Enzymic and immunochemical properties of lysozyme. X. Conformation, enzymic activity and immunochemistry of lysozyme reduced at two carboxyl groups.

Reduction of lysozyme by diborane, followed by air oxidation of the reduced disulfides and chromatography on CM-cellulose, yielded a homogeneous derivative. In the derivative, the carboxyl groups of aspartic acid 119 and the end-chain leucine residue were reduced to their corresponding alcohols. Correct re-forming of the disulfide bonds was demonstrated by peptide mapping of the tryptic hydrolysates of the derivative and lysozyme without breaking the disulfide bonds, followed by identification of the disulfide-containing peptides. Correct disulfide pairing in the two-disulfide peptide in the tryptic hydrolysate was established from its immunochemical behavior. Preparations of the two-disulfide fragment from lysozyme and derivative had equal inhibitory activities (26 or 32%) of the reaction of lysozyme with two homologous antisera. In ORD measurements, lysozyme and the derivative had equal rotatory powers at neutral pH. However, the bo value for the derivative decreased by about 10%. Below pH 6.4 and above pH 8.0, the derivative was less rotatory than native lysozyme. In CD measurements at neutral pH, the negative ellipticity bands at 220 and 208 nm showed little or no decrease in the derivative relative to the native protein. Although conformational differences between the derivative and its parent protein were almost undetectable by ORD and CD measurements, they were readily detected by chemical monitoring of the conformation. In the derivative, both accessibility to tryptic hydrolysis and reducibility of the disulfide bonds increased markedly. The enzymic activity of the derivative was decreased but retained the same pH optimum. With antisera to lysozyme or antisera to the derivative, lysozyme and its derivative possessed equal antigenic reactivities. The immunochemical findings further confirm the correct refolding of the disulfides. Also, they indicate that aspartic acid 119 and the C-terminal leucine residue are not part of an antigenic reactive region in lysozyme.     

Metabolism of aflatoxin B1 by Petroselinum crispum (parsley)

On administration of aflatoxin B₁ to whole parsley (Petroselinum crispum) plants, a derivative was formed, which was shown to be aflatoxicol by its chromatographic properties and mass spectrometry. Optimum conditions for the production of the derivative was on the second day after administration of the toxin to the plants, which were 90 days old after germination. Cell-free preparations of parsley were found not to produce aflatoxicol A from added aflatoxin B₁; instead they formed two new derivatives, which from chromatographic properties, were shown to be more polar than either aflatoxin B₁ or aflatoxicol A.     

Initial Steps in the Degradation of Methoxychlor by the White Rot Fungus Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium mineralized [ring-(sup14)C]methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] and metabolized it to a variety of products. The three most prominent of these were identified as the 1-dechloro derivative 1,1-dichloro-2,2-bis(4-methoxyphenyl)ethane, the 2-hydroxy derivative 2,2,2-trichloro-1,1-bis(4-methoxyphenyl)ethanol, and the 1-dechloro-2-hydroxy derivative 2,2-dichloro-1,1-bis(4-methoxyphenyl)ethanol by comparison of the derivatives with authentic standards in chromatographic and mass spectrometric experiments. In addition, the 1-dechloro-2-hydroxy derivative was identified from its (sup1)H nuclear magnetic resonance spectrum. The 1-dechloro and 2-hydroxy derivatives were both converted to the 1-dechloro-2-hydroxy derivative by the fungus; i.e., there was no requirement that dechlorination precede hydroxylation or vice versa. All three metabolites were mineralized and are therefore likely intermediates in the degradation of methoxychlor by P. chrysosporium.     

14-Dehydro-19-nortestosterone and its 7 -methyl derivative

14-Dehydro-19-nortestosterone and its 7α-methyl derivative were synthesized. The former was found to be approximately 100 and the latter 1000 times as active as testosterone in chick comb (local application) assays. In rat assays (subcutaneous), 14-dehydro-19-nortestosterone was approximately one-half as active as, or equal to testosterone in the ventral prostate or levator ani assays respectively, whereas its 7α-methyl derivative still retained its high potency (100 times as active as testosterone) in either type of assay.     

