Modified Cysteine-Deleted Tachyplesin (CDT) Analogs as Linear Antimicrobial Peptides: Influence of Chain Length,Positive Charge,and Hydrophobicity on Antimicrobial and Hemolytic Activity

Due to increasing resistance of bacteria to traditional antibiotics, antimicrobial peptides are being investigated as a promising alternative. Tachyplesin, an antimicrobial peptide isolated from horseshoe crab, inhibits the growth of many different types of bacteria with its ability to permeabilize the cell membrane. Starting with a previously reported linear tachyplesin analog lacking cysteine (cysteine-deleted tachyplesin, CDT, KWFRVYRGIYRRR-CONH₂), this study examines the systematic deletion of the C-terminal arginines and the N-terminal lysine, addition of positively charged N-and C-terminal residues, replacement of arginine with similarly-charged lysine, and replacement of hydrophobic residues with aliphatic, aromatic, fluoro-substituted aromatic, and bicyclic amino acids to examine effects on activity. The 16 modified CDT analogs were tested for their ability to disrupt model liposomes, and minimum inhibitory concentrations were determined for gram-positive and gram-negative bacterial strains. Hemolytic activity also was assessed. Overall, results indicate that elimination of two C-terminal arginine residues results in a peptide ([des-Arg^12,13]CDT) with preserved antimicrobial activity but a reduction in hemolysis, a selectivity desirable for a therapeutic agent. Additional deletion was not tolerated, nor was addition of residues at the termini. Analysis of the 16 analogs also reveals the importance of hydrophobicity, not necessarily aromaticity, as an analog with hydrophobic isoleucine residues placed throughout the sequence ([Ile^2,3,6,10]CDT) displayed comparable antimicrobial activity to CDT with lower hemolysis, representing a promising antimicrobial peptide with lowered toxicity.     

Charge Segregation and Low Hydrophobicity Are Key Features of Ribosomal Proteins from Different Organisms

Ribosomes are large and highly charged macromolecular complexes consisting of RNA and proteins. Here, we address the electrostatic and nonpolar properties of ribosomal proteins that are important for ribosome assembly and interaction with other cellular components and may influence protein folding on the ribosome. We examined 50 S ribosomal subunits from 10 species and found a clear distinction between the net charge of ribosomal proteins from halophilic and non-halophilic organisms. We found that ∼67% ribosomal proteins from halophiles are negatively charged, whereas only up to ∼15% of ribosomal proteins from non-halophiles share this property. Conversely, hydrophobicity tends to be lower for ribosomal proteins from halophiles than for the corresponding proteins from non-halophiles. Importantly, the surface electrostatic potential of ribosomal proteins from all organisms, especially halophiles, has distinct positive and negative regions across all the examined species. Positively and negatively charged residues of ribosomal proteins tend to be clustered in buried and solvent-exposed regions, respectively. Hence, the majority of ribosomal proteins is characterized by a significant degree of intramolecular charge segregation, regardless of the organism of origin. This key property enables the ribosome to accommodate proteins within its complex scaffold regardless of their overall net charge.     

The Effect of High Temperatures on the Photosynthetic Activity of Intact Barley Leaves at Low and High Irradiance

Light curves of CO₂ fixation by barley seedling leaves preliminarily heated at 30–43°C for 5 min were measured. The slope of the linear part of the light curve decreased after leaf heating at temperatures above 35°C; whereas, at a high light level, the photosynthesis rate decreased only at temperatures of 40°C and higher. The linear relationships between the photosynthetic CO₂-fixation rate and a photon flux density up to 1400 mol/(m² s) were found in leaves preheated at 42°C; this indicates the strong nonphotochemical dissipation of absorbed light quanta. The lowering of the oxygen concentration from 21 to 1% led to a CO₂ fixation maximum quantum yield and a photosynthesis-rate increase at the highest light intensity in leaves preheated at temperatures above 40°C as compared to the control leaves. Nevertheless, the linear relationship between the photosynthetic CO₂ fixation and the light intensity was found in leaves heated at 42°C at O₂ concentrations of both 21 and 1%. The latter fact suggests that the proton gradient of the thylakoid membrane, which causes an increase in the nonphotochemical dissipation of the quanta absorbed, could also be formed due to the cyclic electron transport over photosystem I.     

