Nitrate reductase activity and nitrate accumulation in in vitro produced axillary shoots,plantlets and seedlings of Pinus pinaster

The initial and induced in vivo Nitrate Reductase Activity, the nitrate accumulation by in vitro-produced axillary shoots and plantlets of Pinus pinaster were compared respectively with those of shoots collected from seedlings and whole plants.The usefulness of the nitrate of the medium used for in vitro axillary shoot formation is demonstrated by the occurrence of initial NR activity in the explants. When fed in a non in vitro situation with a 50 mM KNO₃ solution, they have the same induced capacity to reduce nitrate as do shoots from seedlings, even though the latter accumulate less nitrate. Plants regenerated in vitro exhibit an ability to reduce nitrate similar to that of seedlings. In both types of plants, the Nitrate Reductase potential is greater in roots than in shoots.Abbreviation NR Nitrate Reductase - BA 6-Benzyladenine     

Tyrosinase inhibitory flavonoid from Juniperus communis fruits

The fruits of Juniperus communis have been traditionally used in the treatment of skin diseases. In our preliminary experiment, the MeOH extract of J. communis effectively suppressed mushroom tyrosinase activity. Three monoflavonoids and five biflavonoids were isolated from J. communis by bioassay-guided isolation and their inhibitory effect against tyrosinase was evaluated. According to the results of all isolates, hypolaetin 7-O-β-xylopyranoside isolated from J. communis exhibited most potent effect of decreasing mushroom tyrosinase activity with an IC₅₀ value of 45.15 μM. Further study provided direct experimental evidence for hypolaetin 7-O-β-D-xylopyranoside-attenuated tyrosinase activity in α-MSH-stimulated B16F10 murine melanoma cell. Hypolaetin 7-O-β-D-xylopyranoside from the EtOAc fraction of J. communis was also effective at suppressing α-MSH-induced melanin synthesis. This is the first report of the enzyme tyrosinase inhibition by J. communis and its constituent. Therapeutic attempts with J. communis and its active component, hypolaetin 7-O-β-D-xylopyranoside, might be useful in treating melanin pigmentary disorders.     

Papers to Appear in Forthcoming Issues

The Human Trifunctional Enzyme ofde NovoPurine Biosynthesis: Heterologous Expression, Purification, and Preliminary Characterization Mark T. Poch, Wen Qin, and Carol A. CaperelliIsolation and Expression of Murine Carbonic Anhydrase IV Jonathan D. Hurt, Chingkuang Tu, and Philip J. LaipisExpression of Rat Histone H1d inEscherichia coliand Its Purification M. M. Srinivas Bharath, J. R. Khadake, and M. R. S. RaoPolyethylene Glycol Conjugation of Recombinant Methioninase for Cancer Therapy Yuying Tan, Xinghua Sun, Mingxu Xu, Zili An, Xuezhong Tan, Xiuying Tan, Qinghong Han, Dusan A. Miljkovic, Meng Yang, and Robert M. HoffmanExpression inEscherichia coliof the Elongation Factor 1β Gene and Its Nucleotide T160C Mutant from the ArchaeonSulfolobus solfataricusGiuseppe Ianniciello, Mariorosario Masullo, Gennaro Raimo, Paolo Arcari, and Vincenzo BocchiniAn Expression System of Rat Calmodulin using T7 Phage Promoter inEscherichia coliNobuhiro Hayashi, Mamoru Matsubara, Akihiko Takasaki, Koiti Titani, and Hisaaki TaniguchiFunctional Expression of Secreted Mouse BST-1 in Yeast Alamgir M. M. Hussain, Hon Cheung Lee, and Chan Fong ChangEffect of Purification Protocol on the Functional Properties of Erythrocyte Membrane Protein 4.1 Ryan F. Workman and Philip S. LowAcidic Peptide-Mediated Expression of the Antimicrobial Peptide Buforin II as Tandem Repeats inEscherichia coliJae H. Lee, Il Minn, Chan B. Park, and Sun C. Kim.     

Modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity with cAMP and wth protein fractions of rat liver cytosol

3-Hydroxy-3-methylglutaryl coenzyme A reductase activity is diminished in several $\text{in vitro}$ liver systems preincubated in the presence of cAMP. Reductase activity in isolated, washed liver microsomes is inactivated by ATP, Mg⁺⁺, and a protein fraction separated from the liver cytosol. This effect is augmented by 3′–5′ cyclic AMP. Reductase activity in previously inactivated microsomes can be partially restored by incubation with a second protein fraction of the cytosol.     

Halolysin SptA有助于嗜盐古菌Natrinema sp.J7-2长期生存

【背景】嗜盐古菌可以在盐沉积物中存活长达几百万年,是著名的长寿菌。许多嗜盐古菌分泌胞外蛋白酶,大多数分泌的胞外蛋白酶被称为Halolysin,具有以下特征：属于枯草杆菌蛋白酶类蛋白酶;在胞内折叠后经Tat途径高效分泌至胞外;可自加工形成成熟酶;尤其在天然宿主中大多数Halolysin在对数生长后期表达并在稳定期达到最高水平。目前Halolysin的酶学性质、加工成熟及分泌机制已被广泛研究,然而其生理功能的研究较少。Halolysin SptA是嗜盐古菌Natrinema sp.J7-2的主要胞外蛋白酶,前期研究发现多个顺式调控元件协同调节SptA的生长期依赖性表达,使SptA参与J7-2菌株不同生长期之间的转变,而且在衰亡期之后SptA有助于J7-2菌株继续生存。【目的】研究Halolysin SptA对Natrinema sp.J7-2长期生存的作用。【方法】将J7-2菌株和突变体ΔsptA1分别在寡营养、无外源营养物质（液体）及营养丰富（固体）条件下长期培养,通过比较二者的生长、生存和SptA的表达分泌情况进一步探讨SptA的作用。【结果】J7-2菌株在寡营养条件下产生更多SptA,培养后期（33 d） J7-2菌株活细胞数显著高于ΔsptA1。在无外源营养物质情况下长期温育,J7-2菌株和ΔsptA1经历多次细胞分裂和细胞死亡,在延长温育期间（73—200 d）存活的J7-2菌株细胞数量均显著多于存活的ΔsptA1细胞数量。在营养丰富的固体平板上培养的后期（160 d）,由于营养物质消耗,J7-2菌株通过SptA吸收和利用来源于死细胞蛋白的降解产物,帮助其群体长期生存。【结论】SptA介导的细胞死亡和死细胞蛋白降解,促进J7-2菌株利用来源于死细胞的营养物质,从而有助于菌株群体在营养缺乏条件下长期存活。本研究提供了关于Halolysin生理作用的新见解。     

Participation of CD14 in the Phagocytosis of Smooth-Type Salmonella typhimurium by the Macrophage-Like Cell Line,J774.1

The role of CD14 in the phagocytosis and killing of microorganisms was investigated using macrophage-like cell lines, CD14-positive J774.1 cells and CD14-negative mutant J7.DEF3 cells derived from J744.1 cells. The cells were infected with Salmonella typhimurium organisms of the smooth (S)-form LT2, mutant rough (R)-form TV148 or Staphylococcus aureus 248βH. At 30 or 180 min incubation, the cells were washed and disrupted. Colony-forming units (CFUs) liberated from the disrupted cells were determined by quantitative cultivation, and the phagocytic index and killing rate were calculated. Both the phagocytic index and killing rate of J774.1 cells against LT2 organisms were greater than those of J7.DEF.3 cells. However, the index and rate of J774.1 cells against TV148 and 248βH organisms were similar to those of the J7.DEF.3 cells. The phagocytosis of LT2 organisms by J774.1 cells was partially inhibited by S-form LPS (S-LPS) and anti-CD14 antibody, but not by R-chemotype LPS (R-LPS). These results suggest that CD14 participates in the phagocytosis of S-form Salmonella.     