Inotropic activity induced by carbamazepine-alkyne derivative in an isolated heart model and perfused to constant flow

Inotropic activity induced by carbamazepine-alkyne derivative in an isolated heart model and perfused to constant flow Introduction. Few data exist with respect to the effects of carbamazepine and its derivatives at cardiovascular level; furthermore, the molecular mechanisms and cellular site of action are still unclear. Objective. The effects induced by carbamazepine-alquine derivative on perfusion pressure, vascular resistance and left ventricular pressure were evaluated. Materials and methods. The effects of carbamazepine and carbamazepine-alquine on the perfusion pressure, vascular resistance and left ventricular pressure were examined in isolated rat hearts (Langendorff model). Results. Four results were obtained: (1) The carbamazepine-alquine derivative 10-9 mM increased the perfusion pressure and vascular resistance in comparison with the carbamazepine 10-9 mM; (2) the effect of carbamazepine-alquine derivative 10-9-10-4 mM on left ventricular pressure not was inhibited by metoprolol or prazosin at a dose of 10-6 mM; (3) nifedipine 10-6 mM blocked the effects exerted by the carbamazepine-alquine derivative 10-9-10--4 mM on left ventricular pressure, and (4) the carbamazepine-alquine derivative at dose of 10-9 mM increased the concentration of intracellular calcium over a time period of 3-18 min; nevertheless, in presence of nifedipine 10-6 mM this effect was inhibited significantly (p=0.005). Conclusions. The activity exerted by carbamazepine-alquine derivative on perfusion pressure, vascular resistance and left ventricular pressure involved activation of calcium channel type-L, brought indirectly changes in the intracellular calcium levels and subsequently induced a positive inotropic effect.     

Cloning and Expression of the Bacillus subtilis No. 313 γ-Cyclodextrin Forming CGTase Gene in Escherichia coli

All four stereoisomers of pyriculol were synthesized to assist in forming a correlation between their chemical structure and biological activity. The (R,E)-2-hydroxy-3-pentenal derivative was coupled with a lithium acetylide derivative to give a diastereomeric mixture of the acetylenic alcohol, which led to the antipode of pyriculol and its 3′-epimer. Similarly obtained were the natural pyriculol and its 3′-epimer from the (S)-isomer of this aldehyde.     

CRYSTALLINE ACETYL DERIVATIVES OF PEPSIN

Crystalline pepsin has been acetylated by the action of ketene in aqueous solution at pH 4.07–5.5. As acetylation proceeds the activity decreases, the decrease being more rapid at pH 5.0–5.5 than at 4.0–4.5. Three acetyl derivatives have been isolated from the reaction mixture and obtained in crystalline form. The crystal form of these derivatives is similar to that of pepsin. Fractionation and solubility determinations show that these preparations are not mixtures or solid solutions of the original pepsin with an inactive derivative. A compound which contains three or four acetyl groups and which has lost all of its original primary amino groups can be isolated after short acetylation. It has the same activity as the original pepsin. A second derivative containing six to eleven acetyl groups has also been isolated. It has about 60 per cent of the activity of the original pepsin. A third derivative having twenty to thirty acetyl groups and about 10 per cent of the activity of original pepsin can be isolated after prolonged acetylation. The 60 per cent active derivative on standing in strong acid solution loses some of its acetyl groups and at the same time regains the activity of the original pepsin. The compound obtained in this way is probably the same as the completely active three acetyl derivative obtained by mild acetylation. These results show that acetylation of three or four of the primary amino groups of pepsin causes no change in the specific activity of the enzyme but that the introduction of acetyl groups in other parts of the molecule results in a marked loss in activity. The solubilities, amino nitrogen content, acetyl content, isoelectric point, and the specific activity have been determined by a variety of methods and found to be different from the corresponding properties of crystalline pepsin. The pH-activity curves, acid and alkali inactivation, and titration curves were not significantly different from the same respective properties of pepsin.     

