DENATURATION OF PROTEINS. V.* N.M.R. STUDY OF THE ARGININE RESIDUES OF LYSOZYME

The heat denaturation of lysozyme has been studied by proton magnetic resonance spectroscopy at 220 MHz in water at pH 2.8. At 20°C a broad resonance is observed at 6.8δ due to the arginine NH₂ resonances, which becomes a well defined triple resonance at 40°C, without any change in the rest of the spectrum. This sharpening of the arginine NH₂ resonances is due to their increased mobilities and/or some normalisation of their chemical shifts. It is proposed that the surface structure of lysozyme is loosened at 40°C, without any change in the hydrophobic core and this is considered to be an intermediate phase in its heat denaturation. The heat denatured product at 80°C in D₂O retains some noncovalent interactions, since further treatment with guanidine deuterochloride or mercaptoethanol causes sharpening of the aromatic region of the spectrum.     

Solubilization of Liposomes by Weak Electrolyte Drugs. I. Propranolol

The solubilization of dimyristoylphosphatidylcholine (DMPC) liposomes by a weak electrolyte drug, propranolol (PPL) hydrochloride, has been studied as a function of pH, [PPL], [DMPC], and temperature. The solubilization of liposomes at 40°C by 0.2 mM PPL occurred at different rates from 2.9 to 14.4 mM DMPC but converged at complete solubilization after 13 hr at pH 12.0. At the same [PPL], solubilization was complete after 18 days at pH 11.0, but incomplete solubilization occurred at pH 10.0 and not at all at lower pH's. In 14.4 mM DMPC liposomes, solubilization was gradual and proportional to the [PPL] from 0.001 to 0.10 mM up to 95 hr, then rapid thereafter. The [PPL] at which the solubilization efficiency began to increase rapidly was determined to be 0.078 mM. The rate of solubilization was also influenced by the fluidity of the bilayers, a sevenfold increase in the time for complete solubilization being observed upon cooling from 40 to 20°C. Surface tension (st) data confirmed a low critical micelle concentration (CMC) and continued decrease in the st above the CMC. It is concluded that the critical ratio of PPL to DMPC for solubilization occurs in localized regions of the bilayers, with total solubilization at different rates depending on the [PPL] and the physical properties of the liposomes. The processes may be used advantageously to prepare small vesicles or to extract lipids or proteins, more efficiently than detergents, from biological membranes.     

H n.m.r. spectrum from the flexible N-terminal segment of Streptomyces subtilisin inhibitor

The ¹H n.m.r. spectrum of Streptomyces subtilisin inhibitor shows a limited number of unusually sharp signals at room temperature. Some of these signals are assigned uniquely to protons of the side chains of the N-terminal segment, Aspl-Ala2-Pro3-Ser4-Ala5-Leu6-Tyr7-based on experiments of spin decoupling, pH titration, and enzymatic cleavage of the protein. Quantitative examination of these signals indicates that the N-terminal end of this protein is heterogeneous in that the protein contains a considerable fraction whose sequence starts with Ala2 rather than with Asp1. The pK_a values for the amino groups of Asp1 and Ala2 exposed at the N-terminus are determined to be 8.9 ± 0.4 and 9.0 ± 0.1, respectively. Furthermore, examination of the line-widths of the methyl proton resonances of Ala2 and Ala5 residues indicates that the N-terminal peptide segment is free and undergoes rapid segmental motions in the order of 10^?9s.     

Antioxidant Activity of the Essential Oils of Different Parts of Juniperus communis. subsp. hemisphaerica. and Juniperus oblonga.

Abstract

The essential oils of different parts of Juniperus communis. subsp. hemisphaerica. (Presl) Nyman (Cupressaceae) and Juniperus oblonga. M. B. were examined for their potential radical scavenging activity. The compositions of the essential oils of these plants were studied qualitatively and quantitatively by GC and GC-MS. The main components of the essential oils as well as positive controls were subjected to antioxidant testing. A rapid evaluation for antioxidants, using two TLC screening methods, showed that all tested oils and their main components have antioxidant activity. The abilities of the volatile oils to act as nonspecific donors for hydrogen atoms for electron were checked in the diphenylpicrylhydrazyl (DPPH) assay. In the DPPH assay, the strongest effect among the essential oils was measured for the oil of leaves of male J. communis. subsp. hemisphaerica. at a concentration of 4 µL/mL (24.0%) In the deoxyribose degradation assay, the essential oils, pure components, and positive controls were tested at different concentrations. Most of the tested compounds showed some antioxidant effects. The fruit oil of J. oblonga. has the strongest effect among the tested volatile oils.

