Reidentification of cellulolytic enzyme-producing Trichoderma strains W-10 and G-39

Strain W-10, originally identified as Trichoderma koningii, and its supposed mutant G-39, published for production and gene expression of cellulase and xylanase, demonstrated morphological characteristics distinct from those of T. koningii, respectively. To clarify the identification derived from morphological characteristics, several methods were used, including electrophoretic karyotyping, internal transcribed spacer (ITS) analysis of rDNA, and polymerase chain reaction (PCR) fingerprinting using the universal primer L45. All the molecular characteristics showed that strains G-39 and W-10 were identical to T. reesei and T. longibrachiatum, respectively. The results strongly supported that T. koningii G-39 and W-10 should be reassigned as T. reesei and T. longibrachiatum, respectively. Strain G-39 should be considered a mutant from T. reesei QM9414 whose spores were contaminated with those of strain W-10 during a laboratory operation. According to this, we declare that T. koningii G-39 and W-10 must be renamed as T. reesei and T. longibrachiatum, respectively.     

Self-inoculation with Mycobacterium tuberculosis Weiszfeiler W-115.

After intracutaneous self-inoculation with 0.05 mg (wet weight) of Mycobacterium tuberculosis W-115 containing approximately 600 000 viable units, the local reaction was observed during a period of 9 weeks. No adverse effects or general reaction were observed. From the lesion the W-115 strain was recultivated. It was labelled W-115(78)ST vaccine strain and is used for further studies. On the basis of experiments on laboratory animals, monkeys, human new-borns and adults, the strain W-115 is considered as safe for antituberculous vaccination as the strains Mycobacterium bovis BCG and the attenuated "MP" strain of Mycobacterium microti OV 166.     

A novel phage genome integrated into a plasmid in Bacillus thuringiensis strain AF101

Bacillus thuringiensis strain AF101 possesses a single plasmid (pAF101) with a molecular size of 42 MDa (69 kb). During plasmid curing experiments in strain AF101, we found that a phage (J7W-1) was induced by ethidium bromide treatment. Moreover, the phage genome (48 kb) hybridized only with pAF101 on a Southern blot of the DNA of a cleared lysate prepared from strain AF101. Comparison of the restriction patterns of pAF101 and J7W-1 phage DNA revealed that pAF101 contains not only the entire phage DNA but also a plasmid-specific DNA region. These results indicate that the J7W-1 genome has been stably integrated into pAF101 in strain AF101. Integration of the J7W-1 genome into a plasmid was also observed after phage infection of the type strain of B. thuringiensis subsp. israelensis.     

微生物转谷氨酰胺酶的生产菌种诱变和发酵生产分析

对本研究室从土壤分离得到的使霉菌(Streptomyces sp.)WZFF．W-12菌株的斜面孢子预培养处于初萌发状态后,以亚硝基胍(NTG)进行诱变育种试验,并根据诱变处理后菌落的某些形态变化状况与产酶能力相结合的特征,初步判断产酶性能,挑选高酶活菌株,再经过初筛和复筛,获得一性能良好的产酶突变菌株WZFF.W-12.var MN-35,转谷氨酰酶活达0．53U／mL,比原始菌株提高了1．2倍。然后在摇瓶条件下,对其发酵过程中的主要培养基组成及各种培养条件对菌体生长和产酶的影响作用进行了研究,结果表明该菌株发酵生产转谷氨酰酶的适宜破源为可溶性淀粉 葡萄糖,氮源是多价胨外加少量的酵母膏,优化工艺条件为种龄时间24h、接种量10％、初始以值6．5、温度30℃和搅拌速度200r／min,产酶能力显著提高,用小型生化反应器可以稳定生产2.0U／mL以上的酶产品。     

Study on the interaction of taiwaniaquinoids with FTO by spectroscopy and molecular modeling

In this work, an attempt has been made to study the interaction of four taiwaniaquinoids with fat mass and obesity-associated protein (FTO) by UV–vis absorption, fluorescence spectroscopy, and molecular docking techniques. The results indicated that taiwaniaquinoids effectively quenched the intrinsic fluorescence of FTO via static quenching. According to the binding constants and thermodynamic parameters at three different temperatures, the hydrophobic force and electrostatic interactions appeared be the predominant intermolecular forces in stabilizing the complex. Results revealed that W-4 was the strongest quencher and W-3 was the weakest. The results of synchronous and three-dimensional fluorescence spectra showed that the conformation of FTO was changed. In addition, the influence of molecular structure on the quenching effect has been investigated.     

