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Plipastatins: new inhibitors of phospholipase A2, produced by Bacillus cereus BMG302-fF67. II. Structure of fatty acid residue and amino acid sequence.

Plipastatins, new inhibitors of phospholipase A2, were produced by a strain classified as Bacillus cereus BMG302-fF67. The plipastatins are a family of acylated decapeptides which differ from each other by amino acid composition and the nature of the fatty acid side chain. The fatty acids have been shown to be 3(R)-hydroxyhexadecanoic acid (n-C16h3) and 14(S)-methyl-3(R)-hydroxyhexadecanoic acid (a-C16h3) by mass, NMR and optical rotation. Amino acid sequence analysis by secondary ion mass spectrometry and additional physico-chemical evidence indicate that the structures of plipastatinic acids, the lactone-opened peptides are as follows: beta-Hydroxy fatty acid----L-Glu----D-Orn----L-Tyr----D-allo-Thr----L-Glu----D-Ala(Val)---- L-Pro----L-Gln----D-Tyr----L-Ile X OH.

Amino Acid Sequence↗

The structure of manumycin. III. Absolute configuration and conformational studies.

Chromic acid oxidation of manumycin (1), an antibiotic produced by Streptomyces parvulus (strain Tü 64), led to the isolation of 2-(2-methyl-4-oxo-2-pentenoylamino)-5,6-epoxy-1,4-benzoquinone (3) and (-)-(R)-2-methylhexanoic acid (4). From the absolute configuration of 4, determined by comparing its optical rotation with published data, follows the absolute configuration at the center of chirality in the diene side chain of manumycin (1) to be (6'R). Based on the direct comparison of the CD spectra of the two chromic acid oxidation products 2 and 3 with those of the antibiotic G7063-2 (5) and (-)-terreic acid (6) the stereochemistry at C-5 and C-6 of 1 was determined as (5R, 6S). From the negative CD-couplet of manumycin (1) its stereochemistry at C-4 was assigned as (4R).

Alkenes↗

Aldecalmycin, a new antimicrobial antibiotic from Streptomyces. III. Determination of absolute configuration.

The planar structure of aldecalmycin (1) had been determined in the previous paper, together with the conformations of the substituted trans decalin ring having one double bond and the sugar: beta-glucopyranoside type. The absolute configuration of 1 was a following problem to be solved. X-Ray crystallographic analysis of crystalline 4'-6'-O-benzylidenedihydroaldecalmycin (4) revealed the relative configuration. On the other hand, the stereoisomerism of the sugar was determined to be D by the optical rotation value of tetra-O-acetyl-alpha-methylglucopyranoside derived from 1. These results established the absolute configuration of 1.

Anti-Bacterial Agents↗

Novel mammalian cell cycle inhibitors, tryprostatins A, B and other diketopiperazines produced by Aspergillus fumigatus. II. Physico-chemical properties and structures.

Two novel diketopiperazines named tryprostatins A and B and a new natural product belonging to the diketopiperazine series, designated as demethoxyfumitremorgin C, together with four known diketopiperazines, fumitremorgin C, 12,13-dihydroxyfumitremorgin C, fumitremorgin B and verruculogen, are new M phase inhibitors of the mammalian cell cycle, which were isolated from the secondary metabolites of Aspergillus fumigatus. The structures of tryprostatins A, B and demethoxyfumitremorgin C were determined mainly by the use of spectroscopic methods especially by detailed analyses of their 1H and 13C NMR spectra with the aid of 2D NMR techniques including pulse field gradient heteronuclear multiple-bond correlation (PFG-HMBC) spectroscopy. Their absolute configurations were determined on the basis of the optical rotational values and CD spectra.

Aspergillus fumigatus↗

PF1092A, B and C, new nonsteroidal progesterone receptor ligands produced by Penicillium oblatum. II. Physico-chemical properties and structure elucidation.

The structures of PF1092A (1), B (2) and C (3), new nonsteroidal progesterone receptor ligands produced by Penicillium oblatum, were elucidated by spectroscopic analyses. These compounds possess an eremophilane-type sesquiterpene carbon skeleton and differ only in that 1 and 2 are different monoacetates of 3. The absolute configurations of 1-3 were determined by single crystal X-ray diffraction analysis of the 4-bromobenzoyl ester of PF1092A and by measuring the optical rotations of the acetylation products of these compounds.

Furans↗

Erabulenols, inhibitors of cholesteryl ester transfer protein produced by Penicillium sp. FO-5637. II. Structure elucidation of erabulenols A and B.