A High-Resolution Map in the Chromosomal Region Surrounding theLpsLocus

TheLpslocus on mouse chromosome 4 controls host responsiveness to lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria. The C3H/HeJ inbred mouse strain is characterized by a mutantLpsallele (Lps^d) that renders it hyporesponsive to LPS and naturally tolerant of its lethal effects. To identify theLpsgene by a positional cloning strategy, we have generated a high-resolution linkage map of the chromosomal region surrounding this locus. We have analyzed a total of 1604 backcross mice from a preexisting interspecific backcross panel of 259 (Mus spretus× C57BL/6J)F1 × C57BL/6J and two novel panels of 597 (DBA/2J × C3H/HeJ)F1 × C3H/HeJ and 748 (C57BL/6J × C3H/HeJ)F1 × C3H/HeJ segregating atLps.A total of 50 DNA markers have been mapped in a 11.8-cM span overlapping theLpslocus. This positions theLpslocus within a 1.1-cM interval, flanked proximally by a large cluster of markers, including three known genes (Cd30l, Hxb,andAmbp), and distally by two microsatellite markers (D4Mit7/D4Mit178). The localization of theLpslocus is several centimorgans proximal to that previously assigned.     

Stable assembly of PsaE into cyanobacterial photosynthetic membranes is dependent on the presence of other accessory subunits of photosystem I

We studied assembly of the PsaE subunit of photosystem I into photosynthetic membranes of cyanobacterial mutant strains that lack specific photosystem I subunits. Radiolabeled PsaE was incubated with photosynthetic membranes, and their binding and assembly were assayed by resistance to removal by chaotropic agents and proteolytic digestion. PsaE incorporated into the wild-type membranes was resistant to these treatments. In the absence of PsaD, it was resistant to proteolytic digestion, but was removed by NaBr. When the membranes were isolated from a mutant strain in which the psaF and psaJ genes have been inactivated, PsaE assembled in vitro could not be removed. PsaE could associate with the membranes of the strain DF in which the psaD, psaJ and psaF genes have been mutated. However, the radiolabeled PsaE associated with these membranes was removed both by the proteolytic as well as by the chaotropic agents. Characterization of PsaE present in vivo revealed similar results. These observations suggest that PsaD and PsaF/J may interact with PsaE and stabilize it in the photosystem I complex.     

Stability and Folding of Endoglucanase I (CEL7B) from Humicoia Insolens

Cellulases are of economic significance, particularly in the detergent and textile industries, where they are subjected to a wide range of operating conditions affecting their stability. To increase our insight into the properties of this class of enzymes, we have carried out a study of the stability and folding behavior of the 413-residue endoglucanase I (Ce17B) from Humicola insolens. Data from chemical denaturation in guanidinium chloride agree satisfactorily with calorimetric measurements, revealing an optimum stability of ca. 20 kcal mol^?1 around p^H 7 and a peak half-width of 3 -4 p^H units. Stability and activity show very similar p^H-profiles, but this is probably fortuitous. Judging from equilibrium m-values (the dependence of the log of the equilibrium unfolding constant on the denaturant concentration), the denatured state becomes significantly more compact outside p^H 6–9.

Folding and unfolding proceed very slowly with relaxation half times up to 6h. Single- and double-jump kinetic data at p^H 7 suggest a folding scheme involving two intermediates with native-like secondary structure but varying degrees of tertiary structure.     

Characterization of four tetranucleotide and six dinucleotide microsatellite markers for use in the tropical freshwater fish Telmatherina antoniae and related species

Ten primer pairs were designed from two genomic libraries enriched for (GACA)₄ and (GACA)₇ in the sailfin silverside Telmatherina antoniae. Characterization with 57 T. antoniae individuals revealed between three and 30 alleles, with observed and expected heterozygosity values ranging from 0.47 to 0.98 and from 0.46 to 0.93, respectively. Eight of the 10 loci conformed to Hardy–Weinberg equilibrium, with no significant linkage detected between loci pairs. These microsatellite markers are intended for use in population genetic studies of T. antoniae and related fishes of the family Telmatherinidae.     

