Antimicrobial fragments of the pro-region of cathelicidins and other immune peptides

In addition to the numerous cathelicidin peptides that are associated with the antimicrobial activity exhibited by a crude extract from ovine blood, a further three peptides with antimicrobial activity have been identified. These were part of the precursor cathelin domain of cathelicidins, a large fragment of platelet factor 4 and a small peptide similar to signal peptide of the T-cell glycoprotein CD4 precursor. Fragments of proteins that are involved in protecting the host from infection may have a secondary purpose as antimicrobial agents once they have carried out their primary purpose and are cleaved the main protein.     

枯草芽孢杆菌JA-206对皮肤癣菌抑制作用的研究

低能离子诱变筛选获得的枯草芽孢杆菌JA-206的发酵粗提液对红色毛癣菌和须毛癣菌有较强的抑菌或杀菌作用,粗提液经高温、蛋白酶处理后仍然有较高的稳定性,将JA-206粗提液经多步分离纯化,得到三种单一纯化产物抗菌肽AFP1、AFP2和AFP3,其中AFP1和AFP3对两种皮肤癣菌显示出明显的抑菌或杀菌作用。     

Neurohypophyseal hormone-like peptides in the ovine pineal gland using reverse-phase liquid chromatography and radioimmunoassay

A method is described for the determination of the neurohormone contents of ovine pineal tissue by radioimmunoassay (RIA) after successive fractionation on gel filtration in formic acid and reverse-phase liquid chromatography (HPLC). This method gives a good resolution for the neurohormones vasopressin, vasotocin and oxytocin, without a significant interference of aspecific cross-reacting of peptides with the RIA. An acid extract from ovine pineal tissue was found to contain amounts of immunoreactive AVP- and OXT-like peptides, whereas an AVT-like peptide was not detectable over background levels after HPLC with post-column RIA. It is concluded from our results that an AVT-like peptide is not present in ovine pineal tissue, and the pineal AVP- and OXT-like peptides appeared to be associated to neurophysin molecules.     

Avian antimicrobial peptides: the defense role of beta-defensins

Avian antimicrobial peptides, classified as beta-defensins, have been identified from bloods of chicken, turkey, and ostrich; epithelial cells of chicken and turkey; and king penguin stomach contents. Beta-defensins are a family of antimicrobial peptides characterized by six cysteine residues forming beta-defensin motifs that are also found in bovine, ovine, pig, and human. These peptides are active against a wide range of microorganisms including Gram-positive and Gram-negative bacteria, fungi, and yeast. Analysis of evolutionary relationships of vertebrate beta-defensins showed that there might be a common ancestral gene between avian and other mammalian peptides. This ancient gene may have been passed down and evolved from species older than the oldest living birds, forming a beta-defensin-like precursor molecule. This review describes potential applications of these peptides in health care products.     

A novel heterodimeric antimicrobial peptide from the tree-frog Phyllomedusa distincta

We present here the purification and the analysis of the structural and functional properties of distinctin, a 5.4 kDa heterodimeric peptide with antimicrobial activity from the tree-frog Phyllomedusa distincta. This peptide was isolated from the crude extract of skin granular glands by different chromatographic steps. Its minimal inhibitory concentration was determined against pathogenic Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa strains. Amino acid sequencing and mass spectrometric investigations demonstrated that distinctin is constituted of two different polypeptide chains connected by an intermolecular disulphide bridge. Circular dichroism and Fourier-transformed infrared spectroscopy studies showed that this molecule adopts, in water, a structure containing a significant percentage of anti-parallel beta-sheet. A conformational variation was observed under experimental conditions mimicking a membrane-like environment. Database searches did not show sequence similarities with any known antimicrobial peptides. In the light of these results, we can consider distinctin as the first example of a new class of antimicrobial heterodimeric peptides from frog skin.     