Structures of erabulenols A and B, novel fungal inhibitors of cholesteryl ester transfer protein were elucidated by spectroscopic studies including various NMR measurements. Erabulenols consist of a phenalenone skeleton and a 1,2,2-trimethyltetrahydrofuran moiety in common. Erabulenol B possesses an additional 2,6-dihydroxy-5-methyl-3-methylketonyl benzyl moiety. The absolute stereochemistry at the C-2' position of erabulenol A was deduced as S by comparison of the optical rotation with that of other related compounds.

Carrier Proteins↗

Tripropeptins, novel antimicrobial agents produced by Lysobacter sp.

Planar structures of tripropeptins (TPPs) were elucidated by spectroscopic studies including various NMR measurements. Stereochemistry of constituent amino acids of tripropeptin C (TPPC) (3) was identified by marfey's method except hydroxyproline which was determined by studies of NMR and CD spectra. The absolute structure of 3 was determined by analyses of the fragments obtained by Birch reduction and LiBH4 reduction of 3. The configuration of the fatty acid, isolated from acid hydrolysate of 3, was determined to be (3R)-hydroxy-13-methyltetradecanoic acid from MS, NMR spectra and negative sign of the optical rotation.

Aluminum Compounds↗

[Cholecalciferol Reference Standard (Control 971) of National Institute of Health Sciences].

The raw material for cholecalciferol was examined for preparation of the "Cholecalciferol Reference Standard (Control 971)". Analytical data obtained were as follows: melting point, 83.8 degrees C; UV and infrared spectra, the same as those for JP Cholecalciferol Reference Standard (Control 945), respectively; specific absorbance at 265 nm, E1 cm 1% = 485; optical rotation, [alpha]D20 = +107.4 degrees; thin-layer chromatography and high-performance liquid chromatography (HPLC), no impurity was detected; assay, 98.9% by HPLC. Based on the above results, the raw material was authorized as the Cholecalciferol Reference Standard (Control 971) of National Institute of Health Science.

Chemical Phenomena↗

[Ergocalciferol Reference Standard (Control 971) of National Institute of Health Sciences].

The raw material for ergocalciferol was examined for preparation of the "Ergocalciferol Reference Standard (Control 971)". Analytical data obtained were as follows: melting point, 116.7 degrees C; UV and infrared spectra, the same as those for JP Cholecalciferol Reference Standard; specific absorbance, E1 cm 1% = 461(265 nm); optical rotation, [alpha]D20 = +102.5 degrees; thin-layer chromatography and high-performance liquid chromatography (HPLC), no impurity was detected; assay, 102.4% by HPLC. Based on the above results, the raw material was authorized as the Ergocalciferol Reference Standard (Control 971) of National Institute of Health Sciences.

Chemical Phenomena↗

[Sterospecific selection of nucleophilic compounds in the chymotrypsin-catalyzed formation of the peptide bonds].

The course of stereospecific selection of nucleophilic compounds was studied in the reaction of acyl-enzymes interaction with razemic substrate-like nucleophiles, e.g. amino acid esters, by measuring optical rotation or incorporation of labelled D-compounds. It was shown that the acyl-enzymes are not responsible for the stereospecific selection of substrate-like nucleophiles. Since stereospecific selection of nucleophiles occurs in some chymotrypsin-catalyzed reactions, such selection may be produced by chymotrypsin till the formation of an acyl-enzyme compound with the substrate at the enzyme-inhibitor stage (or the Michaelis complex) with nucleophilic compounds. Even under the optimal conditions no absolute stereospecific selection of nucleophiles occurred, as was observed in case of a substrate (a donor of the acyl amino acid residue), undergoing degradation. An essential role of a specific site of nucleophile binding in the reactions of chymotrypsin-catalyzed peptide bond formation, is emphasized.

Amino Acids↗

Immunologically active (1-->3)-(1-->4)-alpha-D-glucan from Cetraria islandica.

A polysaccharide, Ci-3, resembling isolichenan except with a much higher degree of polymerization, has been isolated from the water extract, as well as from the alkali extract, of the lichen Cetraria islandica (L.) using ethanol fractionation, dialysis, ion-exchange chromatography and gel filtration. The mean M(r) of Ci-3 was determined to be 2000 kD, compared to 6-8 kD reported for isolichenan. The structure of Ci-3 was elucidated and found to be composed of (1-->3)- and (1-->4)-alpha-D-glucopyranosyl units in the ratio of 2:1, using methanolysis, methylation analysis, optical rotation and NMR spectroscopy. The immunomodulating activity of Ci-3 was tested in an in vitro phagocytosis assay and anti-complementary, and proved to be active in both tests.

Adjuvants, Immunologic↗

[Binding of sisomicin and gentamicin to serum proteins (author's transl)].