Effect of Heat on the Antimicrobial Activity of Brilliant Green Dye

Antimicrobial activity of brilliant green dye in Trypticase soy broth (BBL) is reduced and ultimately destroyed by prolonged autoclaving at 121 C. Loss of antimicrobial activity is accompanied by decolorization of the dye. This is consistent with other evidence that antimicrobial activity of brilliant green resides in the colored dye ion. The dye is not decolorized when heated in distilled water or peptone, but is decolorized by heating in glucose, glycine, or sodium dodecyl sulfate, showing that decolorization results from reaction with components of the medium. To ensure optimal results, it is recommended that bacteriological media be sterilized by heat prior to addition of brilliant green dye.     

Effect of Carbon Source on the Antimicrobial Activity of the Air Flora

Summary In an attempt to screen for air flora producing new potent antimicrobial substances, Bacillus megaterium NB-3, Bacillus cereus NB-4, Bacillus cereus NB-5, Bacillus subtilis NB-6 and Bacillus circulans NB-7, were isolated and were found to be antagonistic to bacteria and/or fungi. Production of antimicrobial substances by the bacterial strains was greatly influenced by variation of carbon sources. Glycerol strongly enhanced the antimicrobial activity of strains NB-3 and NB-6, whereas glucose increased the antimicrobial activity of strains NB-4 and NB-5. The maximum antibiotic yield of NB-7 was achieved with fructose as a carbon source. Starch (Bacillus megaterium NB-3), maltose (Bacillus cereus NB-5), glycerol (Bacillus circulans NB-7), arabinose, ribose (Bacillus cereus NB-4) and arabinose, fructose, glucose, ribose and sucrose (Bacillus subtilis NB-6) repressed the production of antimicrobial substances by the respective bacterial strains.     

Effect of Covalent Dimer Conjugates of Angiotensin II on Receptor Affinity and Activity in Vitro

Abstract

Angiotensin II [1-8 or 2-8] analogues and [4–8] fragments were dimerized through the amino-or carboxy-terminal groups in order to try to increase their potency as reported for other hormones. The binding affinity to the angiotensin II receptor subtypes A (A II_A) and B (A II_B) was tested and compared to the potency in rabbit aortic ring. The [2–8] dimers coupled through the N-terminus show no significant change in potency in aortic ring. The [4–8] fragments coupled through the N-terminus are inactive in the ring. They have however a significantly increased affinity for the A II^A receptor, the specific function of which has not yet been reported. When angiotensin II analogues or fragments are coupled through the C-terminus, there was a significant drop in affinity and potency, confirming the importance of the free carboxyl group in position 8 for binding and activity. It is concluded that binding to the A II_B receptor correlates well with the effectiveness in aortic ring. However, in contrast to the beneficial effect reported for a large number of other hormones, dimerization of angiotensin II or its fragments is not accompanied by an increased biological activity in aortic ring.     

Synthesis and Antitumor Activity of Conjugates Based on the Phe-D-Trp-Lys-Thr Peptide Fragment of Somatostatin

Russian Journal of Bioorganic Chemistry - New somatostatin analogs containing the fragments of adamantane, coumarin, tetrahydrocarbazole, and palmitic acid of the general formula...     

Antimicrobial Activity of Aflatoxins

Antimicrobial activity of a crude aflatoxin preparation and of aflatoxin B(1) was studied. They were found inactive against common gram-positive and gram-negative bacteria at a concentration of 100 mug/ml. Both samples of aflatoxin did, however, exhibit antimicrobial activity, though narrow and limited, against various strains of Streptomyces and Nocardia. The antibiotic action of aflatoxin B(1) was confirmed by bioautogram after thin-layer chromatography. Among seven strains of microorganisms, including aflatoxin-sensitive and -resistant strains, N. asteroides IFM 8 was found to reduce aflatoxin B(1), in addition to other minor fluorescent components in the crude preparation.     

