Chemical modification of bacitracin A and the biological activity of modified bacitracins

The single side chain amino group of the D-ornithine residue in bacitracin seems to be important for the antibacterial activity of the molecule, since, acetylation, formylation, carbamylation and deamination of the antibiotic caused 90–92% loss of antibacterial activity. In contrast, nearly 80–91% of the antibacterial activity of the parent antibiotic was retained after the esterification, amide formation and acid-chloride formation of the α—and Y -carboxyl groups of D-asparagine and D-glutamic acid residues of the antibiotic, respectively.     

Bacitracin A: Structure-activity relationship

The single imidazole nucleus of L-histidine residue in bacitracin-A seems to be important for the anti-bacterial activity of the molecule, since iodination, carboxymethylation and coupling of diazobenzene sulphonic acid to the histidine residue in the antibiotic caused 90–94% loss of antibacterial activity of the antibiotic. In contrast, the bacitracin sulphone and sulphoxide derivatives are as active as the parent antibiotic.     

N-methylleucine gramicidin-S and (di-N-methylleucine) gramicidin-S conformations with cis L-Orn-L-N-MeLeu peptide bonds.

Conformations containing all trans peptide bonds have previously been proposed for N-methylleucine gramicidin-S and (di-N-methylleucine) gramicidin-S based on an evaluation of proton nuclear magnetic resonance parameters in a series of solvents. These gramicidin-S derivatives exhibit full biological activity despite the fact that the proposed solution conformations differ in backbone topology and relative orientation of the Phe and Orn side chains compared to gramicidin-S. The present authors discuss conformations for N-methylleucine gramicidin-S and (di-N-methylleucine) gramicidin-S which incorporate cis peptide bonds at L -Orn-L -N-MeLeu, where the gramicidin-S backbone is essentially retained, and the relative orientation of the Pro, Orn, Val, and Phe side chains correspond to those observed for gramicidin-S. A novel hydrogen-bond arrangement involving one carbonyl group interacting with two peptide protons (1 ←4 and 1 ←5 types) is proposed to stabilize the backbone conformation in the gramicidin-S derivatives. A recent report on the cyclic heptapeptide antibiotic, Ilamycin B₁, shows the presence of cis peptide bonds at N-CH₃ amino acids, as well as the novel hydrogen-bond arrangement presented above.     

Preparation, characterization, and antibacterial activity of oleic acid-grafted chitosan oligosaccharide nanoparticles

An oleic acid-grafted chitosan oligosaccharide (CSO-OA) with different degrees of amino substitution (DSs) was synthesized by the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction. Fourier transform infrared spectroscopy (FT-IR) suggested the formation of an amide linkage between amino groups of chitosan oligosaccharide and carboxyl groups of oleic acid. The critical aggregation concentrations (CACs) of CSO-OA with 6%, 11%, and 21% DSs were 0.056, 0.042, and 0.028 mg·mL⁻¹, respectively. Nanoparticles prepared with the sonication method were characterized by means of transmission electron microscopy (TEM) and Zetasizer, and the antibacterial activity against Escherichia coli and Staphylococcus aureus was investigated. The results showed that the CSO-OA nanoparticles were in the range of 60–200 nm with satisfactory structural integrity. The particle size slightly decreased with the increase of DS of CSO-OA. The antibacterial trial showed that the nanoparticles had good antibacterial activity against E. coli and S. aureus.     

C-terminal fragment of human laminin-binding protein contains a receptor domain for Venezuelan equine encephalitis and tick-borne encephalitis viruses

Polyclonal and monoclonal antibodies (MABs) to human laminin-binding protein (LBP) can efficiently block the penetration of some alphaand flaviviruses into the cell. A panel of 13 types of MABs to human recombinant LBP was used for more detailed study of the mechanism of this process. Competitive analysis has shown that MABs to LBP can be divided into six different competition groups. MABs 4F6 and 8E4 classified under competition groups 3 and 4 can inhibit the replication of Venezuelan equine encephalitis virus (VEEV), which is indicative of their interaction with the receptor domain of LBP providing for binding with virions. According to enzyme immunoassay and immunoblotting data, polyclonal anti-idiotypic antibodies to MABs 4F6 and 8E4 modeling paratopes of the LBP receptor domain can specifically interact with VEEV E2 protein and tick-borne encephalitis virus (TBEV) E protein. Mapping of binding sites of MABs 4F6 and 8E4 with LBP by constructing short deletion fragments of the human LBP molecule has shown that MAB 8E4 interacts with the fragment of amino acid residues 187–210, and MAB 4F6 interacts with the fragment of residues 263–278 of LBP protein, which is represented by two TEDWS peptides separated by four amino acid residues. This suggested that the LBP receptor domain interacting with VEEV E2 and TBEV E viral proteins is located at the C-terminal fragment of the LBP molecule. A model of the spatial structure of the LBP receptor domain distally limited by four linear loops (two of which are represented by experimentally mapped regions of amino acid residues 187–210 and 263–278) as well as the central β-folded region turning into the α-helical site including residues 200–216 of the LBP molecule and providing for the interaction with the laminin-1 molecule has been proposed.     