The binding of sisomicin and gentamicin to the proteins of human serum was investigated by the dialysis method at 37 degrees C. The antibiotic concentrations were determined by the disc diffusion assay as well as by measurements of the optical rotation of the antibiotics in the buffer solutions. Serum was dialyzed against buffer solutions to obtain the standards for the antibiotic assay with regard to the electrolytical exchange at dialysis. In studies on protein binding of sisomicin and gentamicin no measurable binding to human serum proteins could be found, neither in the therapeutically possible concentrations nor at higher ones. Therefore a different binding of the two antibiotics cannot be the reason of the advantage of sisomicin over gentamicin in tests in vivo with animals described in literature nor of its higher activity against several bacterial strains of different species tested in the presence of serum.

Anti-Bacterial Agents↗

Red blood cell (RBC) deformability, RBC aggregability and tissue oxygenation in hypertension.

Arterial hypertension could be considered a progressive ischaemic syndrome interesting the macro and the microcirculation. In order to improve the clinical and therapeutic approach to the treatment of arterial hypertension, research has centered on blood flow to evaluate the different components and their very intricate relationships influencing the micro- and the macrocirculation. Of course the main problem is to study the link between the blood flow and the peripheral tissue oxygenation. During hypertension very important alterations in rheological, mechanical and biochemical characteristics of erythrocytes and of blood flow have been shown. It is very relevant the increase in blood viscosity, the decrease in red blood cell (RBC) deformability, the formation of RBC "rouleaux" and RBC aggregates. These hemorheological determinants can favour an increase of peripheral resistances and of arterial blood pressure, causing or worsening hypertension, a decrease in oxygen transport to tissue and peripheral perfusion, a decrease of the active exchange surface area in the microvasculature, especially in complicated hypertension. We have studied 320 patients: 123 with Essential Hypertension (EH) (M 59, F 64 aged 50 +/- 25 years); 81 with Secondary Hypertension (SH) without associated other pathologies influencing hemorheology (M 42, F 39 aged 48 +/- 20 years); 116 SH with other pathologies or conditions associated influencing hemorheology such as: diabetes, lipoidoproteinosis, obesity, smoking, HD, elderly, etc. (M 48, F 68 aged 46 +/- 20 years). Using a Laser-assisted Optical Rotational Red Cell Analyzer (LORCA) acc. to Hardeman (1994) we studied Elongation Index (EI) and aggregation kinetics of red blood cells in these patients. We also evaluated TcpO2 and TcpCO2 using a transcutaneous oxymeter (Microgas 7650, Kontron Instruments). In hypertensives we found a decrease in erythrocyte deformability (evaluated with EI), in erythrocyte aggregation time, a fibrinogenaemia increase, an increase of shear rate to disaggregate erythrocytes, a decrease in cellular oxygen delivery and tissue oxygenation, an impairment of microcirculation. These changes may be involved in the development of arterial hypertension and in its pathogenesis. These patterns also are more impaired in hypertensives with diabetes, lipoidoproteinosis, etc. These patterns are not related with the age of the patients but they are significantly and directly related (p < 0.01) with the patient hypertension-age. This could be a new way to realize a better treatment in hypertensives and a prevention of cardiovascular complications (i.e.: myocardial infarction, TIA, etc.).

Adult↗

Measurement of erythrocyte deformability by two laser diffraction methods.

The aim of this work is to study the deformability of red blood cells (RBC) by two laser diffraction methods: the Laser-assisted Optical Rotational Cell Analyser (LORCA, Mechatronics, Amsterdam, Netherlands) and a Shear Stress Diffractometer (RHEODYN SSD, Myrenne, Roetgen, Germany). Experiments were carried out on 46 healthy human subjects. The elongation index EI of normal and hardened RBCs (obtained by heating blood at 49 degrees C or by incubating RBCs in solutions of diamide) was measured. The results showed that the standard deviations of the experimental data for normal RBCs were relatively small, especially at high shear stresses (more than 3.0 Pa), but higher than those reported before. Some correlations between the results given by the two instruments were also found. It should be noted that for hardened RBCs, the standard deviations of the measurements were important compared with the mean values in the two instruments.

Adult↗

Hemorheological aspects in hypertensive menopausal smoker women treated with female hormones.

