Breeding of flocculent industrial alcohol yeast strains by self-cloning of the flocculation gene FLO1 and repeated-batch fermentation by transformants

A nonflocculent industrial polyploid yeast strain, Saccharomyces cerevisiae 396-9-6V, was converted to a flocculent one by introducing a functional FLO1 gene at the URA3 locus. The flocculent strain FSC27 obtained was a so-called self-cloned strain, having no bacterial DNA. FSC27 cells could be easily recovered for reuse from fermentation mash without any physical energy. The strain produced a concentration of alcohol as high as 396-9-6V, although the fermentation rate of FSC27 was slightly lower than that of 396-9-6V. When uracil was added to the medium or when URA3 was reintroduced into FSC27 (named FSCU-L18), the fermentation rate and the growth rate increased, and the ethanol concentration produced was higher than that produced by the parent strain. The stable flocculation and high ethanol productivity were observed by using FSCU-L18 during 10 cycles of repeated-batch fermentation test.     

The protective role of localized nitric oxide production during inflammation may be mediated by the heme oxygenase-1/carbon monoxide pathway

Nitric oxide (NO) is an important part of the host defense mechanism; however, it displays both pro- and anti-inflammatory properties depending on its location and concentration. Importantly, excessive or inappropriate NO production can cause tissue damage. Systemic and local administration of NO synthase (NOS) inhibitors ameliorates and may exacerbate the inflammatory response, respectively. Here, we used a carrageenan-induced pleurisy model of acute inflammation in rats to confirm the location-dependent effects of NO and investigate the underlying mechanisms. As expected, localized suppression of NO production exacerbated inflammation, as evidenced by increased pleural exudate volumes and leukocyte counts and enhanced activity of enzymes related to oxidative stress. In contrast, local NO supplementation reduced leukocyte infiltration, vascular permeability, and the activity of oxidative stress-related enzymes. Interestingly, inhibition of heme oxygenase-1 (HO-1) reversed the anti-inflammatory effects of localized NO production, while the addition of hemin (HO-1 substrate) or carbon monoxide (CO; HO-1 metabolite) decreased leukocyte migration and exudation. Together, these findings confirm a protective role for NO at the inflammatory site, which appears to be mediated via NOS induction of the HO-1/CO pathway. Thus, NO supplementation may be a potential new treatment for oxidative stress-associated inflammatory diseases.     

Purification and characterization of a rat liver membrane tyrosine-protein kinase, the possible protooncogene c-yes product, p60c-yes

A tyrosine-protein kinase was purified more than 270-fold from the rat liver plasma membrane fraction by successive column chromatographies on Sephacryl S-300, wheat germ agglutinin-agarose, casein-Sepharose, and hydroxylapatite, followed by isoelectrofocusing electrophoresis. The enzyme with pI of 6.2 was a 60-kDa single polypeptide which represented 42% of total protein. The enzyme reacted quantitatively with a monoclonal antibody to the amino-terminal sequence (Cys-3 to Ser-66) specific to the human c-yes protein, but not with antibodies to the specific amino-terminal sequences of the c-src, fyn, and lck proteins. The purified enzyme contained almost no phosphotyrosine residue but was autophosphorylated with Mg.ATP exclusively at tyrosine residues with concomitant increase in the kinase activity. The rates of autophosphorylation of the enzyme and phosphorylation of tyrosine-glutamate (1:4) copolymers, catalyzed by the enzyme were proportional to the square of enzyme concentration, suggesting that p60c-yes undergoes autophosphorylation through intermolecular catalysis, resulting in stimulation of the enzyme activity. Although the enzyme reaction showed an essential requirement for Mg2+ or Mn2+ with optimal concentrations of 20 and 3 mM, respectively, autophosphorylation significantly activated the enzyme only in the presence of Mg2+. Autophosphorylation of the enzyme reduced the Km for tyrosine-glutamate copolymers and tubulin, but not for ATP, and increased the Vmax of copolymer and tubulin phosphorylation.     