Growth of human mesenchymal stem cells (MSCs) on films of enzymatically modified chitosan

Mesenchymal stem cells (MSCs) are known to be an attractive cell source for tissue engineering and regenerative medicine. One of the main limiting steps for clinical use or biotechnological purposes is the expansion step. The research of compatible biomaterials for MSCs expansion is recently regarded as an attractive topic. The aim of this study was to create new functional biomaterial for MSCs expansion by evaluating the impact of chitosan derivative films modified by enzymatic approach. First, chitosan particles were enzymatically modified with ferulic acid (FA) or ethyl ferulate (EF) under an eco‐friendly procedure. Then, films of chitosan and its modified derivatives were prepared and evaluated by physicochemical and biological properties. Results showed that the enzymatic grafting of FA or EF onto chitosan significantly increased hydrophobic and antioxidant properties of chitosan films. The MSCs cell viability on chitosan derivative films also increased depending on the film thickness and the quantity of grafted phenols. Furthermore, the cytotoxicity test showed the absence of toxic effect of chitosan derivative films towards MSCs cells. Cell morphology showed a well attached and spread phenotype of MSCs cells on chitosan derivative films. On the other hand, due to the higher phenol content of FA‐chitosan films, their hydrophobic, antioxidant properties and cell adhesion were improved in comparison with those of EF‐chitosan films. Finally, this enzymatic process can be considered as a promising process to favor MSCs cell growth as well as to create useful biomaterials for biomedical applications especially for tissue engineering. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:491–500, 2016     

PARALLEL DNA CONTAINING PYRAZOLO[3,4-D]PYRIMIDINE ANALOGUES OF ISOGUANINE

The phosphoramidites of 8-aza-7-deaza-2′-deoxyisoguanosine (1a) and its bromo derivative 1b as well as of 6-aza-2′-deoxyisocytidine and its 5-methyl derivative (3a,b) were synthesized. Parallel-stranded duplexes containing the nucleosides 1a,b show a significantly enhanced duplex stability compared to those containing 2′-deoxyisoguanosine.     

Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from Trametes versicolor

The hydrolysis of phenolic compounds using an immobilized and highly active and stable derivative of laccase from Trametes versicolor is presented. The enzyme was immobilized on aldehyde supports. For this, the enzyme was enriched in amino groups by chemical modification of its carboxyl groups. The aminated enzyme was immobilized with a high recovered activity (over 60%). Aldehyde derivatives were more stable than soluble or aminated-soluble enzyme and the reference derivatives after incubation in different inactivating conditions (high temperatures, different pH values or presence of organic cosolvents). The most stable derivative was obtained immobilizing the chemically aminated enzyme at pH 10 on aldehyde supports with a stabilization factor approximately 280 fold after incubation at pH 7 and 55 °C. In addition, it was possible to prepare immobilized derivatives with a maximal enzyme loading of 60 mg g^?1 of support. This derivative could be reused for 10 reaction cycles with negligible lost of activity.     

Conformation and stability of the constant fragment of the immunoglobulin light chain containing an intramolecular mercury bridge

The constant fragment of the immunoglobulin light chain in which the intramolecular disulfide bond is reduced (reduced CL fragment) assumes a conformation very similar to that of the intact CL fragment and contains two sulfhydryl groups buried in the interior of the molecule [Goto, Y., & Hamaguchi, K. (1979) J. Biochem. (Tokyo) 86, 1433-1441]. In order to understand the role of the disulfide bond, a derivative in which the disulfide bond is replaced by an S-Hg-S bond was prepared and its conformation and stability were studied. The derivative was prepared by reacting the reduced CL fragment with mercuric chloride. Kinetic studies showed that the reaction is rate-limited by the unfolding process of the reduced CL fragment. The mercury derivative was as compact as the intact CL or reduced CL fragment, and a tryptophyl residue was found to be buried near the S-Hg-S bond in the interior of the protein molecule. Judging from the circular dichroic spectrum, however, the beta-structure characteristic of the immunoglobulin fold was disturbed. The stability of the derivative to guanidine hydrochloride was lower than that of the intact CL fragment, but the unfolding transition was reversible and cooperative. Decreased stability of the mercury derivative is due to its folded conformation being distorted by introduction of the S-Hg-S bond.     

