Drimanes and other terpenoids from the fern Thelypteris hispidula (Decne.) Reed

The drimane-type sesquiterpenoids (-)-polygodial (1), (-)-isopolygodial (4), drimenin (5), and isodrimenin (6) were isolated from a pungent Argentine collection of the fern Thelypteris hispidula, along with other terpenoids. As the mentioned drimanes have been previously found in liverworts, the present results strongly support the theory that Pteridophytes and Bryophytes share the same line of evolution. Our present investigations indicate that two chemotypes of T. hispidula are widespread in the northwest of Argentina. Plants belonging to the pungent chemotype contain drimanes, while nonpungent plants lack drimanes.     

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.     

Conformation, enzymic activity, and immunochemistry of a lysozyme derivative modified at tryptophan 123 by reaction with 2,3-dioxo-5-indolinesulfonic acid.

Reaction of hen egg-white lysozyme with 2,3-dioxo-5-indolinesulfonic acid (DISA) yielded a homogeneous derivative which was modified at a single tryptophan residue. The modification was located at Trp-123. The absorption spectrum of the derivative showed a new peak in the visible range with lambdamax at 365 nm. In addition, the absorption maximum in the ultraviolet which appears in lysozyme at 280 nm was shifted to 270 nm in the derivative and appreciably enhanced. In ORD measurements, the rotatory behaviors of lysozyme and its derivative were identical at the 233 nm negative minimum and the 199 nm positive extremum. CD measurements gave equal [theta] values for lysozyme and derivative at the two negative ellipticity bands at 208 and 220 nm. Although no conformational differences between lysozyme and derivative were observed by ORD and CD measurements, some changes were detectable by chemical methods. Accessibility to tryptic hydrolysis and susceptibility of the disulfide bonds to reduction were increased in the derivative relative to lysozyme. The lytic activity of the derivative, which retained the same pH optimum as native lysozyme, was greatly (50%) decreased, probably as a result of the slight conformational change. With several antisera to lysozyme, the native protein and its derivative had equal antigenic reactivities. The findings were instrumental in further delineation of an antigenic reactive site in lysozyme.     

The synthesis of some fluoro derivatives of sucrose

2,3,1',3'4',6'-Hexa-O-benzylsucrose was obtained by mild acid-catalysed hydrolysis of the 4,6-O-isopropylidene derivative and then converted into its 4,6-di-O-mesyl derivative. Selective displacement of this disulphonate with fluoride anion (from tetrabutylammonium fluoride) then afforded the 6-fluoro-4-mesylate. Removal of the protecting groups yielded 6-deoxy-6-fluorosucrose, which was characterised as its crystalline hepta-acetate. A derivative of 6-deoxy-6-fluoro-galacto-sucrose was formed when the above 6-fluoro-4-mesylate was subjected to nucleophilic displacement with benzoate anion.     

Ile-Ser-bradykinin (T-kinin) and Met-Ile-Ser-bradykinin (Met-T-kinin) are released from T-kininogen by an acid proteinase of granulomatous tissues in rats

An acid proteinase of granulomatous tissues in rats with carrageenin-induced inflammation released kinin from T-kininogen. The kinin isolated by n-butanol extraction was separated by reverse-phase high-performance liquid chromatography into T-kinin and a T-kinin derivative. From determination of its amino acid composition and its immunoreactivity toward anti-bradykinin antiserum, the T-kinin derivative was identified as Met-Ile-Ser-bradykinin (Met-T-kinin).     

Muramyl dipeptide and its synthetic analog as possible inducers of interleukin-2 production

Muramyl dipeptide and its synthetic derivative N-acetyl-glucosaminyl-N-acetyl-muramyl-alanyl - D-isoglutamine induce mitogenic factor production for Con A activated blasts (Con A-blasts) in splenocytes. The maximal factor production was revealed 24 h after stimulation with muramyl dipeptide or its derivative. Addition of the inducers to the culture of Con A-blasts led but to the negligible proliferative response. Therefore, the mitogenic action of muramyl dipeptide on target cells is mediated by the growth factor, evidently by interleukin-2. It has been also shown that muramyl dipeptide and its derivative with Con A exerted a synergic action in interleukin-2 induction.     

Preparation of a stable biocatalyst of bovine liver catalase using immobilization and postimmobilization techniques

Bovine liver catalase was immobilized on different supports. The tetrameric nature of this enzyme was found to cause its rapid inactivation in diluted conditions due to subunit dissociation, a fact that may rule out its industrial use. Multi-subunit immobilization using highly activated glyoxyl agarose was not enough to involve all enzyme subunits. In fact, washing the derivative produced a strong decrease in the enzyme activity. Further cross-linking of previously immobilized enzyme with tailor-made dextran-aldehyde permitted the multimeric structure to be fully stabilized using either multisubunit preparations immobilized onto highly activated glyoxyl-agarose support or one subunit enzymes immobilized onto poorly activated glyoxyl-agarose. The highest stability of the final biocatalyst was observed using the multisubunit immobilized derivative cross-linked with dextran-aldehyde. The optimal derivative retained around 60% of the immobilized activity, did not release any enzyme subunits after boiling in the presence of SDS, and did not lose activity during washing, and its stability did not depend on the dilution. This derivative was used for 10 cycles in the destruction of 10 mM hydrogen peroxide without any decrease in the enzyme activity.     