The deoxyribose assay was modified in three different ways to assess whether the oils exhibited site-specific effects. The results of the current study, which demonstrate the DPPH scavenging activity of the essential oils of the of leaves of male J. communis. subsp. hemisphaerica. and the OH radical scavenging effects of the fruit oil of J. oblong. suggest the use of these two essential oils in very low concentrations for preserving food materials.     

Evaluation of Mucoadhesive Polymers in Ocular Drug Delivery. II. Polymer-Coated Vesicles

Association of Carbopol 934P and Carbopol 1342 (a hydrophobic modified Carbopol resin) with phospholipid vesicles was assessed by photon correlation spectroscopy and microelectrophoresis at pH 7.4 and 5. The precorneal clearance of the polymer-coated vesicles was compared to that of uncoated vesicles by lacrimal dacryoscintigraphy in the rabbit. The mucoadhesive polymer-coated vesicles demonstrated significantly enhanced precorneal retention compared to noncoated vesicles only at pH 5 (P < 0.005). The entrapment and subsequent release of tropicamide from Carbopol 1342-coated and uncoated liposomes were determined in vitro together with an in vivo evaluation of the vesicles formulated at the lower pH. Mucoadhesive polymer-coated vesicles failed to increase significantly the bioavailability of the entrapped tropicamide compared to uncoated vesicles and aqueous solution.     

Randomized, placebo-controlled comparison of famotidine 20 mg b.d. or 40 mg b.d. in patients with erosive oesophagitis

Methods: this US multicentre, randomized, double-blind, placebo-controlled, parallel group study determined the effects of two twice daily oral famotidine regimens on symptom relief and healing of erosive oesophagitis in patients with gastro-oesophageal reflux disease. Three hundred and eighteen patients were enrolled: 66 received placebo, 125 received famotidine 20 mg b.d., and 127 received famotidine 40 mg b.d. Patients maintained diaries of their symptoms. Endoscopy was performed at weeks 0 and 6, and again at week 12 if healing had not occurred. Results: healing at 6 and 12 weeks was (respectively) 48% (P < 0.01 vs. placebo) and 69% (P? 0.01 vs.placebo) for famotidine 40 mg b.d.; 32% and 54% (P? 0.01 vs. placebo) for famotidine 20 mg b.d., and 18% and 29% for placebo. At both 6 and 12 weeks the healing rates of famotidine 40 mg b.d. were significantly greater than placebo and famotidine 20 mg b.d. Compared to placebo, famotidine produced more frequent global symptom improvement and more rapid heartburn relief. There were no significant differences among treatment groups in the incidence of clinical or laboratory adverse events. Conclusions: famotidine 40 mg b.d. was a better regimen than famotidine 20 mg b.d. or placebo. The clinical efficacy paralleled the previously documented effect of the famotidine regimens on decrease of oesophageal acid exposure.     

Isosteric conversion of protein carboxyl groups into carboxamide groups. I. Examination of alkyl and aryl esters as model compounds

With a view to substituting protein carboxyl groups by carboxamide groups the properties of a number of model compounds have been examined. The methyl ester of acetylglycine can be 97% converted into amide in 7 M ammonium acetate/ 7 M ammonia in 10 min. Amidation of the N-ethylsalicylamide ester of benzoyl-glycine, prepared by reaction with N-ethylbenzisoxazolium fluoroborate (EBI), is 100% complete in 2 M ammonium acetate/ammonia, pH 9.2, within 1 min; no rearrangement into imide occurred at all. Under the conditions of protein modification (0.1 M EBI, pH 4.2, 0°) acetyltyrosinamide is 13% converted into a compound, presumably containing the N-ethylsalicylimidate group, exhibiting an absorption maximum at 309 nm (e = 3300). This compound decomposes in ammonium acetate/ammonia, pH 9.1, within 1 min. Nα-acetylhistidinamide is not converted at all. Reaction with aminogroups can either be avoided by protection of these groups or be analyzed by spectroscopy of the amidine produced. Spectral data required for analysis have been measured for N-ethylsalicylamide (Λ^max295 nm, e = 3200 at pH 6, Λ_max324 nm, e = 5100 at pH 10, pK 8.15), for O-acetyl-N-ethylsalicylamide (e = 1200 at 250 nm, e = 180 at 280 nm) and for O-diethylsalicylamidine (Δ_max277nm, e = 2330 at pH 6, Λ_max302 nm, e = 3230 at pH 10, pK 7.90).     