Molecular imaging of single cellulose chains aligned on a highly oriented pyrolytic graphite surface

Individual cellulose macromolecules were successfully visualized on a highly oriented pyrolytic graphite (HOPG) surface by tapping-mode atomic force microscopy under ambient condition. Monomolecular-level dispersion of cellulose chains was achieved through the momentary contact of dilute cellulose/cupri-ethylenediamine (Cu-ED) solution onto the HOPG substrate. Both concentrations of cellulose and Cu-ED provided critical impacts on the topographical images. Single cellulose chains with molecular height of ca. 0.55 nm could be observed under the optimal conditions, showing rigid molecular rods with a unique morphology of hexagonal regularity. It was strongly suggested that the cellulose chains were aligned along the HOPG crystal lattice through a specific attraction, possibly due to a CH-pi interaction between the axial plane of cellulose and the HOPG pi-conjugated system. These phenomena would imply the potential applications of an HOPG substrate for not only nano-level imaging, but also for molecular alignment of cellulose and other structural polysaccharides.     

Structure of the Entire Stalk Region of the Dynein Motor Domain

Dyneins are large microtubule-based motor complexes that power a range of cellular processes including the transport of organelles, as well as the beating of cilia and flagella. The motor domain is located within the dynein heavy chain and comprises an N-terminal mechanical linker element, a central ring of six AAA + modules of which four bind or hydrolyze ATP, and a long stalk extending from the AAA + ring with a microtubule-binding domain (MTBD) at its tip. A crucial mechanism underlying the motile activity of cytoskeletal motor proteins is precise coupling between the ATPase and track-binding activities. In dynein, a stalk region consisting of a long (~ 15 nm) antiparallel coiled coil separates these two activities, which must facilitate communication between them. This communication is mediated by a small degree of helix sliding in the coiled coil. However, no high-resolution structure is available of the entire stalk region including the MTBD. Here, we have reported the structure of the entire stalk region of mouse cytoplasmic dynein in a weak microtubule-binding state, which was determined using X-ray crystallography, and have compared it with the dynein motor domain from Dictyostelium discoideum in a strong microtubule-binding state and with a mouse MTBD with its distal portion of the coiled coil fused to seryl-tRNA synthetase from Thermus thermophilus. Our results strongly support the helix-sliding model based on the complete structure of the dynein stalk with a different form of coiled-coil packing. We also propose a plausible mechanism of helix sliding together with further analysis using molecular dynamics simulations. Our results present the importance of conserved proline residues for an elastic motion of stalk coiled coil and imply the manner of change between high-affinity state and low-affinity state of MTBD.     

Identification of NIPSNAP1 as a nocistatin-interacting protein involving pain transmission

4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) is a molecule of physiologically unknown function, although it is predominantly expressed in the brain, spinal cord, liver, and kidney. We identified NIPSNAP1 as a protein that interacts with the neuropeptide nocistatin (NST) from synaptosomal membranes of mouse spinal cord using high-performance affinity latex beads. NST, which is produced from the same precursor protein as an opioid-like neuropeptide nociceptin/orphanin FQ (N/OFQ), has opposite effects on pain transmission evoked by N/OFQ. The calculated full-length pre-protein of NIPSNAP1 was 33 kDa, whereas the N-terminal truncated form of NIPSNAP1 (29 kDa) was ubiquitously expressed in the neuronal tissues, especially in synaptic membrane and mitochondria of brain. The 29-kDa NIPSNAP1 was distributed on the cell surface, and NST interacted with the 29-kDa but not the 33-kDa NIPSNAP1. Although intrathecal injection of N/OFQ induced tactile allodynia in both wild-type and NIPSNAP1-deficient mice, the inhibition of N/OFQ-evoked tactile allodynia by NST seen in wild-type mice was completely lacking in the deficient mice. These results suggest that NIPSNAP1 is an interacting molecule of NST and plays a crucial role in pain transmission.