Synthesis of a Fluorobenzoxazine Derivative and Its Analogues

Syntheses of a fluorobenzoxazine derivative, fluorobenzothiazine derivative and fluoroquinoxaline derivative are described. These compounds were synthesized by reductive cyclization of the corresponding fluorodinitrobenzene derivatives. The fluorobenzoxazine derivative and its analogues are useful intermediates for agro-chemicals.     

Tomato-produced 7-epizingiberene and R-curcumene act as repellents to whiteflies

How whiteflies (Bemisia tabaci) make the choice for a host plant prior to landing, is not precisely known. Here we investigated whether they respond to specific volatiles of tomato. Zingiberene and curcumene were purified from Solanum habrochaites (PI127826), characterised by NMR and X-ray analysis and identified as 7-epizingiberene and R-curcumene. In contrast, oil from Zingiber officinalis contained the stereoisomers zingiberene and S-curcumene, respectively. Using a combination of free-choice bio-assays and electroantennography, 7-epizingiberene and its dehydrogenated derivative R-curcumene were shown to be active as semiochemicals to B. tabaci adults, whereas the stereoisomers from ginger were not. In addition, R-curcumene elicited the strongest electroantennographic response. Bio-assays showed that a cultivated tomato could be made less attractive to B. tabaci than its neighbouring siblings by the addition of the tomato stereoisomer 7-epizingiberene or its derivative R-curcumene. These sesquiterpenes apparently repel adult whiteflies prior to landing, presumably because it informs them that after landing they, or their offspring, may be exposed to higher and lethal concentrations of the same compounds.     

虫酰肼及其衍生物0593对家蚕的毒性及作用机理

为明确虫酰肼衍生物0593对家蚕Bombyx mori的毒性,本研究采用食下毒叶法测定了虫酰肼及其衍生物0593对家蚕的毒性,观察了亚致死浓度下对家蚕生长发育的影响,并测定了虫酰肼及其衍生物0593对家蚕幼虫体内保护酶的影响,对虫酰肼0593的作用机理进行了初步探索。结果表明：虫酰肼及其衍生物0593对2龄家蚕96 h的LC₅₀值分别为1.2863和0.3364 mg·L^-1,属高毒级药剂;虫酰肼及其衍生物0593在亚致死剂量下对家蚕的生长发育有明显的不利性,可使幼虫历期缩短0.5～2 d;处理组眠期体重、全茧量、蛹重和化蛹率与对照相比均显著降低;对4龄幼虫体内多酚氧化酶和几丁质酶也有较明显影响,虫酰肼及其衍生物0593处理家蚕,处理后6 h对体内多酚氧化酶有明显激活作用,12 h后表现出显著的抑制作用;虫酰肼及其衍生物0593对家蚕体内几丁质酶均有激活作用。0593对保护酶的影响较虫酰肼明显。结果提示虫酰肼及其衍生物0593对家蚕毒性高,对其生长发育和保护酶类均有不利性,不适合在桑园及其周边农田使用。     

Microbial hydroxylation of natural drimenic lactones.

Incubation of confertifolin and isodrimenin with Mucor plumbeus, Aspergillus niger or Rhizopus arrhizus gave in good yields the corresponding 3 beta-hydroxy derivatives. From isodrimenin, the known natural 7 alpha-hydroxy derivative (futronolide) was also obtained and its structure was definitely established by X-ray crystallographic study of its acetate derivative.     