RELATIONSHIPS BETWEEN GROWTH RATES AND NUCLEIC ACID CONTENTS IN THE ROOTS OF INBRED LINES OF CORN

Woodstock , Lowell W., and Folke Skooc . (U. Wisconsin, Madison.) Relationships between growth rates and nucleic acid contents in the roots of inbred lines of corn. Amer. Jour. Bot. 47(9) : 713–716. Illus. 1960.—A study has been made of the rates of elongation and nucleic acid contents of the roots of inbred lines of Zea mays, var. W-158, A-374B, W-58, W-28, 8A, and W-148. A direct proportionality was found between the ribosenucleic acid contents (μg./root) of the apical 3 or 6 mm. of the roots (roughly equivalent to their growing tips) and their rates of elongation. This strict, quantitative relationship to growth rate appears to be specific for RNA; it was not found for DNA, and it was independent of the size of the root. A detailed study of one line (W-58) has shown, furthermore, that the ribosenucleic acid content in the root tip decreases with time in proportion to the decrease in the growth rate of the root.     

Induction of the acrosome reaction in guinea pig spermatozoa by calmodulin antagonist W-7

The effect of the calmodulin antagonist W-7 on the capacitation and the acrosome reaction of guinea pig spermatozoa was examined. The characteristic features of the acrosome reaction induced by W-7 were the dependence on the composition and pH of the medium and on the presence of sodium bicarbonate. The most effective concentration of W-7 for inducing the acrosome reaction was approximately 5 μM, which is far less than the Kd for calmodulin. Moreover, W-7 enhanced the ability of spermatozoa to acquire capacitation in a Ca²⁺-free medium. The spermatozoa induced to undergo the acrosome reaction by W-7 were capable of penetrating the zona-free hamster eggs. W-5, which has a lower affinity for calmodulin than W-7, also induced the acrosome reaction in the same manner as W-7. These results suggest that the naphthalenesulfonamide derivatives W-7 and W-5 can induce the acrosome reaction in guinea pig spermatozoa via capacitation in a pH-dependent, Ca²⁺-calmodulin-independent manner.     

Calmodulin inhibitors, W-7 and TFP, block the calmodulin-independent activation of NADPH-oxidase by arachidonate in a cell-free system

The calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), or trifluoperazine inhibited not only Fc gamma-receptor mediated cytosolic free Ca2+ increase and O2- generation in macrophages, but also an arachidonate-induced activation of NADPH-oxidase in a cell-free system. Although these results suggested the involvement of Ca2+-calmodulin system, the cell-free activation of NADPH-oxidase occurred in the presence of EGTA and addition of calmodulin had no effect. Furthermore W-7 shifted the optimal concentration of arachidonate required for the activation to a higher level, suggesting that W-7 may block the interaction between arachidonate and NADPH-oxidase system rather than inhibiting a Ca2+-calmodulin system.     

Effects of conversion of an invariant tryptophan residue to phenylalanine on the function of human dihydrofolate reductase

The binding site residue Trp-24 is conserved in all vertebrate and bacterial dihydrofolate reductases of known sequence. To determine its effects on enzyme properties, a Trp-24 to Phe-24 mutant (W-24-F) of human dihydrofolate reductase has been constructed by oligodeoxynucleotide site-directed mutagenesis. The W-24-F mutant enzyme appears to have a more open or flexible conformation as compared to the wild-type human dihydrofolate reductase on the basis of results of a number of studies. These studies include competitive ELISA using peptide-specific antibodies against human dihydrofolate reductase, thermal stability, and protease susceptibility studies of both mutant W-24-F and wild-type enzymes. It is concluded that Trp-24 is important for maintaining the structural integrity of the native enzymes. Changes in relative fluorescence quantum yield indicate that Trp-24 is buried and its fluorescence quenched relative to the other two tryptophan residues in the wild-type human reductase. Kinetic studies indicate that kcat values for W-24-F are increased in the pH range of 4.5-8.5 with a 5-fold increase at pH 7.5 as compared to the wild-type enzyme. However, the catalytic efficiency of W-24-F decreases rapidly as the pH is increased from 7.5 to 9.5. The Km values for dihydrofolate are also increased for W-24-F in the pH range of 4.5-9.5 with a 30-fold increase at pH 7.5, while the Km value for NADPH increases only ca. 1.4-fold at pH 7.5 as compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)     