In postmenopausal hypertensive women (PostMHW) the erythrocyte deformability (ED) is reduced if compared with premenopausal hypertensive women (PreMHW). This might partially explain the increased incidence of cardiovascular diseases (CD) in hypertensive women after menopause. Moreover a positive correlation exists between estradiol and rheological patterns in women. If PostMHW smoke cigarettes, there is an important decrease in hemorheological parameters. On the other hand if PostMHW are submitted to an hormonal replacement therapy (HRT) they can show controversial results with an impairment if hemorheological parameters. The aim of this study was to evaluate the influence of smoking and HRT on PostMHW. We studied four groups of subjects: Group 1: PreMHW (10 F aged 35 +/- 3 years) non smokers; Group 2: PostMHW (8 F aged 45 +/- 2 years) non smokers; Group 3: PostMHW (14 F aged 48 +/- 4 years) smokers (20 cigarettes per day); Group 4: PostMHW (16 F aged 50 +/- 2 years) smokers (20 cigarettes per day) submitted to HRT. We evaluated Elongation Index of erythrocytes under torsion force of 30 pascals (EI--30 Pa) using a new computerized instrument Laser assisted Optical rotational Red Cell Analyzer (LORCA) (Mechatronics, Hoorn, NL) acc. to Hardeman (1994) and, also with the same LORCA, Aggregation Index (AI), t(1/2). We measured the transcutaneous oxygen partial pressure (TcpO2) in subclavicular standard area using a Transcutaneous Oximeter (Microgas 7650 Kontron Instruments with Combi Sensor) and total cholesterolaemia. In PostMHW our data showed a significant (p < 0.01) impairment of hemorheological patterns and tissue oxygenation if compared with PreMHW (Group 1). In Group 3 there is a significant (p < 0.01) decrease in EI, a significant (p < 0.01) increase in AI, a significant (p < 0.01) decrease in t(1/2) and TcpO2 if compared with Control Group 1 and Group 2. Finally a further significant (p < 0.01) impairment in hemorheology and tissue oxygenation showed Group 4. In conclusion, it seems necessary, that many studies will be performed to understand really protective action of HRT on cardiovascular diseases in PostMHW and it is necessary to suppress cigarette smoking to prevent cardiovascular complications in these patients.

Adult↗

Tissular oxygenation and erythrocyte deformability in haemodialyzed patients using different dialytic membranes.

We studied the effect of some dialytic membrane on tissular oxygenation (TO) and erythrocyte deformability (ED). Sixteen patients (10 M and 6 F, aged 59 +/- 12 years) have been submitted to bicarbonate dialysis (BD) and subdivided into four groups (GR) of 4 patients each: GR 1 (hemophan membrane, 35 BD), GR 2 (polyacrylonitrile, PAN AN 69, 42 BD), GR 3 (polysulphone, 38 BD) and GR 4 (polycarbonate, 37 BD). The TO has been detected with the transcutaneous oxygen pressure (Tc pO2) using a transcutaneous oxymeter and the ED has been evaluated with the EMI (Erythrocyte Morphometric Index), which results from the ratio between deformable erythrocytes (bowl shape) and rigid erythrocytes (discocyte shape), for every 100 red cells fixed in vitro with 0.3% glutaraldehyde. The ED was also evaluated using a laser instrument: Laser Optical Rotational Cell Analyser. During BD was observed a significant decrease of Tc pO2 in the 1st hour only in the 1st and 2nd GR and, in contrast with results obtained in the 3rd and 4th GR, in the same GRs the EMI showed a significant reduction of ED at the end of BD. Finally the LORCA results, showing a significant decrease of ED only in the 1st and 2nd GR, confirmed the data obtained with EMI. In conclusion, our study has suggested that hemophan and PAN AN 69 are less biocompatible than polysulphone and polycarbonate membranes according to effects on ED and TO.

Blood Gas Monitoring, Transcutaneous↗

Chromatographic resolution and elution order of alkyl aryl and aryl benzyl sulfoxides on cellulose-based chiral stationary phases

Several alkyl aryl and aryl benzyl sulfoxides have been prepared in optically active form via enantioselective Ti-catalyzed oxidation of the corresponding sulfides. The absolute configuration was assigned on the basis of optical rotation while in the case of some new sulfoxides it was determined by the analysis of their circular dichroism spectra. The alkyl aryl sulfoxides have been efficiently resolved by CHIRALCEL OB chiral stationary phase (CSP) while the aryl benzyl sulfoxides were better separated on CHIRALCEL OJ CSP. In both cases the S enantiomer was always eluted first. This finding can then allow to determine the absolute configuration of alkyl aryl and aryl benzyl sulfoxides on the basis of their elution order on these CSPs.

Journal Article↗

[Triamcinolone Acetonide Reference Standard (Control 981) of National Institute of Health Sciences].

The raw material of triamcinolone acetonide was examined for preparation of the "Triamcinolone Acetonide Reference Standard (Control 981)". The analytical data obtained were: melting point, 289 degrees C (decomposition); UV spectrum, lambda max of 238 nm; IR spectrum, same as that of the Triamcinolone Acetonide Reference Standard (Control 834); optical rotation, [alpha]D20 = +106.8 degrees; thin-layer chromatography, no impurities detected; high-performance liquid chromatography, total amount of impurities less than 0.4%; loss on drying, 1.3%; assay by HPLC, 100.1%. Based on the above results, the raw material was authorized as the Triamcinolone Acetonide Reference Standard (Control 981) of the National Institute of Health Sciences.

Government Agencies↗