Candida krusei produces ethanol without production of succinic acid; a potential advantage for ethanol recovery by pervaporation membrane separation

The development of fermentative yeasts secreting no organic acids is highly desirable for ethanol production coupled with membrane separation processes, because the acidic byproduct, succinic acid, significantly inhibits the membrane permeation of ethanol. Of the Pichia and Candida yeasts tested, Candida krusei IA-1 showed the highest ethanol productivity [55 g L(-1) day(-1) from 150 g L(-1) (w/v) of glucose], comparable to the strains of Saccharomyces cerevisiae, and produced much less of the acid (0.6 g L(-1) day(-1)) than the Saccharomyces strains (1.5-1.8 g L(-1) day(-1)) under semi-aerobic conditions. Interestingly, under aerobic conditions, strain IA-1 showed no production of the acid. Stain IA-1 exhibited a good assimilation of the acid, while S. cerevisiae NBRC 0216 showed no assimilation. The activity of succinate dehydrogenase (SDH) in strain IA-1 was 37.5 mU mg(-1), and 7.8-fold higher than that in S. cerevisiae strain NBRC 0216. More significantly, SDH1 was abundantly transcribed in strain IA-1, different from that in strain NBRC 0216, regardless of the culture conditions. From these results, C. krusei IA-1 efficiently takes up succinic acid and metabolizes it in the Krebs cycle, producing an extremely low level of byproducts in the culture medium. Therefore, C. krusei is not only a promising alternative to S. cerevisiae but also a suitable model for metabolic engineering of S. cerevisiae.     

A new method to study the functional response of Scymnus syriacus (Coleoptera: Coccinellidae) to different densities of Aphis gossypii

Functional response is basic to any investigation of predator–prey relationships. In this study, the functional response of female Scymnus syriacus Marseul (Col.: Coccinellidae) to different densities (10, 20, 40, 60, 80, 100) of third instar nymphs of Aphis gossypii Glover as prey was studied in an open patch experiment in a growth chamber (25 °C, 65 ± 5% RH and a photoperiod of 16L:8D h ). Using logistic regression, a type II functional response for female Scymnus syriacus was determined. The searching efficiency (a') and handling time (T_h) of the female predator using non linear least-square regression were estimated as 0.0769 ± 0.0136 h^? 1 and 0.3103 ± 0.0438 h., respectively. Mean times required for the female predator to settle in a patch were 10.20 ± 4.28, 6.58 ± 2.58, 12.58 ± 4.50, 4.53 ± 1.48, 5.14 ± 2.59, 3.87 ± 3.52 min at different prey densities, respectively. Maximum theoretical predation rate (T/T_h) estimated by Rogers' model for the female predator was 77.34. The proportion of female predators remaining in open patches at the end of the experiment was dependent on prey density (R² = 0.876). The type of functional response obtained here agrees with studies on this predator in closed patches.     

Herbicide-resistant tobacco plants expressing the fused enzyme between rat cytochrome P4501A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase.

Transgenic tobacco (Nicotiana tabacum cv Xanthi) plants expressing a genetically engineered fused enzyme between rat cytochrome P4501A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase were produced. The expression plasmid pGFC2 for the fused enzyme was constructed by insertion of the corresponding cDNA into the expression vector pNG01 under the control of the cauliflower mosaic virus 35S promoter and nopaline synthase gene terminator. The fused enzyme cDNA was integrated into tobacco genomes by Agrobacterium infection techniques. In transgenic tobacco plants, the fused enzyme protein was localized primarily in the microsomal fraction. The microsomal monooxygenase activities were approximately 10 times higher toward both 7-ethoxycoumarin and benzo[a]pyrene than in the control plant. The transgenic plants also showed resistance to the herbicide chlortoluron.     

Two types of coagulogen mRNAs found in horseshoe crab (Tachypleus tridentatus) hemocytes: molecular cloning and nucleotide sequences

The complete cDNA sequence coding for the coagulogen present in the horseshoe crab (Tachypleus tridentatus) hemocytes was determined. Clones carrying cDNA fragments for coagulogen were isolated from a cDNA library of the hemocyte mRNA using synthetic oligodeoxyribonucleotides as probes. The nucleotide sequence analyses of the cloned cDNAs revealed that the hemocyte coagulogen consists of 175 amino acids with 20 amino acids in a presegment, and that there are two types of mRNAs for coagulogen. The two mRNAs exhibited three nucleotide substitutions, two of which were in their protein-coding regions, resulting in two amino acid replacements. Subsequently, two molecular species of coagulogen, named coagulogens type I and type II, were identified by tryptic peptide mapping of the mature proteins isolated from the hemocyte lysate. These results suggest that the two types of coagulogens are first synthesized as preproteins and are incorporated into the granules that are abundantly present in the hemocytes with liberation of the signal peptides.     

Free Fatty acids regulate two galactosyltransferases in chloroplast envelope membranes isolated from spinach leaves