Protective effect of Alstonia scholaris Linn. R. Br. against Bleomycin induced chromosomal damage in cultured human lymphocytes,in vitro

It is both interesting and necessary to identify and develop nontoxic radioprotective compounds. Bleomycin (BLM), a known radiomimetic drug was used as a clastogen in the present study. The possible protective effects against BLM (15?μg/ml) induced clastogenicity by aqueous and methanolic extracts from Alstonia scholaris bark, stem and leaves were compared. The treatment of bark extracts significantly (p?2 assay was performed. Lymphocyte cultures from 12 healthy volunteers were exposed to aqueous (50?μg/ml) and methanolic (50?μg/ml) extracts of A. scholaris bark alone as well as in combination with Bleomycin under two different growth phases, G₀ and G₂. There was a statistically significant reduction (p?2 phase as compared to respective cultures exposed at G₀ phase. The highest level (p?2 phase than those at G₀ phase. This indicated that there could be certain compound(s) present in aqueous bark extracts which enhance DNA repair capacity. Therefore, the bark of A. scholaris could be further utilized to identify and bring out front line radio protective agents in the market with effective formulations.     

Acute Head-Only Exposure of Dogs to Phosgene. Part III. Comparison of Indicators of Lung Injury in Dogs and Rats

To better understand the relevance of phosgene-induced changes in bronchoalveolar lavage (BAL) fluid protein observed in acutely exposed rats, groups of beagle dogs were similarly exposed for 30 min to phosgene using a head-only mode of exposure. The actual exposure concentrations were 9, 16.5, and 35 mg/m³, with resultant C × t products of 270, 495, and 1050 mg/m³ × min. In rats, a C × t product of 270 mg/m³ × min caused a significant elevation of protein in the bronchoalveolar lavage (BAL) fluid, while the nonlethal threshold concentration (LCt₀₁) was estimated to be 1075 mg/m³ × min. The endpoints examined in dogs focused on changes in BAL, lung weights, arterial blood gases, and lung histopathology approximately 24 h postexposure. Mortality did not occur at any C × t product. Increased lung weights and elevations in protein, soluble collagen, and polymorphonuclear leukocyte (PMN) counts in BAL were observed at 1050 mg/m³ × min with borderline changes at 495 mg/m³ × min. Following exposure to 1050 mg/m³ × min, the analysis of arterial blood gases provided evidence of a significantly decreased arterial pO₂. Histopathology revealed a mild, although distinctive, inflammatory response at the bronchoalveolar level at 495 mg/m³ × min, whereas serofibrinous exudates and edema were observed at 1050 mg/m³ × min. The magnitude of effects correlated with the individual dogs' respiratory minute volume and breathing patterns (panting). Collectively, phosgene-induced indicators of acute lung injury appeared to be characterized best by protein in BAL fluid. With regard to both the inhaled dose and the associated increase of protein in BAL, the responses obtained in dogs appear to be more similar to humans. In contrast, elevations in BAL protein occurred in rats at three-fold lower concentrations when compared to dogs. The results of this study demonstrate that the magnitude of elevations of plasma exudate in BAL fluid following acute exposure to the pulmonary irritant phosgene is markedly more pronounced in rats when compared to the dog which is considered more human-like than rats. This is believed to be associated with the higher ventilation of small rodents and with rodent-specific sensory bronchopulmonary defense reflexes.     

The action of pesticides on microorganisms. I. parathion

Previous experiments indicate that parathion, DDT, aldrin, and dieldrin induce changes in fatty acid and phospholipid composition in Escherichia coli. The effect of parathion on the growth and viability of E. coli, P. aeruginosa and R. meliloti was studied. When the pesticide was present at a concentration of 8.6 × 10^?6M, the following effects were observed: P. aeruginosa growth was not affected, E. coli was slightly inhibited and R. meliloti was affected adversely in the incubation mixture. We also examined the effects on the cell membrane components such as phospholipids and proteins; both quantitative and qualitative changes were observed. The results varied according to the microorganism utilized. Our data revealed alterations in the phospholipids that may be an important adaptative response of microorganism to growth in the presence of the pesticides.     

Screening of Southeastern Brazilian Mikania. Species on Trypanosoma cruzi.

Abstract

Southeastern Brazilian Mikania. (Asteraceae) species were evaluated for trypanocidal activity against the trypomastigote forms of Trypanosoma cruzi.at a concentration of 4000 µg/mL. Fourteen extracts were examined for in vitro. trypanocidal properties. Of total extracts, 92.9% (13 extracts) exhibited trypanocidal effects. The dichloromethane extract of Mikania camporum. B. Robinson and the methanol extract of Mikania micrantha. H. B. K. caused 100% lysis of the parasites.     

