Chickpea Defensive Proteinase Inhibitors Can Be Inactivated by Podborer Gut Proteinases1

Developing chickpea (Cicer arietinum L.) seeds 12 to 60 d after flowering (DAF) were analyzed for proteinase inhibitor (Pi) activity. In addition, the electrophoretic profiles of trypsin inhibitor (Ti) accumulation were determined using a gel-radiographic film-contact print method. There was a progressive increase in Pi activity throughout seed development, whereas the synthesis of other proteins was low from 12 to 36 DAF and increased from 36 to 60 DAF. Seven different Ti bands were present in seeds at 36 DAF, the time of maximum podborer (Helicoverpa armigera) attack. Chickpea Pis showed differential inhibitory activity against trypsin, chymotrypsin, H. armigera gut proteinases, and bacterial proteinase(s). In vitro proteolysis of chickpea Ti-1 with various proteinases generated Ti-5 as the major fragment, whereas Ti-6 and -7 were not produced. The amount of Pi activity increased severalfold when seeds were injured by H. armigera feeding. In vitro and in vivo proteolysis of the early- and late-stage-specific Tis indicated that the chickpea Pis were prone to proteolytic digestion by H. armigera gut proteinases. These data suggest that survival of H. armigera on chickpea may result from the production of inhibitor-insensitive proteinases and by secretion of proteinases that digest chickpea Pis.

Trans-dominant inhibitory human immunodeficiency virus type 1 protease monomers prevent protease activation and virion maturation.

Production of infectious human immunodeficiency virus (HIV) requires proper polyprotein processing by the dimeric viral protease. The trans-dominant inhibitory activity of a defective protease monomer with the active site Asp-25 changed to Asn was measured by transient transfection. A proviral plasmid that included the drug-selectable Escherichia coli gpt gene was used to deliver the wild-type (wt) or mutant proteases to cultured cells. Coexpression of the wt proviral DNA (HIV-gpt) with increasing amounts of the mutant proviral DNA (HIV-gpt D25N) results in a concomitant decrease in proteolytic activity monitored by in vivo viral polyprotein processing. The viral particles resulting from inactivation of the protease were mostly immature, consisting predominantly of unprocessed p55gag and p160gag-pol polyproteins. In the presence of HIV-1 gp160 env, the number of secreted noninfectious particles correlated with the presence of increasing amounts of the defective protease. Greater than 97% reduction in infectivity was observed at a 1:6 ratio of wt to defective protease DNA. This provides an estimate of the level of inhibition required for effectively preventing virion processing. Stable expression of the defective protease in monkey cells reduced the yield of infectious particles from these cells by 90% upon transfection with the wt proviral DNA. These results show that defective subunits of the viral protease exert a trans-dominant inhibitory effect resulting from the formation of catalytically compromised heterodimers in vivo, ultimately yielding noninfectious viral particles.

Antibacterial Activity of Extracellular Products from Cyanobacteria.

Cyanobacteria are photosynthetic prokaryotes that can be found in freshwater and marine environments as well as in soil. These organisms produce a variety of different biologically active compounds exhibiting anti-bacterial, anti-fungal and anti-cancer properties among others. In this study, cyanobacterial isolates were screened for their ability to produce extracellular antibacterial products. Cyanobacteria were isolated from fresh water and soil samples collected in the Pembroke Pines, FL area. Twenty- seven strains of cyanobacteria were isolated belonging to the following genera: Limnothrix, Nostoc, Fischerella, Anabaena, Pseudoanabaena, Lyngbya, Leptolyngbya, Tychonema, and Calothrix. Individual strains were grown in liquid culture in laboratory conditions. Following 14-day cultivation, the culture liquid was filtered and tested for activity against the following bacteria: Escherichia coli, Bacillus megatarium, Staphylococcus aureus, and Micrococcus luteus. Among all genera of cyanobacterial strains tested, Fischerella exhibited the greatest inhibitory activity. An attempt was made to isolate the active compound from the culture liquid of the active strains. Lipophilic extracts from culture liquid were obtained from three selected Fischerella strains. The extracts proved to have varying levels of activity against the tested bacteria. Inhibitory activity from all three Fischerella strains was detected against B. megatarium and M luteus. The only strain that was active against S. aureus was Fischerella sp. 114-12 while none of the extracts showed activity against E. coli. This kind of screening has potential pharmaceutical and agricultural benefits, including possible discovery of novel antibiotics.

