Inhibition of muscarinic receptor-linked phospholipase D activation by association with tubulin

Mammalian phospholipase D (PLD) is considered a key enzyme in the transmission signals from various receptors including muscarinic receptors. PLD activation is a rapid and transient process, but a negative regulator has not been found that inhibits signal-dependent PLD activation. Here, for the first time, we report that tubulin binding to PLD2 is an inhibition mechanism for muscarinic receptor-linked PLD2 activation. Tubulin was identified in an immunoprecipitated PLD2 complex from COS-7 cells by peptide mass fingerprinting. The direct interaction between PLD2 and tubulin was found to be mediated by a specific region of PLD2 (amino acids 476-612). PLD2 was potently inhibited (IC50 <10 nM) by tubulin binding in vitro. In cells, the interaction between PLD2 and tubulin was increased by the microtubule disrupting agent nocodazole and reduced by the microtubule stabilizing agent Taxol. Moreover, PLD2 activity was found to be inversely correlated with the level of monomeric tubulin. In addition, we found that interaction with and the inhibition of PLD2 by monomeric tubulin is important for the muscarinic receptor-linked PLD signaling pathway. Interaction between PLD2 and tubulin was increased only after 1-2 min of carbachol stimulation when carbachol-stimulated PLD2 activity was decreased. The expression of the tubulin binding region of PLD2 blocked the later decrease in carbachol-induced PLD activity by masking tubulin binding. Taken together, these results indicate that an increase in local membrane monomeric tubulin concentration inhibits PLD2 activity, and provides a novel mechanism for the inhibition of muscarinic receptor-induced PLD2 activation by interaction with tubulin.     

Gram-negative bacteria-binding protein, a pattern recognition receptor for lipopolysaccharide and beta-1,3-glucan that mediates the signaling for the induction of innate immune genes in Drosophila melanogaster cells

Pattern recognition receptors, non-clonal immune proteins recognizing common microbial components, are critical for non-self recognition and the subsequent induction of Rel/NF-kappaB-controlled innate immune genes. However, the molecular identities of such receptors are still obscure. Here, we present data showing that Drosophila possesses at least three cDNAs encoding members of the Gram-negative bacteria-binding protein (DGNBP) family, one of which, DGNBP-1, has been characterized. Western blot, flow cytometric, and confocal laser microscopic analyses demonstrate that DGNBP-1 exists in both a soluble and a glycosylphosphatidylinositol-anchored membrane form in culture medium supernatant and on Drosophila immunocompetent cells, respectively. DGNBP-1 has a high affinity to microbial immune elicitors such as lipopolysaccharide (LPS) and beta-1,3-glucan whereas no binding affinity is detected with peptidoglycan, beta-1,4-glucan, or chitin. Importantly, the overexpression of DGNBP-1 in Drosophila immunocompetent cells enhances LPS- and beta-1,3-glucan-induced innate immune gene (NF-kappaB-dependent antimicrobial peptide gene) expression, which can be specifically blocked by pretreatment with anti-DGNBP-1 antibody. These results suggest that DGNBP-1 functions as a pattern recognition receptor for LPS from Gram-negative bacteria and beta-1, 3-glucan from fungi and plays an important role in non-self recognition and the subsequent immune signal transmission for the induction of antimicrobial peptide genes in the Drosophila innate immune system.     

Structures of ovine corticotropin-releasing factor and its Ala32 mutant as studied by CD and NMR techniques

The corticotropin-releasing factor (CRF) is a 41-amino acid peptide-amide hormone, which mediates a general stress-response. It has been reported that the substitution of His-32 in the ovine CRF (oCRF) with Ala brings about a 4.5-fold increase in activity [Kornreich et al. (1992) J. Med. Chem. 35, 1870-76]. Here, we have determined the secondary structure of this Ala-substituted ovine CRF ([Ala32]oCRF) and compare it with that of oCRF using circular dichroism (CD) and NMR techniques in trifluoroethanol (TFE) solution, which is known to stabilize the alpha-helix formation. In contrast to an earlier report, it was observed the alpha-helical structure extends to the C-terminus of oCRF. By analyzing the CalphaH and NH chemical shifts, the properties of local structures of oCRF were elucidated. The oCRF and [Ala32]oCRF have stable alpha-helical structures in the middle region, regardless of pH and temperature, and the alpha-helix initiation regions of these peptides are stabilized as the pH is decreased. However, the [Ala32]oCRF has a more stable alpha-helical structure than oCRF in the vicinity of the substitution region, and it is thought that this is the cause of the increased activity of [Ala32]oCRF.     

Thrombin induces NO release from cultured rat microglia via protein kinase C, mitogen-activated protein kinase, and NF-kappa B

Microglia, brain resident macrophages, become activated in brains injured due to trauma, ischemia, or neurodegenerative diseases. In this study, we found that thrombin treatment of microglia induced NO release/inducible nitric-oxide synthase expression, a prominent marker of activation. The effect of thrombin on NO release increased dose-dependently within the range of 5-20 units/ml. In immunoblot analyses, inducible nitric-oxide synthase expression was detected within 9 h after thrombin treatment. This effect of thrombin was significantly reduced by protein kinase C inhibitors, such as Go6976, bisindolylmaleimide, and Ro31-8220. Within 15 min, thrombin activated three subtypes of mitogen-activated protein kinases: extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase/stress-activated protein kinase. Inhibition of the extracellular signal-regulated kinase pathway and p38 reduced the NO release of thrombin-treated microglia. Thrombin also activated nuclear factor kappaB (NF-kappaB) within 5 min, and N-acetyl cysteine, an inhibitor of NF-kappaB, reduced NO release. However, thrombin receptor agonist peptide (an agonist of protease activated receptor-1 (PAR-1)), could not mimic the effect of thrombin, and cathepsin G, a PAR-1 inhibitor, did not reduce the effect of thrombin. These results suggest that thrombin can activate microglia via protein kinase C, mitogen-activated protein kinases, and NF-kappaB but that this occurs independently of PAR-1.     

