protein added. We conclude that pOTCase is indeed the bona fide precursor of mitochondrial OTCase and that use of this simplified cell-free system will facilitate analysis of OTCase biogenesis at both the cellular and the molecular level.
LDHk, an unusual oxygen-sensitive lactate dehydrogenase expressed in human cancer.
Authors: Anderson G R , Kovacik W P
Published: May 2006

An unusual isozyme of lactate dehydrogenase (LDH; L-lactate:NAD+ oxidoreductase, EC 1.1.1.27), LDHk, has been described in cells transformed by the Kirsten murine sarcoma virus (KiMSV). This isozyme appears to contain one or more subunits encoded by the transforming gene of KiMSV and is readily distinguished from other isozymes of LDH. Specifically, it is more basic than other LDH isozymes, has an apparent subunit structure of (35,000)4(22,000)1, is essentially inactive if assayed under a normal atmosphere, and is strongly inhibited by GTP and various related compounds. We have examined human cancer and normal tissue controls for expression of an activity like LDHk. In 11 out of 16 human carcinomas, LDHk activity was increased 10- to 500-fold over the level seen in adjoining nontumor tissue. In contrast, other LDH isozymes were increased by only 2- to 5-fold.
Adenosine receptors and behavioral actions of methylxanthines.
Authors: Snyder S H , Katims J J , Annau Z , Bruns R F , Daly J W
Published: May 2006

Central stimulant actions of 10 methylxanthines in mice correlate with affinities for adenosine receptors labeled with N6-[3H]cyclohexyladenosine. Affinities of methylxanthines for adenosine receptors are consonant with central levels attained at behaviorally effective doses. The much higher concentrations of methylxanthines required to influence benzodiazepine receptor binding do not correlate with behavioral potency. N6-(L-Phenylisopropyl)adenosine (L-PIA), a metabolically stable analog of adenosine with high affinity for adenosine receptors, is an extremely potent behavioral depressant, reducing locomotor activity of mice at doses as little as 0.05 mumol/kg. The D isomer, which has much less affinity for adenosine receptors, is much less active as a central depressant. Theophylline stimulates locomotor activity and reverses depressant effects of L-PIA. Caffeine or 1,7-dimethylxanthine, when administered alone, elicits biphasic effects, with locomotor depression at lower doses and stimulation at higher doses. When administered with L-PIA, even low doses of caffeine produce marked stimulation. 3-Isobutyl-1-methylxanthine given alone elicits only behavioral depression. However, like theophylline and caffeine, isobutylmethylxanthine reverses the L-PIA-evoked depression, converting it into pronounced locomotor stimulation. The data strongly suggest that the behavioral stimulant effects of methylxanthines involve a blockade of central adenosine receptors.
Galloping induced by pontine tegmentum damage in rats: a form of "Parkinsonian festination" not blocked by haloperidol.
Authors: Cheng J T , Schallert T , De Ryck M , Teitelbaum P
Published: May 2006

Localized lesions or local applications of gamma-aminobutyric acid (GABA) in the nucleus reticularis tegmenti pontis (NRTP) of rats cause rapidly accelerating forward locomotion. Such "festination" can coexist with blockade of the dopamine system. We suggest that (i) the akinesia produced by dopamine deficiency results at least in part from release of excessive inhibition of locomotion by a neural system whose final common inhibitory path includes the region of the NRTP and (ii) when it occurs in addition to nigrostriatal damage, destruction in the region of the NRTP might be the cause of a form of festination seen in some patients suffering from Parkinsonism.
Nonlinear shear viscosity and long time tails
Authors: Zwanzig R.
Published: May 2006

A theoretical connection between nonlinear shear viscosity and the long time tail of the equilibrium stress-stress correlation function is pointed out. The connection is a consequence of the Goddard-Miller rheological equation of state which takes into account the angular rotation of a fluid in steady uniform shear.
Molecular cloning and comparative analyses of the genomes of simian sarcoma virus and its associated helper virus.
Authors: Gelmann E P , Wong-Staal F , Kramer R A , Gallo R C
Published: May 2006