Synthesis and X-ray crystal structure study of the hydroxyurea and hydantoin derivatives of L-valine.

The novel hydroxyurea 5 derivative of L-valine was prepared by aminolysis of N-(1-benzotriazolecarbonyl)-L-valine cyclohexanemethylamide 4 with hydroxylamine. The corresponding hydantoin derivative 6 was synthesized by base catalyzed cyclization of the amide 4. The exact stereostructure of hydantoin derivative 6 has been determined by X-ray crystal structure analysis. The chiral atom of the hydantoin ring in 6 has S configuration what is in agreement with its configuration in the starting L-valine. The molecules of 6 are joined into infinite chains by N-H...O intermolecular hydrogen bond. The infinite chains are additionally linked by two C-H...O hydrogen bonds, thus forming two-dimensional network. The hydantoin derivative of L-valine 6 and its L-leucine analogue LH have similar packing arrangements, so they are homostructural.     

Study of embryotoxic and teratogenic effects of amphotericin B and a methyl derivative of amphotericin B in rats after their intravenous and intra-amniotic administration]

The embryotoxic action of amphotericin B and its methyl derivative was compared in rats after their intravenous and intraamniotic administration. The concentrations of amphotericin B and its methyl derivative in the amniotic cavity on days 13, 14 and 15 of pregnancy were 1.5 and 36 micrograms/ml, respectively. When administered intravenously during the preimplantation period the antibiotics had no embryotoxic action. Intravenous administration of amphotericin B in a dose of 500 micrograms/kg and its derivative in a dose of 2000 micrograms/kg during organ genesis induced a decrease in the craniocaudal size. In a dose of 3000 micrograms/kg administered intravenously the methyl derivative of amphotericin B induced an increase in postimplantation death rates. Administration of amphotericin B to the amniotic cavity had no damaging action. Administration of the methyl derivative on day 15 of pregnancy led to anomalous development of the lower extremities and slower ossification. The threshold doses by the embryotoxic action for intravenous administration are 500 micrograms/kg for amphotericin B and 2000 micrograms/kg for the methyl derivative. Administration of the antibiotics to the amniotic cavity revealed potential teratogenic properties of the amphotericin B methyl derivative.     

A crystalline A14-(2-nitro-4-trimethylammoniophenyl) derivative of bovine insulin

[O-(2-Nitro-4-trimethylammoniophenyl)-TyrA 14]insulin (bovine) is a product formed on reaction of bovine insulin with the hydrophilic reagent 1-fluoro-2-nitro-4-trimethyl-ammoniobenzene iodide (TAN-F) in an aqueous buffer at pH 8.00. The derivative was isolated and its purity established by standard procedures. The identity of the derivative was determined by degrative studies with alpha-chymotrypsin. The addition of zinc to the above reaction decreases the yield of the title derivative, but increases the yield of the [N alpha-TAN-GlyA1] derivative. [N alpha-Boc-GlyA1]insulin was also reacted with the above mentioned reagent in an attempt to improve the yield of the A14-tyrosine derivative. The biological activity of this microcrystalline derivative was found to be 12.4 units/mg as measured by the mouse convulsion assay.     

Competitive binding of various beta-lactam compounds to penicillin-binding proteins of Escherichia coli

Competing interaction of two novel N-acyl derivatives of ampicillin i.e. N'-benzylchlorbenzimidazole (No. 48) and N-pyrazolytiazole (No. 72) derivatives and 14C-benzylpenicillin with penicillin-binding proteins (PBP) of E. coli was studied. It was shown that ampicillin and its derivative No. 48 markedly differed in their affinity to various PBPs. Derivative No. 72 did not prevent binding of the labeled benzylpenicillin to any PBP which corresponded to its low antimicrobial activity. Analogous experiments with new cephalosporin structures i.e. active and inactive N-acyl derivatives of cephalosporin showed that the active derivative No. 94 i.e. N-methyltiobenzimidazole derivative had the highest affinity to PBP-2 and PBP-5. The inactive derivative No. 68 i.e. N-chlorbenzimidazole derivative also had high affinity to PBP-1b, PBP-2 and PBP-3 essential for the cell. No activity of the latter compound against intact cells of E. coli was probably due to its low penetration through the outer membrane of the bacterial cell. Estimation of affinity of the beta-lactam structures to various PBPs not only provided data on the mechanism of their action but also made it possible to explain in some cases the peculiarities of their antimicrobial spectrum.     