Effects of MgCl₂ and free fatty acids (FFA) on galactolipid:galactolipid galactosyltransferase (GGGT) and UDP-galactose: 1,2-diacylglycerol galactosyltransferase (UDGT) in chloroplast envelope membranes isolated from spinach (Spinacia oleracea L.) leaves were examined. GGGT activity was sigmoidally stimulated by MgCl₂ with a saturated concentration of more than 5 millimolar. Free α-linolenic acid (18:3) caused a drastic increase in GGGT activity under limiting concentrations of MgCl₂, without affecting its maximum activity at higher MgCl₂ concentrations. Free 18:3 alone did not affect the GGGT activity. The effective species of FFA for the stimulation of GGGT activity in the presence of MgCl₂ were unsaturated 16- and 18-carbon fatty acids. GGGT activity was also stimulated by 18:3 in the presence of MnCl₂, CaCl₂ and a high concentration of KCl in place of MgCl₂. UDGT activity was hyperbolically enhanced by MgCl₂ with a saturated concentration of 1 to 2 millimolar. In contrast to GGGT, UDGT was severely inhibited by 18:3, and MgCl₂-induced stimulation was completely abolished by 18:3. Unsaturated 16- and 18-carbon fatty acids were more inhibitory to UDGT than the saturated acids. The dependence of GGGT activity on monogalactosyldiacylglycerol (MGDG) and MgCl₂ concentrations was identical in the envelope membranes isolated from non- and ozone (0.5 microliter/liter)-fumigated spinach leaves, indicating that GGGT remained active in the leaves during ozone fumigation. The results are discussed in relation to the regulation of galactolipid biosynthesis by the endogenous FFA in the envelopes and to the involvement of GGGT in the triacylglycerol synthesis from MGDG in ozone-fumigated leaves.     

Autocrine Regulation of Macrophage Activation via Exocytosis of ATP and Activation of P2Y11 Receptor

It is important to understand the mechanisms that regulate macrophage activation to establish novel therapies for inflammatory diseases, such as sepsis; a systemic inflammatory response syndrome generally caused by bacterial lipopolysaccharide (LPS). In this study, we investigated the involvement of extracellular ATP-mediated autocrine signaling in LPS-induced macrophage activation. We show here that ATP release via exocytosis, followed by activation of P2Y11 receptor, is a major pathway of the macrophage activation, leading to release of cytokines. Treatment of human monocyte THP-1 cells with LPS induced rapid ATP release from cells, and this release was blocked by knockdown of SLC17A9 (vesicular nucleotide transporter, VNUT), which is responsible for exocytosis of ATP. ATP-enriched vesicles were found in cytosol of THP-1 cells. These data suggest the involvement of vesicular exocytosis in the release of ATP. Knockdown of SLC17A9, the P2Y11 antagonist NF157 or knockdown of P2Y11 receptor significantly suppressed both M1-type polarization and IL-6 production in THP-1 cells, indicating an important role of activation of P2Y11 receptor by released ATP in macrophage activation. Next, the effect of NF157 on LPS-induced immune activation was examined in vivo. Administration of LPS to mice caused increase of serum IL-1ß, IL-6, IL-12 and TNF-alpha levels at 3–24 h after the administration. Pre-treatment of LPS-treated mice with NF157 suppressed both elevation of proinflammatory cytokines in serum and M1 polarization of peritoneal/spleen macrophages. Moreover, post-treatment with NF157 at 30 min after administration of LPS also suppressed the elevation of serum cytokines levels. We conclude that vesicular exocytosis of ATP and autocrine, positive feedback through P2Y11 receptors is required for the effective activation of macrophages. Consequently, P2Y11 receptor antagonists may be drug candidates for treatment of inflammatory diseases such as sepsis.     

Crystal structure of CYP105A1 (P450SU-1) in complex with 1alpha,25-dihydroxyvitamin D3

Vitamin D 3 (VD 3), a prohormone in mammals, plays a crucial role in the maintenance of calcium and phosphorus concentrations in serum. Activation of VD 3 requires 25-hydroxylation in the liver and 1alpha-hydroxylation in the kidney by cytochrome P450 (CYP) enzymes. Bacterial CYP105A1 converts VD 3 into 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) in two independent reactions, despite its low sequence identity with mammalian enzymes (<21% identity). The present study determined the crystal structures of a highly active mutant (R84A) of CYP105A1 from Streptomyces griseolus in complex and not in complex with 1alpha,25(OH) 2D 3. The compound 1alpha,25(OH) 2D 3 is positioned 11 A from the iron atom along the I helix within the pocket. A similar binding mode is observed in the structure of the human CYP2R1-VD 3 complex, indicating a common substrate-binding mechanism for 25-hydroxylation. A comparison with the structure of wild-type CYP105A1 suggests that the loss of two hydrogen bonds in the R84A mutant increases the adaptability of the B' and F helices, creating a transient binding site. Further mutational analysis of the active site reveals that 25- and 1alpha-hydroxylations share residues that participate in these reactions. These results provide the structural basis for understanding the mechanism of the two-step hydroxylation that activates VD 3.     

Purification and characterization of the Ca2+ plus Mg2+-dependent endodeoxyribonuclease from calf thymus chromatin

Ca2+ plus Mg2+-dependent endodeoxyribonuclease was extracted from calf thymus chromatin and purified to a state free from contamination by other DNases. This DNase required both Ca2+ and Mg2+, or Mn2+ alone for its activity and the optimum pH for activity was at 6.5-7.5. No specificity for the 5'-base was observed. The molecular weight of the DNase was estimated to be about 25,000-30,000 by glycerol gradient centrifugation. Actin and antibody for pancreatic DNase (DNase I) did not inhibit the enzyme, whereas both strongly inhibited DNase I, suggesting that these two DNases are different enzymes.     

The 717Val----Ile substitution in amyloid precursor protein is associated with familial Alzheimer's disease regardless of ethnic groups

Alzheimer's disease (AD) is a devastating neurological disorder and the leading cause of dementia among aged individuals. The human amyloid beta protein, which is a cleavage product of amyloid precursor protein (APP), is a major component of the amyloid deposited in the brain of patients with AD. By using PCR direct sequencing of exon 17 (encoding part of the beta protein) of the APP gene, we have found that a Japanese AD patient harbours a C to T substitution, responsible for a valine to isoleucine change at position 717, heterogeneously. The mutation is exactly the same as that found in a Caucasian AD family by Goate et al. (1). Furthermore, the mutation was shown to co-segregate with AD in his family. These results suggest that the Val----Ile change in the APP causes AD, regardless of ethnic background.