Closed circular viral DNA of simian sarcoma virus (SSV) and simian sarcoma-associated virus (SSAV) obtained from acutely infected dog cells was purified on preparative agarose gels, cleaved with EcoRI, and cloned in the phage lambda vector Charon 21A. The cloned 9-kilobase SSAV genome (B11) has the same restriction map as the bulk of the unintegrated linear SSAV DNA intermediate. Heteroduplex analysis between an SSV clone (lambda-C60) and an SSAV clone (lambda-B11) showed two substitution loops and one deletion loop. By using detailed restriction enzyme mapping and electron microscopic analysis, we showed that one of the substitution loops corresponds to an inversion of one of the two long terminal repeat units and adjacent cellular sequences in C60. The other substitution loop mapped close to the 3' long terminal repeat. At least part of this region was shown to contain SSV-specific sequences not shared by SSAV. The 1.9-kilobase deletion mapped at 3.5-5.5 kilobases of the linear SSAV genome, corresponding to most, if not all, of the pol gene.
Control region for adenovirus VA RNA transcription.
Authors: Guilfoyle R , Weinmann R
Published: May 2006

Plasmids containing the VA RNA genes of adenovirus are faithfully transcribed by a crude cytoplasmic extract containing DNA-dependent RNA polymerase III [Wu, G.-J. (1978) Proc. Natl. Acad. Sci. USA 75, 2175-2179]. By subjecting these DNA templates to in vitro site-directed mutagenesis with a novel enzyme of Pseudomonas and recloning in pBR322, we have constructed an ordered series of deletions which affect the in vitro transcription of the major RNA polymerase III viral product, VAI RNA. Three regions that are required for specific synthesis of VAI RNA can be defined. One, inside the gene at nucleotides +10 to +76, affects the transcription in an all-or-none fashion. Transcription is initiated on plasmid sequences that replace up to 10 nucleotides downstream from the 5' end of the gene. Variants with deletions past nucleotide +15 do not support the transcription of VAI RNA. Removal of 3'-end sequences downstream from +76 allows correct initiation. A second region, upstream from the initiation site, affects the exact alignment of the first nucleotide of the transcript [Thimmapaya, B., Jones, N. & Shenk, T. (1979) Cell 18, 947-959]. A third region, downstream from +76, encodes signals for termination of transcription, and new signals were brought in with other viral DNA sequences. Transcription competition experiments indicate that the primary site for binding of a transcriptional regulation factor is located between nucleotides +55 and +70 and suggest that the control region is bifunctional. An internal control region for VAI RNA, approximately 60 bases long and 11 bases downstream from the 5' end of the gene, can be defined.
Resolution of cointegrates between transposons gamma delta and Tn3 defines the recombination site.
Authors: Reed R R
Published: May 2006

Transposition of the genetically related insertion elements gamma delta and Tn3 is thought to involve two steps. In the case of transposition from one replicon to another, the first step is fusion of the parent and target replicons with the element appearing in direct orientation at the two junctions. In a subsequent reaction, the cointegrate structure is resolved via a site-specific recombination event. I have constructed two plasmids, each carrying segments of gamma delta and Tn3, that contain the internal resolution site. The tnpR gene product encoded by either Tn3 or gamma delta mediates intramolecular recombination between these two sites. The product of this recombination is a hybrid region that contains gamma delta and Tn3 sequences fused at the point of crossover. DNA sequence analysis of such recombinants indicates that the recombination occurs within a 19-base-pair (bp) region of exact homology between gamma delta and Tn3. The site lies in the 160-bp center intercistronic region, 50 bp before the beginning of the tnpA gene. My results therefore suggest a model for the coupled regulation of the repressor (tnpR) and the transposase (tnpA) genes and site-specific recombination of transposition intermediates. The Tn3/gamma delta recombination system and bacteriophage lambda integration are compared.