Isolation and characterisation of proline/arginine-rich cathelicidin peptides from ovine neutrophils

Cathelicidins are a family of gene-encoded antimicrobial peptides found in mammals. Seven cathelicidin genes have been identified in sheep, but up to now only two variants of one of these predicted peptides (OaBac5) have been purified from ovine neutrophils. In this work numerous proline/arginine-rich cathelicidin peptides were purified, including the originally predicted OaBac5 and another OaBac5 variant. As well as this, the C-terminus of the predicted OaBac7.5 and various truncated forms of OaBac11 were purified. Even though these peptides were much smaller than those predicted, they still displayed antimicrobial activity.     

Purification and characterization of high-molecular-weight forms of adrenocorticotropic hormone of ovine pituitary glands.

A highly purified preparation of high-molecular-weight adrenocorticotropic hormone (ACTH) was prepared from ovine pituitary glands by dilute acetic acid extraction, oxycellulose fractionation. Sephadex gel filtration, and affinity chromatography on immobilized alphap(1-39)ACTH antibodies. Two ACTH peptides of molecular weights of 24 000 and 34 000 were detected by sodium dodecyl sulfate-acrylamide gel electrophoresis in this preparation. It appeared that the immobilized antibodies adsorbed two forms equally well and could not distinguish between them under the conditions used. These two ACTH peptides were found to be present in crude extracts of ovine pituitary glands, indicating that they were not artifacts produced by the purification procedure. The high-molecular-weight forms of ACTH were found to be susceptible to degradation by tissue enzymes. They could be easily destroyed during the extraction, if precautions were not taken. Moreover, they were poorly adsorbed by oxycellulose which had been used for the adsorption of ACTH activity from crude preparations by most investigators. These properties probably accounted for the fact that high-molecular-weight forms of ACTH remained undetected until very recently.     

Induction of N-acetyllactosamine synthetase activity in the mouse mammary gland by prolactin and placental lactogen hormone]

The authors show that the ovine prolactine promote induction of N. acetyl lactosamine synthetase in tissue culture of mammary glands of pregnant mice. A crude extract of human placenta has also a lactogenic activity as tested by the same method, but in this case the blank values are very high for large concentration of crude extract. The molecular forms of HCS are tested: the slow band has a lactogenic activity, the intermediate band has no activity and the rapid band seems to be inhibitory.     

Analysis of antimicrobial peptides from porcine neutrophils

Cationic host defence peptides are important components of innate immunity in pigs and other mammalians. Most of these peptides have a direct antimicrobial activity and they also have a broad spectrum of effects on the host immune system, which may be taken into account in the introduction of novel therapeutics. Our method permits simultaneous isolation of six antibacterial peptides, i.e. prophenin-1, prophenin-2, PR-39, and protegrins 1-3 from a porcine neutrophil crude extract and characterisation of them. Among the obtained peptides the greatest bactericidal activity expressed as MBC was seen in protegrins (10 μg/ml), whereas in the other studied peptides MBC was on the level of 20 μg/ml. Minimal inhibitory concentrations (MIC) reached 10 μg/ml for protegrins 1-3 and 20 μg/ml for prophenins, and PR-39. Within the bactericidal range all isolated peptides didn't show cytotoxicity on cell lines used in our experiment.     

A one-enzyme strategy to release an antimicrobial peptide from the LFampin-domain of bovine lactoferrin

Antimicrobial peptides have been found throughout living nature, yet antimicrobial sequences may still lie hidden within a wide variety of proteins. A rational strategy was developed to select interesting domains, based on the presumed common features of antimicrobial peptides, and to release these from accessible and safe proteins. In silico proteolysis simulations of bovine lactoferrin (bLF) with selected endoproteinases predicted the liberation of peptides that encompasses a cationic amphipathic alpha-helix. Three predicted peptides were synthesized and tested for their biological activity, demonstrating that one single enzyme was sufficient to obtain an antimicrobial peptide. The proof of principle demonstrated that a 32-mer fragment isolated from the endoproteinase AspN digestion of bLF possessed strong antimicrobial activity. Moreover, desalted crude digest had improved activity over native bLF. Hence, selective digestion of bLF increases its antimicrobial activity by release of antimicrobial stretches.     