Callinectin, an Antibacterial Peptide from Blue Crab, Callinectes sapidus, Hemocytes

This paper describes the isolation of an approximately 3.7 kDa, basic, antibacterial peptide (designated callinectin), which represents the major antibiotic activity in blue crab, Callinectes sapidus, hemocytes. A single-step purification using low-pressure cation-exchange chromatology yielded a highly purified (>95%) peptide. Purity was confirmed by C₄ reverse-phase high-performance liquid chromatography (RP-HPLC), native gel electrophoresis, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), capillary electophoresis, and mass spectral analysis. The partial amino acid sequence obtained via Edman degradation revealed no significant homology to other reported peptides in the Basic Local Alignment Search Tool (BLAST) program database. Received April 21, 1998; accepted July 3, 1998.     

Purification and properties of alcohol oxidase from <Emphasis Type="Italic">Pichia putida</Emphasis>

Alcohol oxidase (AO) was extracted from the methylotrophic yeast Pichia putida and purified using various methods. AO purified by crystallization was homogeneous based on analytical centrifugation with subsequent gel filtration and SDS-PAGE. The molecular weight of the enzyme was around 600 kDa. SDS-PAGE revealed a single protein band (74 ± 4 kDa), and 8–9 bands of native protein with similar specific AO activities and substrate specificities were identified by PAGE without SDS. Electron microscopy of a single molecule revealed eight subunits located on the top of a regular tetragon with dotted symmetry of 422D4 providing evidence that AO consists of eight subunits. Apparently, each molecule of AO has two types of subunits with very similar molecular weights and differing from each other by the number of acidic and basic amino acid residues. Each subunit includes one molecule of FAD and 2–3 cysteine residues. The pH optimum was within 8.5–9.0. Specific activity of the enzyme varied from 10 to 50 μmol methanol/min per mg protein from batch to batch depending on separation methods and had linear relationship with protein concentration. The AO was quickly inactivated at 20°C and seemed to be stable in phosphate-citrate buffer with 30–50% (w/v) of sucrose. Different forms of 0.1–1 mm crystals of the enzyme were obtained. However the crystals did not yield X-ray reflections, apparently as a result of their molecular microheterogeneity.     

Optimizing the biodegradation of two keratinous wastes through a Bacillus subtilis recombinant strain using a response surface methodology

Statistical optimization of the biodegradation of two keratinous wastes directed by Bacillus subtilis recombinant cells was carried out by means of a response surface methodology. A Box–Behnken design was employed to predict the optimal levels of three variables namely, keratin percent, incubation time and inoculum size. Analysis of variance revealed that, only keratin percent had the highest significant effect. Canonical analysis and ridge max analysis were used to get the optimal levels of the three predictors along with the optimum levels of the responses. The optimal sets of predicted and validated levels of the three variables were [7.69% (w/v) feathers, 96.58 h and 1.28% (v/v) inoculum size] and [8% (w/v) feathers, 98.45 h, 3.9% (v/v) inoculum size] to achieve the highest levels of soluble proteins (1.25–1.7 mg/ml) and NH₂-free amino groups (245.82–270.0 μmol leucine/ml), respectively upon using three optimized feathers-based media. These values represented 83.67–100% and 100% adequacy for the models of soluble proteins and NH₂-free amino groups, respectively. While, [8.23% (w/v) sheep wool, 5.52% (v/v) inoculum size and 46.58 h] and [8.33% (w/v) sheep wool, 5.89% (v/v) inoculum size and 63.46 h] were the optimal sets of predicted and validated levels of the above variables to achieve the highest yields of soluble proteins (3.4–4.6 mg/ml) and NH₂-free amino groups (290.9–302.0 μmol leucine/ml), respectively upon using three optimized sheep wool-based media. These values represented 100% adequacy for the models of soluble proteins and NH₂-free amino groups. By the end of the optimization strategy, a fold enhancement (2.14–2.43 and 1.78–2.12) in the levels of released soluble proteins and NH₂-free amino groups, respectively was obtained upon using three optimized feathers-based media. However, a fold enhancement (4.25–5.75 and 2.42–2.5) in the levels of soluble proteins and NH₂-free amino groups, respectively was obtained upon using three optimized sheep wool-based media. Data would encourage pilot scale optimization of the biodegradation of these wastes.     