新疆家蚕抗菌肽基因突变及其抗菌活性的研究

为了改变抗菌肽的结构参数,探讨其结构与活性的关系,采用PCR扩增和人工合成基因的方法,对新疆家蚕抗菌肽基因进行改造及原核表达蛋白的抑菌活性研究.结果表明,α-螺旋、两亲性、疏水性、净正电荷数和关键氨基酸的替换等参数是相互依赖、相互影响,协同发挥作用,任何一个参数的改变都会影响抗菌肽整体的活性.α-螺旋是抗菌肽功能有效性的结构基础,但其所处的位置可能并不影响抗菌活性:两亲性结构是抗菌肽与生物膜相互作用的重要结构;疏水性程度必须保持在一定的范围内;在一定范围内增加多肽的阳离子能够增加抗菌活性,但正电荷数和抗菌活性之间无绝对正相关性;色氨酸的存在及抗菌肽的C-末端酰胺化能增强抗菌活性.     

重庆低海拔虫草发酵条件及其发酵液抑菌活性研究

从重庆低海拔地区采得一株虫草,分离得到其无性型菌株.以海绵抑菌圈大小为活性指标,考核了无机盐、碳氮比值、发酵温度及培养基初始pH等因素对其发酵液抑菌活性的影响,筛选出抑菌活性物质的发酵培养基为:NaNO3 0.05%,KCl 0.05%,蔗糖3.0%,蚕蛹粉0.05%.摇瓶最佳发酵条件为:pH 6.5,温度25 ℃,转速250 r/min.在此优化条件下,发酵活性在第6 d最高,对枯草芽孢杆菌的抑菌圈为30.4 mm.用海绵抑菌圈法,测定虫草真菌发酵液对不同供试菌的抑菌活性,对真菌酿酒酵母无抑菌作用,对供试的所有革兰氏阴、阳性细菌都有抑菌活性,且对枯草芽孢杆菌的活性最强,其抑菌圈为(26±4) mm.     

Ionic Charge, Hydrophobicity and Tryptophan Fluorescence of the Folate Binding Protein Isolated from Cow's Milk

A high-affinity folate binding protein was isolated and purified from cow's milk by a combination of cation exchange chromatography and methotrexate affinity chromatography. Chromatofocusing studies revealed that the protein possessed isoelectric points in the pH-interval 8–7. Polymers of the protein prevailing at pH values close to the isoelectric points seemed to be more hydrophobic than monomers present at pH 5.0 as evidenced by hydrophobic interaction chromatography and turbidity (absorbance at 340 nm) in aqueous buffer solutions (pH 5–8). Ligand binding seemed to induce a conformation change that decreased the hydrophobicity of the protein. In addition, Ligand binding quenched the tryptophan fluorescence of folate binding protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. There was a noticeable discordance between the ability of individual folate analogues to compete with folate for binding and the quenching effect.     

黄酮类化合物抑制微生物活性及其作用机制

本文综述了黄酮类化合物抑制细菌、病毒和真菌的活性及可能的作用机制,黄酮类化合物结构与抑菌活性之间的关系。结果表明,黄酮类化合物主要通过抑制细菌DNA旋转酶,抑制细菌细胞质膜的功能,抑制细菌能量代谢等方面发挥抑菌功效。指出黄酮类化合物是今后抗病源微生物药物开发新的研究方向。     