家蝇蛆抗菌肽提取工艺研究

蝇蛆抗菌肽多有广谱抗菌、抗癌等功能,是很好的天然抗菌药物来源,但由于得率较低,目 前对其产品开发的研究较少。以家蝇Musca domestica干蝇蛆为原料,利用加热-层析法和海藻酸吸附法2种工艺提取蝇蛆抗菌肽。结果表明：加热-层析法快速、简便,抗菌肽提取得率达0.26％,是海藻酸吸附法提取抗菌肽得率的5.2倍。提取的家蝇抗菌肽主要是分子量6.2～17.2 kD、等电点5.59～5.91的弱酸性小分子多肽,其热稳定性高,能杀灭枯草杆菌Bacillus subtilis等多种革兰氏阳性菌。加热-层析法能有效去除外源性蛋白酶,保证肽类产品的稳定性,同时还能提取出非蛋白类的抗菌成分,提示其对开发具有高附加值的抗菌产品将会有良好的应用前景。     

Allelopathic effects of water hyacinth [Eichhornia crassipes

Eichhornia crassipes (Mart) Solms is an invasive weed known to out-compete native plants and negatively affect microbes including phytoplankton. The spread and population density of E. crassipes will be favored by global warming. The aim here was to identify compounds that underlie the effects on microbes. The entire plant of E. crassipes was collected from El Zomor canal, River Nile (Egypt), washed clean, then air dried. Plant tissue was extracted three times with methanol and fractionated by thin layer chromatography (TLC). The crude methanolic extract and five fractions from TLC (A-E) were tested for antimicrobial (bacteria and fungal) and anti-algal activities (green microalgae and cyanobacteria) using paper disc diffusion bioassay. The crude extract as well as all five TLC fractions exhibited antibacterial activities against both the gram positive bacteria; Bacillus subtilis and Streptococcus faecalis; and the gram negative bacteria; Escherichia coli and Staphylococcus aureus. Growth of Aspergillus flavus and Aspergillus niger were not inhibited by either E. crassipes crude extract nor its five fractions. In contrast, Candida albicans (yeast) was inhibited by all. Some antialgal activity of the crude extract and its fractions was manifest against the green microalgae; Chlorella vulgaris and Dictyochloropsis splendida as well as the cyanobacteria; Spirulina platensis and Nostoc piscinale. High antialgal activity was only recorded against Chlorella vulgaris. Identifications of the active antimicrobial and antialgal compounds of the crude extract as well as the five TLC fractions were carried out using gas chromatography combined with mass spectroscopy. The analyses showed the presence of an alkaloid (fraction A) and four phthalate derivatives (Fractions B-E) that exhibited the antimicrobial and antialgal activities.     

Antifungal activity of crude extract produced by Bacillus sp. associated with entomopathogenic nematode from media formulated by six nitrogen sources with fructose against Penicillium expansum

A specific symbiotic Bacillus species isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp. was found to produce a number of bioactive compounds. The present study was conducted to determine the effect of six different nitrogen sources in combination with fructose on the production of antifungal crude extract by Bacillus sp. against Penicillium expansum. The yield of crude extract and antifungal activity against the test fungi differed significantly when the nitrogen sources in the fermentation media were changed. The highest yield was recorded for beef extract plus fructose (921?mg/L). The antifungal activity was higher in yeast extract plus fructose [P. expansum (46.5?±?2.12?mm)], followed by beef extract. High performance liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention times indicating that they produced different compounds. When a carbon source was not included in the fermentation medium, the antimicrobial production was substantially reduced almost eight times. Carbon source in the fermentation medium plays a vital role in the production of antimicrobial substances. Yeast extract and fructose as nitrogen and carbon sources in the fermentation medium produced maximum antimicrobial activity.     