Recent progress in biomolecular engineering

During the next decade or so, there will be significant and impressive advances in biomolecular engineering, especially in our understanding of the biological roles of various biomolecules inside the cell. The advances in high throughput screening technology for discovery of target molecules and the accumulation of functional genomics and proteomics data at accelerating rates will enable us to design and discover novel biomolecules and proteins on a rational basis in diverse areas of pharmaceutical, agricultural, industrial, and environmental applications. As an applied molecular evolution technology, DNA shuffling will play a key role in biomolecular engineering. In contrast to the point mutation techniques, DNA shuffling exchanges large functional domains of sequences to search for the best candidate molecule, thus mimicking and accelerating the process of sexual recombination in the evolution of life. The phage-display system of combinatorial peptide libraries will be extensively exploited to design and create many novel proteins, as a result of the relative ease of screening and identifying desirable proteins. Even though this system has so far been employed mainly in screening the combinatorial antibody libraries, its application will be extended further into the science of protein-receptor or protein-ligand interactions. The bioinformatics for genome and proteome analyses will contribute substantially toward ever more accelerated advances in the pharmaceutical industry. Biomolecular engineering will no doubt become one of the most important scientific disciplines, because it will enable systematic and comprehensive analyses of gene expression patterns in both normal and diseased cells, as well as the discovery of many new high-value molecules. When the functional genomics database, EST and SAGE techniques, microarray technique, and proteome analysis by 2-dimensional gel electrophoresis or capillary electrophoresis in combination with mass spectrometer are all put to good use, biomolecular engineering research will yield new drug discoveries, improved therapies, and significantly improved or new bioprocess technology. With the advances in biomolecular engineering, the rate of finding new high-value peptides or proteins, including antibodies, vaccines, enzymes, and therapeutic peptides, will continue to accelerate. The targets for the rational design of biomolecules will be broad, diverse, and complex, but many application goals can be achieved through the expansion of knowledge based on biomolecules and their roles and functions in cells and tissues. Some engineered biomolecules, including humanized Mab's, have already entered the clinical trials for therapeutic uses. Early results of the trials and their efficacy are positive and encouraging. Among them, Herceptin, a humanized Mab for breast cancer treatment, became the first drug designed by a biomolecular engineering approach and was approved by the FDA. Soon, new therapeutic drugs and high-value biomolecules will be designed and produced by biomolecular engineering for the treatment or prevention of not-so-easily cured diseases such as cancers, genetic diseases, age-related diseases, and other metabolic diseases. Many more industrial enzymes, which will be engineered to confer desirable properties for the process improvement and manufacturing of high-value biomolecular products at a lower production cost, are also anticipated. New metabolites, including novel antibiotics that are active against resistant strains, will also be produced soon by recombinant organisms having de novo engineered biosynthetic pathway enzyme systems. The biomolecular engineering era is here, and many of benefits will be derived from this field of scientific research for years to come if we are willing to put it to good use.     

Separation of Alcaligenes eutrophus cells containing Poly(3-hydroxybutyrate) from fermentation broth with pretreatment using Al- and Fe-based coagulants

Alcaligenes eutrophus containing intracellular poly(3-hydroxybutyrate) was recovered from fermentation broth by centrifugation and filtration after pretreatment with Al- and Fe-based coagulants. Coagulation efficiency was largely affected by pH, and the optimum pH's for cell recovery were about 4.6-5.6 for the Al-based coagulants and about 5-8 for the Fe-based coagulants. Ammonium ions that combined with metals to form complex compounds increased the coagulant requirement, and the additional requirement of coagulant was found to be proportional to the ammonium concentration. In addition, various ligands in addition to ammonium ions contained in the culture medium interfered with the coagulation reaction and increased the coagulant requirement also. The coagulant requirement increased with the cell concentration regardless of coagulant type. The polymeric coagulants such as PACS, Hi-PAX, and Ferix-3 were more effective than nonpolymeric coagulants of aluminum sulfate and ferrous sulfate. The optimum dosages of the coagulants tested were determined over a broad range of cell concentration of 20.5-210 g/L. It was observed that the energy requirement for centrifugation could be greatly reduced with cell coagulation.     

Purification and partial characterization of a novel glucanhydrolase from Lipomyces starkeyi KSM 22 and its use for inhibition of insoluble glucan formation

A novel glucanhydrolase from a mutant of Lipomyces starkeyi ATCC 74054 was purified. The single protein (100 kDa) showed either dextranolytic or amylolytic activity. We referred to the glucanhydrolase as a DXAMase. The DXAMase was produced in a starch medium and it was 3.75-fold more active for hydrolysis of the purified insoluble-glucan of Streptococcus mutans than Penicillium funiculosum dextranase. Aggregation of S. mutans cells with dextran and adherence to glass were eliminated by incubating with the DXAMase. The addition of DXAMase (0.1 IU/ml) to the mutansucrase reaction digest with sucrose reduced the formation of insoluble-glucan about 80%. Also the DXAMase (0.5 IU/ml) removed 80% of the pre-formed sucrose-dependent adherent film. These in vitro properties of L. starkeyi KSM 22 DXAMase are desirable for its application as a dental plaque control agent.