Kunitzins: Prototypes of a new class of protease inhibitor from the skin secretions of European and Asian frogs

Amphibian skin secretions contain biologically-active compounds, such as anti-microbial peptides and trypsin inhibitors, which are used by biomedical researchers as a source of potential novel drug leads or pharmacological agents. Here, we report the application of a recently developed technique within our laboratory to “shotgun” clone the cDNAs encoding two novel but structurally-related peptides from the lyophilized skin secretions of one species of European frog, Rana esculenta and one species of Chinese frog, Odorrana schmackeri. Bioanalysis of the peptides established the structure of a 17-mer with an N-terminal Ala (A) residue and a C-terminal Cys (C) residue with a single disulphide bridge between Cys 12 and 17, which is a canonical Kunitz-type protease inhibitor motif (-CKAAFC-). Due to the presence of this structural attribute, these peptides were named kunitzin-RE (AAKIILNPKFRCKAAFC) and kunitzin-OS (AVNIPFKVHLRCKAAFC). Synthetic replicates of these two novel peptides were found to display a potent inhibitory activity against Escherichia coli but were ineffective at inhibiting the growth of Staphylococcus aureus and Candida albicans at concentrations up to 160 μM, and both showed little haemolytic activity at concentrations up to 120 μM. Subsequently, kunitzin-RE and kunitzin-OS were found to be a potent inhibitor of trypsin with a Ki of 5.56 μM and 7.56 μM that represent prototypes of a novel class of highly-attenuated amphibian skin protease inhibitor. Substitution of Lys-13, the predicted residue occupying the P1 position within the inhibitory loop, with Phe (F) resulted in decrease in trypsin inhibitor effectiveness and antimicrobial activity against Esherichia coli, but exhibits a potential inhibition activity against chymotrypsin.

Purificação, caracterização e avaliação da atividade antiproliferativa de um inibidor de quimotripsina tipo kunitz de semente de Eryhrina velutina

A chymotrypsin inhibitor was purified from Erythrina velutina seeds by ammonium sulphate fractionation, affinities chromatographies on Trypsin-Sepharose, Quimotrypsin-Sepharose and reversed phase C-18 FPLC/AKTA system. The inhibitor, named EvCI, shown molecular mass of 17 kDa, as determined by SDSPAGE. 2D-PAGE showed four isoinhibitors with pI values of 4,42, 4,63, 4,83 and 5,06, with molecular mass of 17 kDa each. The aminoacid sequence of EvCI was determined by MALDI-TOF-MS and showed a high similarity with other Kunitz-type inhibitor of Erythrina variegata. EvCI competitively inhibited chymotrypsin, with Ki of 4 x10-8 M, but did not inhibited trypsin, pancreatic elastase, bromelain and papain. The inhibitory activity of EvCI was stable over wide pH and temperature ranges. In the presence of DTT 100 mM for 120 min, EvCI lost 50 % of activity. Cytotoxicity was studied in HeLa, MDA, HepG2, K562 and PC3 cells after 72-h incubation period. EvCl inhibited HeLa cells growth with an IC50 value of 50 μg/ml. Subsequent studies in HeLa cells analysis of cell death by annexin V/PI double-staining and cell cycle, using flow cytometry. The results provide evidence for a cytostatic activity of EvCl and support further studies on potential application of this inhibitors as an antiproliferative agent in combined therapy against cervical cancer

Design, synthesis and antiproliferative activity of hydroxyacetamide derivatives against HeLa cervical carcinoma cell and breast cancer cell line