The circular dichroism properties of phi W-14 DNA containing alpha-putrescinylthymine

The circular dichroism properties of phi W-14 DNA containing alpha-putrescinylthymine and its acetylated derivative have been examined in a number of aqueous solvents. Native phi W-14 DNA exhibits a B-type CD spectrum whose characteristics do not entirely conform to what would be expected for its GC content (51%). The conformationally sensitive positive band above 260 nm has a rotational strength greater than that normally found in prokaryotic DNAs of comparable GC content, such as Escherichia coli DNA. The rotational strength of this band in the spectrum of the heat-denatured form of phi W-14 DNA, however, is similar to that of heat denatured E. coli DNA. Abolition of the positive charge on the putrescine residues of native phi W-14 DNA by reaction with CH2O or by acetylation reduces the rotational strength to a level appropriate for its GC content. Increases in the electrolyte content of the solvent have the same effect, although the rotational strength of this band in phi W-14 DNA does not become comparable to that of E. coli DNA until 6-7 M LiCl. Titration to pH 10.6 in solvents of modest electrolyte content, however, fails to appreciably affect the CD spectral properties of either native phi W-14 DNA or the derivative in which half of the secondary and all of the primary amino groups have been acetylated. On the basis of these results we have concluded that the enhanced rotational strength of the positive band above 260 nm in the CD spectrum of phi W-14 DNA is due to a conformational difference caused by an ion-pair interaction of the positively charged primary amino groups of putrescine with the phosphate backbone. The CD spectral properties, however, reveal that these differences, averaged over the entire basepair population, appear to be relatively small. The average conformation, at least in dilute aqueous solutions, seems to be an unexceptional B variant with conformational properties which would be more appropriate for a DNA of higher CG content.     

Dynamic secondary structural changes in Ca²⁺-saturated calmodulin upon interaction with the antagonist, W-7

Although the 3D structure of the Ca(2+)-bound CaM (Ca(2+)/CaM) complex with the antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7), has been resolved, the dynamic changes in Ca(2+)/CaM structure upon interaction with W-7 are still unknown. We investigated time- and temperature-dependent dynamic changes in Ca(2+)/CaM interaction with W-7 in physiological conditions using one- and two-dimensional Fourier-transformed infrared spectroscopy (2D-IR). We observed changes in the α-helix secondary structure of Ca(2+)/CaM when complexed with W-7 at a molar ratio of 1:2, but not at higher molar ratios (between 1:2 and 1:5). Kinetic studies revealed that, during the initial 125s at 25°C, Ca(2+)/CaM underwent formation of secondary coil and turn structures upon binding to W-7. Variations in temperature that induced significant changes in the structure of the Ca(2+)/CaM complex failed to do so when Ca(2+)/CaM was complexed with W-7. We concluded that W-7 induced stepwise conformational changes in Ca(2+)/CaM that resulted in a rigidification of the complex and its inability to interact with target proteins and/or polypeptides.     

Influence of W-7, a calmodulin antagonist on phospholipid biosynthesis in Candida albicans

AIM: This study was undertaken to investigate the role of calmodulin in phospholipid biosynthesis in Candida albicans using W-7, a calmodulin antagonist. METHODS: Cells were grown as shake cultures in the absence and presence of W-7 at different concentrations. Changes in cell mass, phospholipid content and incorporation of labelled precursor into phospholipid and activities of respective enzymes have been studied. RESULTS: Decreased incorporation of labelled acetate into total lipids and phospholipids was observed in the presence of 40 microm of W-7 which was not as a consequence of altered growth of Candida in the presence of calmodulin antagonist. Further, a significant decrease in the levels of calmodulin and CaM dependent protein kinase activity was observed in cells grown with different concentrations of W-7. This was accompanied by decreased/increased activity of phosphatidic acid phosphatase and phospholipase A, respectively in W-7 grown cells as compared to controls. CONCLUSIONS: These findings suggest definite involvement of calmodulin in the regulation of phospholipid metabolism in Candida albicans.     