Synthesis and photophysical properties of L-N ε-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-lysine,dabcyl-like chromophore for peptide studies

Summary A synthesis ofl-N^ɛ-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-lysine [Lys(AQN)], the dabcyl-like chromophore, and its derivatives useful in peptide chemistry is described. N^α-tert-butoxycarbonyl derivative of the title compound was obtained in a good yield using aromatic nucleophilic substitution reaction. In this form, or after conversion to the N^α-fluorenylmethoxycarbonyl derivative, it can be directly used in the solid phase peptide synthesis using either Boc- or Fmoc-strategy.     

Synthesis and biological evaluation of water-soluble derivatives of chiral gossypol as HIV fusion inhibitors targeting gp41

A series of novel or known water-soluble derivatives of chiral gossypol were synthesized and screened in vitro for their anti-HIV-1 activity. (?)-gossypol derivative was more active against HIV-1 than the corresponding (+)-gossypol derivative, respectively. Among these derivatives, d-glucosamine derivative of (?)-gossypol, oligopeptide derivative of (?)-gossypol and taurine derivative of (?)-gossypol, such as compounds 1a, 3a and 14a, showed significant inhibitory activities against HIV-1 replication, HIV-1 mediated cell-cell fusion and HIV gp41 6-helix bundle formation as some amino acid derivatives of (?)-gossypol.     

Translocation of antibiotic resistance markers of a plasmid-free Streptococcus pyogenes (group A) strain into different streptococcal hemolysin plasmids

Summary Wild-type strain A454 (Streptococcus pyogenes) transferred en bloc its erythromycin (Em) and tetracycline (Tc) resistance markers into several plasmid-free streptococcal recipients. No plasmid DNA was detected in either the wild-type or the transconjugant strains. Crosses were performed between A454 and S. faecalis Rec⁺ or Rec^- recipients carrying hemolysin-bacteriocin plasmids, pIP964 or pAD1. The Em Tc-resistant transconjugants obtained harbored either the parental plasmid or an Em Tc resistance plasmid derived from pIP964 or pAD1. The restriction endonuclease analysis of 12 derivative plasmids showed insertions of various sizes into different fragments of pIP964 or pAD1. A454 and the Em Tc-resistant plasmid-free transconjugants were found to contain two EcoRI DNA fragments, that shared homology with ³²P-labeled pIP1077, one of the Em Tc resistance derivative plasmids, but not with ³²P-labeled pIP964. No homology was detected between pIP1077 and the cellular DNA of the antibiotic-susceptible recipients.Previously Thea Horodniceanu     

Comparison of DNA in the core component from Schmidt-Ruppin RSV transforming virus and non-transforming virus.

Viral core fractions from transforming Schmidt-Ruppin sarcoma virus (SR-RSV-t) and its transformation-defective derivative (SR-RSV-td) were examined for DNA. The transforming virus contained DNA that was easily detected, but the transformation-defective virus had little or no DNA associated with the core fraction. The buoyant density of the DNA from SR-RSV-t in CsC1 was 1.715 g/cm³.     

Novel glycosylation sites localized in Campylobacter jejuni flagellin FlaA by liquid chromatography electron capture dissociation tandem mass spectrometry

Glycosylation of flagellin in Campylobacter jejuni is essential for motility and virulence. It is well-known that flagellin from C. jejuni 81-176 is glycosylated by pseudaminic acid and its acetamidino derivative, and that Campylobactor coli VC167 flagellin is glycosylated by legionaminic acid and its derivatives. Recently, it was shown, by use of a metabolomics approach, that C. jejuni 11168 is glycosylated by dimethyl glyceric acid derivatives of pseudaminic acid, but the sites of glycosylation were not confirmed. Here, we apply an online liquid chromatography electron capture dissociation (ECD) tandem mass spectrometry approach to localize sites of glycosylation in flagellin from C. jejuni 11168. Flagellin A is glycosylated by a dimethyl glyceric acid derivative of pseudaminic acid at Ser181, Ser207 and either Thr464 or Thr 465; and by a dimethyl glyceric acid derivative of acetamidino pseudaminic acid at Ser181 and Ser207. For comparison, on-line liquid chromatography collision-induced dissociation of the tryptic digests was performed, but it was not possible to assign sites of glycosylation by that method.