Purpose: To design and develop a new series of histone deacetylase inhibitors (FP1 - FP12) and evaluate their inhibitory activity against hydroxyacetamide (HDAC) enzyme mixture-derived HeLa cervical carcinoma cell and MCF-7. Methods: The designed molecules (FP1 - FP12) were docked using AUTODOCK 1.4.6. FP3 and FP8 showed higher interaction comparable to the prototypical HDACI. The designed series of 2-[[(3- Phenyl/substituted Phenyl-[4-{(4-(substituted phenyl)ethylidine-2-Phenyl-1,3-Imidazol-5-One}](-4H- 1,2,4-triazol-5-yl)sulfanyl]-N-hydroxyacetamide derivatives (FP1-FP12) was synthesized by merging 2- [(4-amino-3-phenyl-4H- 1, 2, 4-triazol-5-yl) sulfanyl]-N-hydroxyacetamide and 2-{[4-amino-3-(2- hydroxyphenyl)-4H-1,2, 4-triazol-5-yl]sulfanyl}-N hydroxyacetamide derivatives with aromatic substituted oxazolone. The biological activity of the synthesized molecule (FP1-FP12) was evaluated against HDAC enzyme mixture-derived HeLa cervical carcinoma cell and breast cancer cell line (MCF-7). Results: HDAC inhibitory activity of FP10 showed higher IC50 (half-maximal concentration inhibitory activity) of 0.09 μM, whereas standard SAHA molecule showed IC50 of 0.057 μM. On the other hand, FP9 exhibited higher GI50 (50 % of maximal concentration that inhibited cell proliferation) of 22.8 μM against MCF-7 cell line, compared with the standard, adriamycin, with GI50 of (-) 50.2 μM. Conclusion: Synthesis, spectral characterization, and evaluation of HDAC inhibition activity and in vitro anticancer evaluation of novel hydroxyacetamide derivatives against MCF-7 cell line have been achieved. The findings indicate the emergence of potentialanticancer compounds.

Exploring the role of the α-carboxyphosphonate moiety in the HIV-RT activity of α-carboxy nucleoside phosphonates

As α-carboxy nucleoside phosphonates (α-CNPs) have demonstrated a novel mode of action of HIV-1 reverse transcriptase inhibition, structurally related derivatives were synthesized, namely the malonate 2, the unsaturated and saturated bisphosphonates 3 and 4, respectively and the amide 5. These compounds were evaluated for inhibition of HIV-1 reverse transcriptase in cell-free assays. The importance of the α-carboxy phosphonoacetic acid moiety for achieving reverse transcriptase inhibition, without the need for prior phosphorylation, was confirmed. The malonate derivative 2 was less active by two orders of magnitude than the original α-CNPs, while displaying the same pattern of kinetic behavior; interestingly the activity resides in the “L”-enantiomer of 2, as seen with the earlier series of α-CNPs. A crystal structure with an RT/DNA complex at 2.95 Å resolution revealed the binding of the “L”-enantiomer of 2, at the polymerase active site with a weaker metal ion chelation environment compared to 1a (T-α-CNP) which may explain the lower inhibitory activity of 2.

Propolis components accountable for bactericidal accomplishment and antibiofilm activity

This study aimed at evaluating antimicrobial and antibiofilm activity of phenolic compounds present in propolis ethanol extracts (PEE). Seventy per cent ethanol extracts from seven types of propolis, one Green, two Red and four Brown collected in four Brazilian States were prepared and total phenolics, flavonoids, tannins and anthocyanins were assessed by high-performance liquid chromatography (HPLC). Minimum bactericidal concentration (MBC) and inhibitor effect on Staphylococcus aureus biofilm formation and capacity to disrupt established biofilms were assessed towards eight S. aureus isolates from milk of small ruminants with mastitis, one methicillin-resistant S. aureus (MRSA) and S. aureus ATCC 25923. To evaluate different propolis components accountability for bactericidal accomplishment and antibiofilm activity, the results were analysed by the non-parametric Spearman coefficient. Results of phenolic compounds were 216,21 to 312,08 gallic acid milligram equivalent per extract gram (mg EGA/g) of total phenolics, 55,08 to 140,6 quercetin milligram equivalent per extract gram (mg EQ/g) of flavonoids, 118,51 to 3766,16 catechin milligram equivalent per extract gram (mg EC/g) of tannins and 1,03 to 8,39 milligram per extract gram (mg/g) of anthocyanins. Red1 and Red2 showed higher tannin contents, while Red2 exhibited superior amount of anthocyanins and total phenolics. Brown3 presented higher flavonoid quantity. Green, Red1 and Red2 PEE showed the lowest levels of flavonoids, but the higher antimicrobial activity. Most PEE exhibit bactericidal activity at a concentration of 1.6 mg/mL. Brown4 PEE showed the worst capacity to inhibit S. aureus. Green PEE showed to be the most efficient in both preventing and disrupting biofilm. All PEE studied exhibited a better inhibitory activity prior-to than post-biofilm formation. According to non-parametric Spearman correlation analysis, there seems to be a significant negative correlation between the ability to disrupt biofilm and both tannins and anthocyanins contents.