Oxygen-17 n.m.r. of peptides

Peptide-¹⁷O chemical shifts of linear dipeptides with and without protecting groups in H₂O, CH₃OH, CH₂Cl₂, CHCl₃, CCl₄, CH₃CN and DMSO were between 256–350 ppm downfield from external water. Increasing solvent H-bond donating ability correlated with shifts to higher field. The ¹⁷O resonance of several cyclic dipeptides appeared at higher field relative to comparable linear dipeptides (303–317 p.p.m. vs. 327–337 p.p.m.). Separate signals were simultaneously observed by ¹³C and ¹⁷O n.m.r. for cis and trans N-tert.-butyl-formamide in binary mixtures with H₂O, (CH₃)₂CO, and CCl₄. The differences in the ¹⁷O nuclear screening of the amide isomers and most probably for cis and trans peptides were independent of contributions from H-bonding at the amide or peptide linkage, apparently reflecting differences between geometric isomers in electron distribution and through space effects. Peptide-¹⁷O of Gly-Ala, Gly-Leu and Gly-Glu in aqueous solution experienced upfield shifts of 6–12 p.p.m. and 12–16 p.p.m. upon deprotonation of the C-terminal COOH and of the N-terminal NH+₃ groups respectively. These observations were rationalized in terms of the attendant changes in substituent effects, especially on the ± electron donating ability of the N atom at the peptide linkage and increased partial negative charge on the peptide oxygen. Temperature studies of peptide-¹⁷O of Gly-Ala between pH 1.5–9.0 revealed a chemical shift coefficient of 0.08 p.p.m./K and similar behavior of T₁ and T₂ relaxation times. Ea for molecular rotation was 5 kcal/mol between 301–331·K. Rotational correlation times, _c, were within the range expected from the Stokes-Einstein relation.     

THE PREFERRED CONFORMATIONS OF PROTECTED HOMODI-TO HOMOHEPTAMETHIONINE PEPTIDES A 1H N.M.R. Study in Deuterochloroform Medium

Detailed analyses of the conformations of the homo-oligopeptide series, Boc-(L-Met)_n-OMe n = 2–7, in deuterochloroform have been carried out with proton n.m.r. and IR spectroscopy. Well-resolved high field n.m.r. spectra with assignments for the NH and α-CH resonances of these homo-methionine peptides are presented. Extensive n.m.r. concentration-dependent chemical shift studies are combined with IR results to delineate the involvement of the various methionine NH protons in intra- and/or intermolecular hydrogen bonding. N.m.r. chemical shift dependencies with temperature and solvent, DMSO-d₆, are used to explore the strength of the hydrogen bonds for the various oligopeptides. At low concentrations, where peptide aggregation is absent, the dipeptide is found to be disordered. The tetra- to heptapeptides possess intramolecular hydrogen bonded seven-membered rings at internal residues. The number of internal rings and the oligopeptide self-association increase with increasing peptide chainlength. At intermediate concentrations associations of peptide molecules with folded structures occur with initial association at the C-terminal region. At high concentrations, “in-register” associated extended β structures are formed.     

Untersuchungen an 1.3-Thiazinen, 34. Mitt. Oxidationen an Thiourethanen, 8. Mitt. Oxidative Desulfurierung und nitrosierende N-Desalkylierung durch salpetrige Säure oder Salpetersäure

1.3-Thiazines, XXXIV; Oxidations of Thiourethanes, VIII Oxidative Desulfurisation and Nitrosating N-Dealkylation by Nitrous or Nitric Acid Depending on the reaction conditions, 3-amino-2-thioxotetrahydro-4H-1.3-thiazin-4-ones 1 are desulfurized by nitrous or nitric acid to yield the 2-oxoanalogues 2 or in addition are dealkylated at the nitrogen atom with liberation of aldehydes and subsequent N-nitrosation to 3 .     

Antioxidant Potential of an Extract of Calendula officinalis. Flowers in Vitro. and in Vivo.