Characterisation of Streptococcus thermophilus CNRZ1205 and its cured and re-lysogenised derivatives

Streptococcus thermophilus CNRZ1205 is the lysogenic host for the temperate phage phi O1205. A derivative of CNRZ1205 was isolated which was cured of phi O1205 and this strain was used to construct a re-lysogenised derivative. Pulse field gel electrophoresis and sequencing of the attachment site regions confirmed that excision and re-integration of the phage was a site-specific event. Interestingly, cells from the cured, as well as its re-lysogenised derivative, were found to have a very long chain length.     

Isolation and activities of the trypsin-modified Vicia angustifolia proteinase inhibitor lacking carboxyl-terminal hexapeptide.

The Vicia angustifolia proteinase inhibitor was incubated with p-toluenesulfonyl-L-phenylalanine chloromethyl ketone-trypsin (EC 3.4.21.4) and a main product was isolated. The purified product was different to the first trypsin-modified V. angustifolia inhibitor. The C-terminal residues of the new derivative were arginine, which was also the C-terminal of the cleaved antitryptic site; lysine was a newly exposed C-terminal. These results suggest that the new derivative lacks the C-terminal portion of the native inhibitor, which has asparagine at its C-terminus. The liberated C-terminal peptide had the following amino acid sequence: H-Glu-Glu-Val-Ile-Lys-Asn-OH. The derivative lacking the C-terminal hexapeptide still possesses inhibitory activities against trypsin and alpha-chymotrypsin (EC 3.4.21.1), however, its antichymotryptic activity was inactivated by incubation with chymotrypsin at pH 8.0.     

Synthesis and antihyperlipidemic activity of acetylated derivative of ulvan from Ulva pertusa

In this study, acetylated ulvan (AU) was prepared with acetic anhydride in N,N-dimethylacetamide, and the antihyperlipidemic activity of natural ulvan and its acetylated ulvan derivative (AU) in mice was determined. Obvious differences in antihyperlipidemic activity between natural ulvan and its derivative were observed, moreover, AU showed stronger antihyperlipidemic activity on triglyceride (TG) and low density lipoprotein cholesterol (LDL-C).     

Unwinding of supercoiled Col E1-DNA after covalent binding of the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene and its 7-iodo derivative

The unwinding of superhelical Col E1-DNA was studied by means of gel electrophoresis and electron microscopy after covalent binding of N-acetoxy-N-2-[14C]acetylaminofluorene (N-Aco-[14C]AAF) and its 7-iodo derivative (N-Aco-[14C]AAIF). Studies with both compounds indicated that complete unwinding of the supercoiled DNA required the binding of hydrocarbon residue to about 3% of the bases. Thus the unwinding angle per residue of N-2-acetylaminofluorene (AAF) and its 7-iodo derivative was of 22 degrees +/- 3 and 18 degrees +/- 3 respectively. Our results are in good agreement with those obtained by Drinkwater et al. [9]. Precedent studies from this laboratory have shown that N-Aco-AAF and its 7-iodo derivative induce different local conformation change in native DNA (insertion-denaturation model and outside binding model respectively). The unexpected ability of the 7-iodo derivative to unwind supercoiled DNA is discussed.     

Photoaffinity labeling of insulin receptor of rat adiopocyte plasma membrane

A photosensitive insulin derivative was synthesized by reacting radioactive iodinated bovine insulin with N-hydroxysuccinimide ester of 4-azidobenzolic acid. The photo-sensitivity and specificity of this insulin derivative were established by its covalent nonspecific cross-link to albumin and its covalent specific cross-link to the heavy and light chains of anti-insulin immunoglobulin. Plasma membrane preparations of rate adipocytes were incubated with the photosensitive insulin derivative and irradiated with light. Sodium dodecyl sulfate gel electrophoresis of these plasma membrane preparations after solubilization with sodium dodecyl sulfate and reduction with beta-mercaptoethanol showed that a protein having a molecular weight of 130,000 was specifically labeled by the radioactive photosensitive insulin, suggesting that this protein may be the insulin receptor.     

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.     

Synthesis and biological properties of new phosmidosine analogs having an N-acylsulfamate linkage

A new phosmidosine analog 10, in which the proline and 8-oxoadenosine moieties were linked by an N-acyl sulfamate linkage, was successfully synthesized by the sulfamoylation of an 8-oxoadenosine derivative 5 followed by coupling with an L-proline derivative 8. An L-alanine-substituted derivative 13 and its derivative 14 without the alanyl residue were also synthesized. The morphological reversion activity of these synthetic compounds in v-src(ts) NRK cells and their antitumor activity in L1210 and KB cells were studied. As the result, neither L-proline- nor L-alanine-substituted phosmidosine analogs 10 and 13 showed any antitumor activity. Contrary to these results, the derivative 14 lacking the amino acid residue showed potent antitumor activities against cancer cells.     

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.