Antifungal activity of 7-hydroxycalamenene-rich essential oil nanoemulsion.

The aim of this study was to evaluate the inhibitory activity of 7-hydroxycalamenene-rich essential oil nanoemulsion against filamentous fungi and yeasts.

Screening of Trypanosoma cruzi glycosomal glyceraldehyde-3-phosphate dehydrogenase enzyme inhibitors

The inhibitory activity of crude extracts of Meliaceae and Rutaceae plants on glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) enzyme from Trypanosoma cruzi was evaluated at 100 μg/mL. Forty-six extracts were tested and fifteen of them showed significant inhibitory activity (IA % > 50). The majority of the assayed extracts of Meliaceae plants (Cedrela fissilis, Cipadessa fruticosa and Trichilia ramalhoi) showed high ability to inhibit the enzymatic activity. The fractionation of the hexane extract from branches of C. fruticosa led to the isolation of three flavonoids: flavone, 7-methoxyflavone and 3',4',5',5,7-pentamethoxyflavone. The two last compounds showed high ability to inhibit the gGAPDH activity. Therefore, the assayed Meliaceae species could be considered as a promising source of lead compounds against Chagas' disease.

Antibiosis and dark-pigments secretion by the phytopathogenic and environmental fungal species after interaction in vitro with a Bacillus subtilis isolate

In this work, different reactions in vitro between an environmental bacterial isolate and fungal species were related. The Gram-positive bacteria had terminal and subterminal endospores, presented metabolic characteristics of mesophilic and acidophilic growth, halotolerance, positive to nitrate reduction and enzyme production, as caseinase and catalase. The analysis of partial sequences containing 400 to 700 bases of the 16S ribosomal RNA gene showed identity with the genus Bacillus. However, its identity as B. subtilis was confirmed after analyses of the rpoB, gyrA, and 16S rRNA near-full-length sequences. Strong inhibitory activity of environmental microorganisms, such as Penicillium sp, Aspergillus flavus, A. niger, and phytopathogens, such as Colletotrichum sp, Alternaria alternata, Fusarium solani and F. oxysporum f.sp vasinfectum, was shown on co-cultures with B. subtilis strain, particularly on Sabouraud dextrose agar (SDA) and DNase media. Red and red-ochre color pigments, probably phaeomelanins, were secreted by A. alternata and A. niger respectively after seven days of co-culture.

Evaluation of Antiproliferative, Anti-Type 2 Diabetes, and Antihypertension Potentials of Ellagitannins from Strawberries (Fragaria x ananassa Duch.) Using In Vitro Models

Strawberries represent the main source of ellagic acid derivatives in the Brazilian diet, corresponding to more than 50% of all phenolic compounds found in the fruit. There is a particular interest in the determination of the ellagic acid content in fruits because of possible chemopreventive benefits. In the present study, the potential health benefits of purified ellagitannins from strawberries were evaluated in relation to the antiproliferative activity and in vitro inhibition of alpha-amylase, alpha-glucosidase, and angiotensin I-converting enzyme (ACE) relevant for potential management of hyperglycemia and hypertension. Therefore, a comparison among ellagic acid, purified ellagitannins, and a strawberry extract was done to evaluate the possible synergistic effects of phenolics. In relation to the antiproliferative activity, it was observed that ellagic acid had the highest percentage inhibition of cell proliferation. The strawberry extract had lower efficacy in inhibiting the cell proliferation, indicating that in the case of this fruit there is no synergism. Purified ellagitannins had high alpha-amylase and ACE inhibitory activities. However, these compounds had low alpha-glucosidase inhibitory activity. These results suggested that the ellagitannins and ellagic acid have good potential for the management of hyperglycemia and hypertension linked to type 2 diabetes. However, further studies with animal and human models are needed to advance the in vitro assay-based biochemical rationale from this study.