Abstract

An extract of Calendula officinalis. Linn. (Compositae) was evaluated for its antioxidant potential in vitro. and in vivo.. Calendula officinalis. extract was found to scavenge superoxide radicals generated by photoreduction of riboflavin and hydroxyl radicals generated by Fenton reaction and inhibited in vitro. lipid peroxidation. Concentrations needed for 50% inhibition (IC₅₀) were 500, 480, and 2000 µg/mL, respectively. Extract scavenged ABTS radicals and DPPH radicals and IC₅₀ were 6.5 and 100 µg/mL, respectively. IC₅₀ values were compared with that of ginger extract, which is a standard antioxidant extract. The drug also scavenged nitric oxide, and the IC₅₀ was found to be 575 µg/mL. Extract also produced dose-dependent scavenging of nitric oxide in culture. The oral administration of Calendula. extract inhibited superoxide generation in macrophages in vivo. by 12.6% and 38.7% at doses of 100 and 250 mg/kg b.wt. Oral administration of Calendula officinalis. to mice for 1 month significantly increased catalase activity. The extract produced significant increase in glutathione levels in blood and liver. Glutathione reductase was found to be increased, whereas glutathione peroxidase was found to be decreased after administration of Calendula. extract. These results indicated Calendula officinalis. has significant antioxidant activity in vitro. and in vivo..     

Hypolipidemic Agents of Phthalimide Derivatives 6. Effects of Aromatic vs. Non-Aromatic Imides

A number of substituted phthalimide, 1, 8-naphthalimide, succinimide and glutarimide derivatives demonstrated significant hypolipidemic activity at 20 mg/kg/ day, I.P. after 16 days dosing. The N-(n-pentyl) succinimide proved to be the most potent analogue of the new compounds, lowering serum triglyceride levels 51 % and serum cholesterol 47 % after 16 days dosing in mice. For the N-substituted derivatives, i. e., n-butyl, butanone, and propionic acid, of these four cyclic imides, there appeared to be no obvious trend in ability to reduce serum lipid levels. In general, the 1,8-naphthalimide and glutarimide derivatives appeared to be less active than phthalimide and succinimide. However, the -phenylsuccinimide afforded less activity than the -phenylglutarimide. Most of the derivatives at 20mg/kg/day demonstrated improved activity over clofibrate at 150mg/kg/day.     

Nature of the B10 amino acid residue.

Human [10-asparagine-B] insulin ([Asn¹⁰-B] insulin), an analogue which differs from the parent molecule in that the histidine residue at position 10 of the B chain (B¹⁰) is replaced by asparagine, has been synthesized and isolated in purified form. In vitro biological assays indicated a potency of ca. 35% compared to insulin. We have previously shown that the replacement of histidine at position B¹⁰ by lysine resulted in an analogue displaying ca. 15% of the biological activity of natural hormone, while the substitution of leucine in this position produced a molecule exhibiting ca. 45% potency in in vivo assays. The data indicate that molecular size of the amino acid residue at position B¹⁰ may be important in the maintenance of a structure commensurate with high biological activity. Polarity at this position appears to be rather unimportant while a strongly basic group appears to be deleterious.     

N.M.R. OF ‘ORIENTED’ MONOSUBSTITUTED AMIDES—CIS AND TRANS N-METHYL FORMAMIDE

The n.m.r. spectrum of N-methyl formamide oriented in a lyotropic liquid crystal solvent shows the presence of the cis as well as the trans forms. Analysis of the spectra due to both the species has been carried out. Possible non-planar distortions at the nitrogen atom for the trans species have been computed using the derived dipolar couplings and the available structural data.     

Antiprotozoal Activity Against Plasmodium falciparum. and Trypanosoma cruzi. of Aeroplysinin-1 Isolated from the New Sponge Aplysina chiriquensis.

ABSTRACT

Crude methanol extracts from the recently discovered sponge, Aplysina chiriquensis. and Aplysina gerardogreeni., collected at the southwest Pacific Coast of Panama, showed moderate antiplasmodial activity. Fractionation of the crude extracts from A.. chiriquensis. led to the isolation of the known dibromotyrosine derivatives 1–5. Compound 3, aeroplysinin-1, displayed antiprotozoal activity against chloroquine-resistant Plasmodium falciparum. and Trypanosoma cruzi.. Comparative analysis by HPLC of the five specimens collected showed the presence of the same metabolites but in different relative proportions. Compounds 1–5 are reported for the first time as constituents of A.. chiriquensis. and A.. gerardogreeni.. This is the first report of the antiprotozoal activity of aeroplysinin-1 (3).     

In Vitro. Screening of Cleome Viscosa. Extract for Anthelmintic Activity

Abstract

The crude alcohol and aqueous extracts of the seeds of Cleome viscosa. Linn. (Capparidaceae) were investigated for their anthelmintic activity against Pheretima posthuma. and Ascardia galli.. Various concentrations (10–100 mg/mL) of each extract were tested in the bioassay, which involved determination of time of paralysis and time of death of the worms. Both extracts exhibited considerable anthelmintic activity in a dose-dependent manner. The most significant activity was observed at the highest concentration of 100 mg/mL against both types of worms. Piperazine citrate (10 mg/mL) was included as standard reference and distilled water as control.