Androgen‐metabolizing enzymes show region‐specific changes across the breeding season in the brain of a wild songbird

The Lapland longspur (Calcarius lapponicus) is an arctic‐breeding songbird that shows rapid behavioral changes during a short breeding season. Changes in plasma testosterone (T) in the spring are correlated with singing but not territorial aggression in males. Also, T treatment increases song but not aggression in this species. In contrast, in temperate‐zone breeders, song and aggression are highly correlated, and both increase after T treatment. We asked whether regional or temporal differences in androgen‐metabolizing enzymes in the longspur brain explain hormone‐behavior patterns in this species. We measured the activities of aromatase, 5α‐reductase and 5β‐reductase in free‐living longspur males. Aromatase and 5α‐reductase convert T into the active steroids 17β‐estradiol (E₂) and 5α‐dihydrotestosterone (5α‐DHT), respectively. 5β‐Reductase deactivates T via conversion to 5β‐DHT, an inactive steroid. We examined seven brain regions at three stages in the breeding season. Overall, aromatase activity was high in the hypothalamus, hippocampus, and ventromedial telencephalon (containing nucleus taeniae, the avian homologue to the amygdala). 5β‐Reductase activity was high throughout the telencephalon. Activities of all three enzymes changed over time in a region‐specific manner. In particular, aromatase activity in the rostral hypothalamus was decreased late in the breeding season, which may explain why T treatment at this time does not increase aggression. Changes in 5β‐reductase do not explain the effects of plasma T on aggressive behavior. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 176–188, 1999     

Reversible inhibition of bacterial bioluminescence by long-chain fatty acids

Long-chain unsaturated fatty acids, as well as certain saturated fatty acids such as lauric acid, are inhibitors of the in vivo luminescence of wild-type strains of four species of luminous bacteria (Beneckea harveyi, Photobacterium phosphoerum, P. fischeri, andP. leiognathi) as well as the myristic acid-stimulated luminescence in the aldehyde dim mutant M17 ofB. harveyi. Based on studies with the system in vivo, the principal site of action of all the fatty acids appears to be the reductase activity that converts myristic acid to myristyl aldehyde. This was confirmed by in vitro studies: Reductase activity in crude cell-free extracts is strongly inhibited by oleic acid.     

Purification and characterization of recombinant spider silk expressed in Escherichia coli

A partial cDNA clone, from the 3′ end of the dragline silk gene was isolated from Nephila clavipes major ampullate glands. This clone contains a 1.7-kb insert, consisting of a repetitive coding region of 1.4-kb and a 0.3-kb nonrepetitive coding region; 1.5-kb of the 1.7-kb fragment was cloned into Escherichia coli and a␣43-kDa recombinant silk protein was expressed. Characterization of the purified protein by Western blot, amino acid composition analysis, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry confirms it to be spider dragline silk. Received: 7 April 1997 / Received revision: 24 July 1997 / Accepted: 25 August 1997     

Two new and two resurrected species of the sciaenid genusJohnius (Johnius) from the West Pacific

Two new species of the sciaenid genusJohnius (Johnius) are described:J. trewavasae from Taiwan, Hong Kong, and Singapore, differs from all other congeners in having 24–27 dorsal soft rays, 5–6 scales above and 7–10 scales below the lateral line, 6–8 obtuse lower gill rakers, the last pleural rib on the 11th vertebra, and a shorter lower jaw (33.8–38.4% HL);J. latifrons from Thailand and Java is characterized by 25–29 dorsal soft rays, 7–9 scales above and 11–14 scales below the lateral line, 7–9 obtuse lower gill rakers, a wide interorbital width (26.1–30.6% HL), a small eye (16.7–26.4% HL), and a short, second anal spine (25.9–37.1% HL). Two related species,J. heterolepis Bleeker from “Suriname” andJ. cantori Bleeker from Malaya, are resurrected as valid West Pacific species of Johnius (Johnius).     