Antimicrobial Activity of Propolis on Oral Microorganisms

Formation of dental caries is caused by the colonization and accumulation of oral microorganisms and extracellular polysaccharides that are synthesized from sucrose by glucosyltransferase of Streptococcus mutans. The production of glucosyltransferase from oral microorganisms was attempted, and it was found that Streptococcus mutans produced highest activity of the enzyme. Ethanolic extracts of propolis (EEP) were examined whether EEP inhibit the enzyme activity and growth of the bacteria or not. All EEP from various regions in Brazil inhibited both glucosyltransferase activity and growth of S. mutans, but one of the propolis from Rio Grande do Sul (RS2) demonstrated the highest inhibition of the enzyme activity and growth of the bacteria. It was also found that propolis (RS2) contained the highest concentrations of pinocembrin and galangin. Received: 8 June 1997 / Accepted: 7 July 1997     

The Effect of High CO2 and Low O2 Concentrations in Simulated Landfill Gas on the Growth and Nodule Activity of Leucaena leucocephala

When roots of Leucaena leucocephala seedlings (White Popinac,a tropical legume tree belongs to the Family Mimosaceae) werefumigated with simulated landfill gas (CO₂ above 10% and O₂from 10% to atmospheric level), the stem elongation rate andstomatal conductance were inhibited at the absence of any apparentleaf water deficit. When compared with a treatment where rootsystem was flooded, the effect of gas fumigation on the shootphysiology was relatively mild and appeared later. On the otherhand, nodule activity (measured as rate of acetylene reductionactivity, ARA) was much more severely inhibited by gas fumigation.Although nodule dry weight and carbohydrate storage in noduleswere reduced, the inhibition was not likely a result of theshortage of carbohydrate reserve in the nodules. This was becausethe ARA of untreated fresh nodules was also inhibited immediatelyfollowing exposure to the simulated landfill gas. In furtherexperiments where CO₂ and O₂ were manipulated separately, althougha reduction of O₂ concentration to half of the atmospheric levelmight account for up to 30% loss of ARA with considerable variation,the high CO₂ alone showed a much more severe inhibition. ThisCO₂-induced inhibition was not reversible one hour after thehigh CO₂ gas was removed. There was some recovery of activity5 day after plants were fumigated, suggesting that the legumeplant can maintain some nitrogen-fixation activity under theinfluence of landfill gas. (Received April 10, 1995; Accepted August 22, 1995)     

Antimicrobial Activity of Sodium n-Alkylsalicylates

The activities are reported of sodium salts of several n-alkylsalicylic acids against Staphylococcus aureus, Pseudomonas aeruginosa, Mycobacterium phlei, and Candida albicans. The acids had alkyl substituents of 1 to 18 carbon atoms in the 3, 4, or 5 positions relative to the carboxyl group of salicylic acid. Generally, the antimicrobial properties were typical of anionic surface-active agents although the salicylates are more potent than most of these, particularly against S. aureus.     

Antimicrobial Activity of Several Bis-Methanethiolsulfonates

We determined the inhibitory end point of a series of bis-methanethiolsulfonates, CH(3)SO(2)S(CH(2))(n)SSO(2)CH(3) (n = 2 to 6), and correlated the structures with antimicrobial activity. Eleven microorganisms were used in the evaluation, and the maximal activity occurred when the methylene chain length was five or six. The effect of CH(3)SO(2)S(CH(2))(5)SSO(2)CH(3) (PMTS) on respiration of Aerobacter aerogenes and Staphylococcus aureus was studied at various stages of their growth cycles. The effect on viability was correlated with that of respiration in the case of A. aerogenes. Respiratory inhibition caused by PMTS varied with the growth phase of both organisms, being most pronounced when the cells tested were taken during the period of transition from the log to stationary phase. The lethal effect on A. aerogenes was also greatest at this time. During the period of increased sensitivity, the antimicrobial activity of PMTS appears to be closely related to cell respiration. The delayed rate of kill otherwise observed may reflect an impairment of protein metabolism through reaction with sensitive SH groups.     