Inhibition of the acrosome reaction of sea urchin spermatozoa by a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7)

A calmodulin antagonist, W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), inhibited induction of the acrosome reaction of sea urchin spermatozoa by egg jelly at 20-50 microM, but W-5 did not. The latter, a chlorine-deficient derivative of W-7, has a lower affinity for calmodulin than does W-7. These results suggest that a Ca2+-calmodulin-dependent process plays an important role in the triggering process of the acrosome reaction of sea urchin spermatozoa.     

Membrane-permeable calmodulin inhibitors (e.g. W-7/W-13) bind to membranes, changing the electrostatic surface potential: dual effect of W-13 on epidermal growth factor receptor activation

Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca2+/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca2+/CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR; W-13 inhibits epidermal growth factor-dependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due to W-13 inhibition of Ca2+/CaM, but the latter results could be due to binding of W-13 to the plasma membrane.     

The circular dichroism properties of φW-14 DNA containing α-putrescinylthymine

The circular dichroism properties of φW-14 DNA containing α-putrescinylthymine and its acetylated derivative have been examined in a number of aqueous solvents. Native φW-14 DNA exhibits a B-type CD spectrum whose characteristics do not entirely conform to what would be expected for its GC content (51%). The conformationally sensitive positive band above 260 nm has a rotational strength greater than that normally found in prokaryotic DNAs of comparable GC content, such as Escherichia coli DNA. The rotational strength of this band in the spectrum of the heat-denatured from of φW-14 DNA, however, is similar to that of heat denatured E. coli DNA. Abolition of the positive charge on the putrescine residues of native φW-14 DNA by reaction with CH₂O or by acetylation reduces the rotational strength to a level appropriate for its GC content. Increases in the electrolyte content of the solvent have the same effect, although the rotational strength of this band in φW-14 DNA does not become comparable to that of E. coli DNA until 6–7 M LiCl. Titration to pH 10.6 in solvents of modest electrolyte content, however, fails to appreciably affect the CD spectral properties of either native φW-14 DNA or the derivative in which half of the secondary and all of the primary amino groups have been acetylated. On the basis of these results we have concluded that the enhanced rotational strength of the positive band above 260 nm in the CD spectrum of φW-14 DNA is due to a conformational difference caused by an ion-pair interaction of the positively charged primary amino groups of putrescine with the phosphate backbone. The CD spectral properties, however, reveal that these differences, averaged over the entire basepair population, appear to be relatively small. The average conformation, at least in dilute aqueous solutions, seems to be an unexceptional B variant with conformational properties which would be more appropriate for a DNA of higher CG content.     

Participation of protein kinase C in the activation of human neutrophils by phorbol ester

The calmodulin antagonist N(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) has been examined as an inhibitor of superoxide anion production and granule exocytosis in phorbol ester (PMA)-activated neutrophils. Inhibition of the respiratory burst was observed at a concentration of W-7 identical to that required for inhibition of native protein kinase C (PKC), whereas the concentration required to inhibit the secretory response was found to correspond to that required for inhibition of the proteolytically converted fully active PKC. The IC50 of W-7 was in both cases 5 and 12 fold higher than that required for inhibition of calmodulin dependent kinases. The results confirm the essential role for the membrane-bound PKC in the production of O2- radicals and provide a clear evidence of the direct participation of the proteolytically activated cytosolic PKC to the secretory response of PMA activated neutrophils.     