Isolation and biochemical characterization of peptides presenting antimicrobial activity from the skin of Phyllomedusa hypochondrialis

Amphibian antimicrobial peptides have been known for many decades and several of them have been isolated from anuran species. Dermaseptins are among the most studied antimicrobial peptides and are found in the skin secretion of tree frogs from the Phyllomedusinae subfamily. These peptides exert a lytic action on bacteria, protozoa, yeast, and filamentous fungi at micromolar concentrations, but unlike polylysines, present little hemolytic activity. In this work, two antimicrobial peptides were isolated from the crude skin secretion of Phyllomedusa hypochondrialis and tested against Gram-positive and Gram-negative bacteria, presenting no hemolytic activity at the tested concentrations. One of them was identified with the recently reported peptide PS-7 belonging to the phylloseptin family, and another was a novel peptide, named DPh-1, which was fully purified, sequenced by ‘de novo’ mass spectrometry and grouped into Dermaseptins (DPh-1).     

Antimicrobial activity of 2,5-dihydroxy-3-methyl-1,4-benzoquinone from Embelia schimperi

Chromatographic separation of an ethyl acetate extract from Embelia schimperi led to the isolation of a new compound identified as 2,5-dihydroxy-3-methyl-1,4-benzoquinone (1) on the basis of spectroscopic and physical data. The plant's crude extract and pure compound 1 were assayed for in vitro antimicrobial activity against clinical strains of Salmonella spp., Proteus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Cryptococcus neoformans, Shigella dysentriae and Staphylococcus aureus. Disc diffusion method was used and zones of inhibition, after respective incubation periods, were used to quantify antimicrobial activity. Standard antibiotics namely: augmentin, cotrimoxazole, gentamycin, tetracycline and lyncomycin were used as controls. The crude extract was inactive while the pure compound 1 showed significant activities against Salmonella spp., Proteus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Cryptococcus neoformans, Shigella dysentriae and Staphylococcus aureus with zones of inhibition ranging from 10-20 mm. The most sensitive microorganism was P aeruginosa while C. neoformans was insensitive to both the crude extract and compound 1.     

Post-secretory events alter the peptide content of the skin secretion of Hypsiboas raniceps

A novel family of antimicrobial peptides, named raniseptins, has been characterized from the skin secretion of the anuran Hypsiboas raniceps. Nine cDNA molecules have been successfully cloned, sequenced, and their respective polypeptides were characterized by mass spectrometry and Edman degradation. The encoded precursors share structural similarities with the dermaseptin prepropeptides from the Phyllomedusinae subfamily and the mature 28-29 residue long peptides undergo further proteolytic cleavage in the crude secretion yielding consistent fragments of 14-15 residues. The biological assays performed demonstrated that the Rsp-1 peptide has antimicrobial activity against different bacterial strains without significant lytic effect against human erythrocytes, whereas the peptide fragments generated by endoproteolysis show limited antibiotic potency. MALDI imaging mass spectrometry in situ studies have demonstrated that the mature raniseptin peptides are in fact secreted as intact molecules within a defined glandular domain of the dorsal skin, challenging the physiological role of the observed raniseptin fragments, identified only as part of the crude secretion. In this sense, stored and secreted antimicrobial peptides may confer distinct protective roles to the frog.     

Brazilian Kefir-Fermented Sheep’s Milk,a Source of Antimicrobial and Antioxidant Peptides