Antimicrobial Activity and Molecular Mechanism of the CRES Protein

Cystatin-related epididymal spermatogenic (CRES) protein, a member of the cystatin superfamily of cysteine protease inhibitors (also known as CST8), exhibits highly specific, age-dependent expression in mouse testis and epididymis. The CRES protein possesses four highly conserved cysteine residues which govern the overall conformation of the cystatins through the formation of two disulfide bonds. Previous studies have revealed that other cystatin family members, such as cystatin 3 and cystatin 11, show antibacterial activity in vitro. This prompted us to investigate the potential antimicrobial activity of the CRES protein. Colony forming assays and spectrophotometry were used to investigate the effects of recombinant CRES protein on Escherichia coli (E. coli) and Ureaplasma urealyticum (Uu), respectively, in vitro. After incubation of E. coli with CRES recombinant protein fused with glutathione-S-transferase (GST), a substantial decrease in colony forming units was observed, and the effect was dose and time dependent. Furthermore, it took longer for Uu to grow to plateau stage when incubated with GST-CRES recombinant protein compared with the control GST. The antibacterial and Anti-Uu activities were not impaired when the cysteine residues of CRES protein were mutated, indicating that the antimicrobial effect was not dependent on its disulfide bonds. Functional analysis of three CRES polypeptides showed that the N-terminal 30 residues (N30) had no antimicrobial activity while N60 showed similar activity as full-length CRES protein. These results suggest that the active center of CRES protein resides between amino acid residues 31 and 60 of its N-terminus. Mechanistically, E. coli membrane permeabilization was increased in a dose-dependent manner, and macromolecular synthesis was inhibited on treatment with GST-CRES. Together, our data on the antimicrobial activities of CRES protein suggest that it is a novel and innate antimicrobial protein which protecting the male reproductive tract against invading pathogens.     

Protein Modifications in High Protein-Oil and Protein-Oil-Sugar Systems at Low Water Activity

Physicochemical and thermal properties of high protein systems during storage at 20 and 40 °C were investigated for 14 weeks. Component interactions of whey protein isolate (WPI)-olive oil (OO), WPI-sunflower oil (SO) (75:25), WPI-(glucose-fructose; G-F) (45:40), WPI-OO-(G-F), and WPI-SO-(G-F) (45:15:40) systems at low water contents during storage were derived from differential scanning calorimetry (DSC), colorimetric, water activity (a_w), reducing and nonreducing SDS-PAGE electrophoresis data. The degree of unsaturation of oil affected color (yellowness) and microstructure of the systems as well as variations in water migration and nonenzymatic browning kinetics (NEB) during storage. These effects were evident in the SO systems. All systems at 40 °C showed changes in protein conformation to those favoring hydrophobic interactions with oil. These systems showed decreased a_w, insolubilization, hardening as a result of carbonyl-amine polymerization and covalent cross-linking of proteins in the NEB. The DSC data showed a protein hydration transition for rehumidified-WPI, WPI-oil, WPI-sugar, and WPI-oil-sugar. The rehumidified-WPI and WPI-oil also showed a_w-dependent denaturation endotherms (irreversible transition) for α-lactalbumin and β-lactoglobulin at higher temperatures (T). The WPI-sugar and WPI-oil-sugar showed an exotherm for the browning reaction (irreversible transition) at T_onset ~ 90 °C. An exothermic protein hydration in the systems containing sugar was storage time-dependent, and indicated changes of protein conformation. The presence of oil in WPI-oil-sugar caused an increase in the glass transition of sugars during storage, especially for SO. The WPI-(G-F) and WPI-oil-(G-F) showed broadened glass transition during a reheating scan in DSC that was a result of polymerization in protein, oil, and sugar components mixture. Stability of high protein systems is dependent on hydration and reactions in both hydrophilic and hydrophobic phases.     

Effect of the Fluorescein to Protein Ratio on Anti-Treponema pallidum Conjugates

The effect of using different labeling techniques and various amounts of fluorescein-isothiocyanate was tested with several pools of antisera to Treponema pallidum. Optimal labeling occurred in all serum pools at a fluorescien to protein ratio of 15. The use of pure dye and the dialysis method of labeling is recommended as the method of choice because of its effectiveness in the reduction of nonspecific staining.