Reciprocal Hemizygosity Analysis of Mouse Hepatic Lipase Reveals Influence on Obesity

Objectives: We previously demonstrated coincident quantitative trait loci (QTLs) for percentage body fat, plasma hepatic lipase (HL) activity, and plasma cholesterol on mouse chromosome 7. In the present study, we investigated whether hepatic lipase (Lipc) is an obesity gene, whether Lipc interacts with an unknown gene on chromosome 7, and how HL activity is linked to the chromosome 7 locus. Research Methods and Procedures: BSB mice are a model of complex obesity due to interactions among genes from C57BL/6J and Mus spretus (SPRET) in (C57BL/6J × SPRET) × C57BL/6J backcross mice. Five crosses tested the impact on obesity of combinations of inactive (knockout) and wild‐type Lipc alleles from C57BL/6J or SPRET in a reciprocal hemizygosity analysis. Results: The combined data from this allelic series suggest that Lipc alleles, and not alleles from a gene linked to Lipc, influence obesity. No interaction between Lipc and chromosome 7 was demonstrated. We confirmed the chromosome 7 QTLs for obesity, HL activity, and cholesterol. Because obesity and HL activity are not consistently associated in the BSB model, linkage of HL activity to chromosome 7 is not secondary to obesity per se. We also report, for the first time to our knowledge, a QTL in mammals for food intake. Discussion: This use of reciprocal hemizygosity analysis in mammals, which, to our knowledge, is the first reported, reveals its power to detect previously unknown effects of Lipc on obesity.     

Comparison of OspA Serotypes for Borrelia burgdorferi Sensu Lato from Japan,Europe and North America

Sixty-one Borrelia burgdorferi sensu lato strains from various sources (ticks, human, and wild animals) in Japan and two strains from ticks in Far Eastern Russia were classified on the basis of reactivity with 16 monoclonal antibodies (mAb) to outer surface protein A (OspA) and by DNA-DNA hybridization assay. Eleven OspA serotypes (J1 to J11) were recognized among the Japanese and the Far East Russian isolates (serotypes J1 to J9 were identified as B. garinii, serotype J10 was identified as B. afzelii, and serotype J11 corresponded to B. japonica), whereas 7 OspA serotypes for North American and European isolates previously reported (Bettina Wilske et al, J. Clin. Microbiol. 31:340-350, 1993) were not observed except for OspA serotype 2 which showed identical reactivity with OspA serotype J10. This finding provides helpful information for understanding the geographical distribution of Lyme disease borrelia and the development of vaccine and diagnostic tests. In conclusion: 1. B. burgdorferi sensu stricto has not been observed in Japan, 2. Japanese B. afzelii isolates are closely related to those from Europe, 3. B. garinii isolates from Japan are highly heterogeneous and apparently different from European B. garinii isolates.     

Topology,glycosylation and conformational changes in the membrane domain of the vacuolar H+‐ATPase a subunit

Published topological models of the integral membrane a subunit of the vacuolar proton‐translocating ATPase complex have not been in agreement with respect to either the number of transmembrane helices within the integral membrane domain, or their limits and orientations within the lipid bilayer. In the present work we have constructed a predictive model of the membrane insertion of the yeast a subunit, Vph1p, from a consensus of seven topology prediction algorithms. The model was tested experimentally using epitope tagging, green fluorescent protein fusion, and protease accessibility analysis in purified yeast vacuoles. Results suggest that a consensus prediction of eight transmembrane helices with both the amino‐terminus and carboxyl‐terminus in the cytoplasm is correct. Characterization of two glycosylation sites within the homologous mouse a subunit membrane domain further corroborates this topology. Moreover, the model takes into account published data on cytoplasmic and luminal accessibility of specific amino acids. Changes in the degree of protease accessibility in response to the V‐ATPase substrate, MgATP, and the V‐ATPase‐specific inhibitor, concanamycin A, suggest that functional conformational changes occur in the large cytoplasmic loop between TM6 and TM7 of Vph1p. These data substantially confirm one topological model of the V‐ATPase a subunit and support the notion that conformational changes occur within the membrane domain, possibly involving previously proposed axial rotation and/or linear displacement of TM7 in the proton transport cycle. J. Cell. Biochem. 114: 1474–1487, 2013. © 2013 Wiley Periodicals, Inc.     