Evaluation of Indigenous Grains from the Peruvian Andean Region for Antidiabetes and Antihypertension Potential Using In Vitro Methods

The health-relevant functionality of 10 thermally processed Peruvian Andean grains (five cereals, three pseudocereals, and two legumes) was evaluated for potential type 2 diabetes-relevant antihyperglycemia and antihypertension activity using in vitro enzyme assays. Inhibition of enzymes relevant for managing early stages of type 2 diabetes such as hyperglycemia-relevant alpha-glucosidase and alpha-amylase and hypertension-relevant angiotensin I-converting enzyme (ACE) were assayed along with the total phenolic content, phenolic profiles, and antioxidant activity based on the 1,1-diphenyl-2-picrylhydrazyl radical assay. Purple corn (Zea mays L.) (cereal) exhibited high free radical scavenging-linked antioxidant activity (77%) and had the highest total phenolic content (8 +/- 1 mg of gallic acid equivalents/g of sample weight) and alpha-glucosidase inhibitory activity (51% at 5 mg of sample weight). The major phenolic compound in this cereal was protocatechuic acid (287 +/- 15 mu g/g of sample weight). Pseudocereals such as Quinoa (Chenopodium quinoa Willd) and Kaniwa (Chenopodium pallidicaule Aellen) were rich in quercetin derivatives (1,131 +/- 56 and 943 +/- 35 mu g [expressed as quercetin aglycone]/g of sample weight, respectively) and had the highest antioxidant activity (86% and 75%, respectively). Andean legumes (Lupinus mutabilis cultivars SLP-1 and H-6) inhibited significantly the hypertension-relevant ACE (52% at 5 mg of sample weight). No alpha-amylase inhibitory activity was found in any of the evaluated Andean grains. This in vitro study indicates the potential of combination of Andean whole grain cereals, pseudocereals, and legumes to develop effective dietary strategies for managing type 2 diabetes and associated hypertension and provides the rationale for animal and clinical studies.

Antimicrobial activities of Garcinia kola on oral Fusobacterium nucleatum and biofilm

The extracts from the root, bark and seed of Garcinia kola are currently used in traditional medicine in Nigeria. The aim of this study was to evaluate the inhibitory activity of crude extracts of G. kola on Fusobacterium nucleatum isolated from the oral cavity. Methanol and aqueous extracts were prepared from the seed and the minimal inhibitory concentration was evaluated by the agar dilution method, using a Wilkins-Chalgren agar supplemented with horse blood (5%), hemin (5 mu g/ml) and menadione (1 mu g/ml). Antimicrobial activity of plant extracts on microbial biofilms was determined in microtiter plates. The seed of G. kola demonstrated significant inhibitory action on F. nucleatum isolates at a concentration of 1.25 and 12.5 mg/ml for amoxicillin resistant strain. It was able to inhibit the microbial biofilm formed by the association of F. nucleatum with Porphyromonas gingivalis ATCC 33277, Aggregatibacter actinomycetemcomitans ATCC 33384 and Prevotella intermedia ATCC 2564 at a concentration of 25 mg/ml. The in-vitro inhibitory effect of G. kola on F. nucleatum population suggests a potential role for its use in oral hygiene.

Molecular determinants of improved cathepsin B inhibition by new cystatins obtained by DNA shuffling

Background: Cystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis. Results: Starting from the sequences of oryzacystatin-1 and canecystatin-1, a shuffling library was designed and a hybrid clone obtained, which presented higher inhibitory activity towards cathepsin B. This clone presented two unanticipated point mutations as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously abolishes the inhibitory activity towards cathepsin B. Homology modeling together with experimental studies of the reverse mutants revealed the likely molecular determinants of the improved inhibitory activity to be related to decreased protein stability. Conclusion: A combination of experimental approaches including gene shuffling, enzyme assays and reverse mutation allied to molecular modeling has shed light upon the unexpected inhibitory properties of certain cystatin mutants against Cathepsin B. We conclude that mutations disrupting the hydrophobic core of phytocystatins increase the flexibility of the N-terminus, leading to an increase in inhibitory activity. Such mutations need not affect the inhibitory site directly but may be observed distant from it and manifest their effects via an uncoupling of its three components as a result of increased protein flexibility.