Stage-specific response of preimplantation mouse embryos to W-7, a calmodulin antagonist

Involvement of calmodulin-dependent processes in preimplantation development of mouse embryos was studied with the use of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a specific antagonist of calmodulin. At 25 microM, W-7 interfered with compaction of eight-cell embryos, caused decompaction of compacted eight-cell embryos, inhibited cavitation of late morulae, and caused collapse and degeneration of blastocysts. These effects of W-7 appear to be due to specific inhibition of calmodulin-dependent processes, because W-5, a less active analogue of W-7, was less effective in interfering with development; at 25 microM, W-5 had only a slight effect on compaction and had no effect on blastocyst formation, maintenance of blastocoels, or post-blastocyst development. In addition to the developmental effects just described, W-7 inhibited cell proliferation in four-cell embryos and reduced cell numbers of morulae after treatment at the two- to eight-cell stages. There was a marked increase in embryos' sensitivity to W-7 at the late morula stage, and the sensitivity increased further as embryos developed into blastocysts; the effects of W-7 were largely reversible after treatment at the two-cell through the compacted eight-cell stages, but not after treatment at the late morula or blastocyst stage. At the blastocyst stage, inner cell mass cells appeared to be slightly more resistant to W-7 than trophectoderm cells. This differential sensitivity became more pronounced at the late blastocyst stage: after 3.5-4-h exposure of late blastocysts to 25 microM W-7, all trophectoderm cells degenerated but most of the inner cell masses survived. From these results it appears that calmodulin-dependent processes are involved in development of mouse embryos at all of the preimplantation stages examined.     

Biochemical and physiological evidence that calmodulin is involved in the taste response of the sugar receptor cells of the blowfly, Phormia regina

The gustatory system is essential for almost all animals. However, the signal transduction mechanisms have not yet been fully elucidated. We isolated labellar chemosensilla from blowfly, Phormia regina, and purified calcium binding proteins from the water soluble fraction. The most abundant calcium-binding protein was calmodulin. To investigate the role of calmodulin in taste transduction, electrophysiological responses were recorded with the calmodulin inhibitor, W-7. When we stimulated the labellar chemosensillum with sucrose plus W-7, a dose-dependent decrease of impulse frequency was observed when the concentration was <50 microM. In addition, when W-7 at 50 microM or higher concentration was added, an initial short-term impulse generation from the sugar receptor cell was observed, but this was followed by a silent period. When the sensillum was stimulated with W-7 plus a membrane-permeable cGMP analog, dibtyryl-cGMP or 8-bromo-cGMP, impulses of the sugar receptor cell were induced but the frequency was decreased. By the sidewall-recording method, we observed that the receptor potential induced by sucrose stimulation was decreased by W-7 in the sugar receptor cell, and corresponded with a disappearance of impulses. These data strongly suggest that the cGMP-gated channel generating receptor potential in the sugar receptor cell requires calmodulin for its gating.     

Cross-resistance to insecticides in a malathion-resistant strain of Ceratitis capitata (Diptera: Tephritidae)

Resistance to malathion has been reported in field populations of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), in areas of Spain where an intensive use of this insecticide was maintained for several years. The main goal of this study was to determine whether resistance to malathion confers cross-resistance to different types of insecticides. Susceptibility bioassays showed that the malathion-resistant W-4Km strain (176-fold more resistant to malathion than the susceptible C strain) has moderate levels of cross-resistance (three- to 16-fold) to other organophosphates (trichlorphon, diazinon, phosmet and methyl-chlorpyrifos), the carbamate carbaryl, the pyrethroid lambda-cyhalothrin, and the benzoylphenylurea derivative lufenuron, whereas cross-resistance to spinosad was below two-fold. The W-4Km strain was selected with lambda-cyhalothrin to establish the lambda-cyhalothrin-resistant W-1Klamda strain (35-fold resistant to lambda-cyhalothrin). The synergistic activity of the esterase inhibitor DEF with lambda-cyhalothrin and the increase in esterase activity in the W-1Klamda strain suggests that esterases may be involved in the development of resistance to this insecticide. Our results showed that resistance to malathion may confer some degree of cross-resistance to insecticides currently approved for the control of Mediterranean fruit fly in citrus crops (lambda-cyhalothrin, lufenuron, and methyl-chlorpyrifos). Especially relevant is the case of lambda-cyhalothrin, because we have shown that resistance to this insecticide can rapidly evolve to levels that may compromise its effectiveness in the field.