Papers to Appear in Forthcoming Issues

Preparation of Recombinant Bovine, Porcine, and Porcine W4R/R5K Leptins and Comparison of Their Activity and Immunoreactivity with Ovine, Chicken, and Human LeptinsNina Raver, Eugene E. Gussakovsky, Duane H. Keisler, Radha Krishna, Jehangir Mistry, and Arieh GertlerPurification of Histidine-Tagged Mitochondrial ADP/ATP Carrier: Influence of the Conformational States of the C-Terminal RegionChristelle Fiore, Véronique Trézéguet, Pierre Roux, Agnès Le Saux, Florence Noël, Christine Schwimmer, Delphine Arlot, Anne-Christine Dianoux, Guy J.-M. Lauquin, and Gérard BrandolinExpression and Purification of Recombinant Human Indoleamine 2,3-DioxygenaseTamantha K. Littlejohn, Osamu Takikawa, Daniel Skylas, Joanne F. Jamie, Mark J. Walker, and Roger J. W. TruscottComparative Characterization of Two Forms of Recombinant Human SPC1 Secreted from Schneider 2 CellsJean-Bernard Denault, Claude Lazure, Robert Day, and Richard LeducFunctional and Immunological Analysis of Recombinant Mouse H and L Ferritins from E. coliPaolo Santambrogio, Anna Cozzi, Sonia Levi, Ermanna Rovida, Fulvio Magni, Alberto Albertini, and Paolo ArosioExpression and Purification of Soluble and Inactive Mutant Forms of Membrane Type-1 Matrix MetalloproteinaseHeli Valtanen, Kaisa Lehti, Jouko Lohi, and Jorma Keski-OjaFunctional Human Insulin-Degrading Enzyme Can Be Expressed in BacteriaValérie Chesneau and Marsha Rich RosnerHeterologous Expression in Pseudomonas aeruginosa and Purification of the 9.2 kDa c-Type Cytochrome Subunit of p-Cresol MethylhydroxylaseCiarán N. Cronin and William S. McIntire     

Origins of the specificity of inhibitor P218 toward wild-type and mutant PfDHFR: a molecular dynamics analysis

Molecular dynamics simulations were performed to evaluate the origin of the antimalarial effect of the lead compound P218. The simulations of the ligand in the cavities of wild-type, mutant Plasmodium falciparum Dihydrofolate Reductase (PfDHFR) and the human DHFR revealed the differences in the atomic-level interactions and also provided explanation for the specificity of this ligand toward PfDHFR. The binding free energy estimation using Molecular Mechanics Poisson-Boltzmann Surface Area method revealed that P218 has higher binding affinity (~ ?30 to ?35 kcal/mol) toward PfDHFR (both in wild-type and mutant forms) than human DHFR (~ ?22 kcal/mol), corroborating the experimental observations. Intermolecular hydrogen bonding analysis of the trajectories showed that P218 formed two stable hydrogen bonds with human DHFR (Ile7 and Glu30), wild-type and double-mutant PfDHFR’s (Asp54 and Arg122), while it formed three stable hydrogen bonds with quadruple-mutant PfDHFR (Asp54, Arg59, and Arg122). Additionally, P218 binding in PfDHFR is stabilized by hydrogen bonds with residues Ile14 and Ile164. It was found that mutant residues do not reduce the binding affinity of P218 to PfDHFR, in contrast, Cys59Arg mutation strongly favors inhibitor binding to quadruple-mutant PfDHFR. The atomistic-level details explored in this work will be highly useful for the design of non-resistant novel PfDHFR inhibitors as antimalarial agents.