Fermented milks are a source of bioactive peptides and may be considered as functional foods. Among these, sheep’s milk fermented with kefir has not been widely studied and its most relevant properties need to be more thoroughly characterized. This research study is set out to investigate and evaluate the antioxidant and antimicrobial properties of peptides from fermented sheep’s milk in Brazil when produced by using kefir. For this, the chemical and microbiological composition of the sheep’s milk before and after the fermentation was evaluated. The changes in the fermented milk and the peptides extracted before the fermentation and in the fermented milk during its shelf life were verified. The antimicrobial and antioxidant activities of the peptides from the fermented milk were evaluated and identified according to the literature. The physicochemical properties and mineral profile of the fermented milk were like those of fresh milk. The peptide extract presented antimicrobial activity and it was detected that 13 of the 46 peptides were able to inhibit the growth of pathogenic microorganisms. A high antioxidant activity was observed in the peptides extracted from fermented milk (3.125 mg/mL) on the 28th day of storage. Two fractions displayed efficient radical scavenging properties by DPPH and ABTS methods. At least 11 peptides distributed in the different fractions were identified by tandem mass spectrometry. This sheep’s milk fermented by Brazilian kefir grains, which has antioxidant and antimicrobial activities and probiotic microorganisms, is a good candidate for further investigation as a source for bioactive peptides. The fermentation process was thus a means by which to produce potential bioactive peptides.     

Arnebins and antimicrobial activities of Arnebia hispidissima DC. cell cultures.

The whole plant of Arnebia hispidissima DC. (Boraginaceae) is used for the treatment of tongue and throat ailments in Indian traditional medicine. The present paper deals with the plants phytochemical constituents, the arnebins, and antimicrobial activities of its root extract. The antimicrobial activities were tested against Gram-positive and Gram-negative bacteria and fungi. The crude hexane extract demonstrated a potent antimicrobial effect against bacteria and a mild effect against fungi. Likewise, the hexane extract of cell cultures of A. hispidissima also showed mild bioefficacy against the select microorganisms.     

Impact of beef extract and six carbon source on antifungal metabolites production by bacterium associated with entomopathogenic nematode against Fusarium oxysporum

A specific symbiotic Bacillus species isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp., was found to produce a number of bioactive compounds. The present study was conducted to determine the effect of six different carbon sources in combination with beef extract on the production of antifungal substances by Bacillus sp. The yield of crude antimicrobial substances and antimicrobial activity against the test microorganism also differed significantly when the carbon sources in the fermentation media were changed. The highest yield was recorded for fructose plus beef extract (956?mg/l). The antifungal activity was significantly high in beef extract plus maltose (21?±?1.5?mm) followed by beef extract plus glucose and beef extract plus fructose. Antifungal activity was significantly reduced in beef extract plus lactose and sucrose. High pressure liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention times indicating that they produced different compounds. When a carbon source was not included in the fermentation media, the antifungal production was substantially reduced. Carbon source in the fermentation medium plays a vital role in the production of antimicrobial substances. Beef extract and maltose as nitrogen and carbon sources in the fermentation medium produced maximum antifungal activity. It is concluded that Beef extract and maltose as nitrogen and carbon sources produced maximum activity which can effectively control the Fusarium oxysporum which causes vascular fusarium wilt in tomato, tobacco, legumes, cucurbits, sweet potatoes, banana, etc.     

Influence of carbon and nitrogen sources on antifungal metabolite production by bacterium associated with entomopathogenic nematode against Penicillium expansum

A specific symbiotic Bacillus sp. isolated from a rhabditid entomopathogenic nematode, Rhabditis (Oscheius) sp. was found to produce large number of bioactive compounds. The present study was conducted to determine the effect of carbon and nitrogen sources for the production of antimicrobial substances by Bacillus sp. The yield of the crude antimicrobial substances and antimicrobial activity against the test micro-organism also differed significantly when carbon and nitrogen sources in the fermentation media were changed. The antifungal activity was significantly high in yeast extract plus fructose (46.5?±?2.12?mm) followed by yeast extract plus maltose, beef extract plus fructose and meat infusion plus glucose. High pressure liquid chromatography analysis of the crude antimicrobial substances revealed different peaks with different retention time indicating that they produced different compounds. When the carbon source was not included in the fermentation media, the antimicrobial production was substantially reduced. The results indicate that carbon source in the fermentation media plays a vital role in the production of antifungal substances. It is concluded that yeast extract and fructose as nitrogen and carbon sources produced maximum activity, which can effectively control the blue mould caused by Penicillium expansum in apples and pears.