Characterization of high rate composting of vegetable market waste using Fourier transform-infrared (FT-IR) and thermal studies in three different seasons

Fourier transform-infrared (FT-IR), Thermogravimetry (TG), Differential thermal analyses (DTA) and Differential Thermogravimetric (DTG) studies of a mixture of vegetable waste, saw dust, tree leaves and cow dung for microbial activity (feedstock) and their compost were reported in three different seasons i.e. winter, spring and summer. The correlation between spectral studies and compost composition provide information regarding their stability and maturity during composting. FT-IR spectra were conferred the functional groups and their intensity and TG, DTG and DTA for wt. loss, rate of wt. loss and enthalpy change in compost. Weight loss in feedstock and compost at two different temperatures 250–350 and 350–500°C was found 38.06, 28.15% for inlet and 14.08, 25.67% for outlet zones in summer and 50.59, 29.76% for inlet and 18.08, 25.67% in outlet zones in spring season, higher (5–10%) than winter. The corresponding temperatures in DTA in the samples from inlet to outlet zone were; endotherm (100–200°C), due to dehydration, exotherm (300–320°C), due to peptidic structure loss and exotherm (449–474°C) due to the loss of polynuclear aromatic structures, which were higher by 4°C and 10–20°C and rate of wt. loss was higher by 5–10% in spring and summer season, respectively than winter season composting, reported regardless of the maturation age of the compost. Relative intensity of exotherms (300–320/449–474°C) gave the thermally more stable fractions of organic compound. Our results indicated that the rotary drum composting of organic matters in spring and summer season gave higher molecular complexity and stability than the winter season.     

Comparative activity against pathogenic bacteria of the root,stem, and leaf of <Emphasis Type="Italic">Raphanus sativus</Emphasis> grown in India

Aqueous, methanol, ethyl acetate, and chloroform extracts of the root, stem, and leaf of Raphanus sativus were studied for antibacterial activity against food-borne and resistant pathogens. All extracts except the aqueous extracts had significant broad-spectrum inhibitory activity. The ethyl acetate extract of the root had the potent antibacterial activity, with a minimum inhibitory concentration (MIC) of 0.016–0.064 mg/ml and a minimum bactericidal concentration (MBC) of 0.016–0.512 mg/ml against health-damaging bacteria. This was followed by the ethyl acetate extracts of the leaf and stem with MICs of 0.064–0.256 and 0.128–0.256 mg/ml, respectively and MBCs of 0.128–2.05 and 0.256–2.05 mg/ml, respectively. The ethyl acetate extracts of the different parts of R. sativus retained their antibacterial activity after heat treatment at 100°C for 30 min, and their antibacterial activity was enhanced when pH was maintained in the acidic range. Hence this study, for the first time, demonstrated that the root, stem, and leaf of R. sativus had significant bactericidal effects against human pathogenic bacteria, justifying their traditional use as anti-infective agents in herbal medicines.     

Action of neotelomycin derivatives on bacillus magaterium cells]

The effect of neotelomycin derivatives on the cells of Bac. megaterium was studied. Derivatives with modification of one of the two active centers of the antibiotic molecule, i.e. the free alpha-amine group of the residue of asparaginic acid or hydrophobic triptophanic structure were studied. The derivative with modified indol rings of the residues of beta-methyl-and dehydrotriptophane induced the same though lower damages as the natural antibiotic: increased permeability of the cytoplasmic membranes, protoplast lysis, suppression of the dehydrogenase activity. The activity of this derivative was due to the free amino group and amounted approximately to 3 per cent of the activity of neotelomycin. The derivative with the free amino group of the asparaginic acid residue replaced by the benzoylic group showed a high antibacterial activity but had almost no effect on the membrane permeability and a very low lytic effect. The capacity of this derivative to inhibit the bacterial dehydrogenase activity remained relatively high. Possibly the free amino group of the asparaginic acid residue provided neotelomycin with the capacity for damaging the structure of the bacterial cytoplasmic membranes. No detectable damages in the membrane state after exposure to the benzoylic derivative, as well as its high antibacterial activity are evident of the fact that the mechanism of action of the benzoylic derivative on the cells was in principal different from that of the derivative preserving the free amino group. The triptophane structure was probably not only the center actively affecting the cell but also the factor that provided the antibiotic molecule with conformation most favourable for the action of the free amino group on the membrane structures.     

Nickel toxicity inhibits ribonuclease and protease activities in rice seedlings: protective effects of proline

Seedlings of two rice cvs Malviya-36 and Pant-12, when grown under increasing levels of nickel (Ni²⁺: 200 and 400 μM) in the nutrient medium, showed increased levels of RNA, soluble proteins and free amino acids, especially proline over a total growth period of 5–20 days. Ribonuclease (RNase) and protease activities decreased in both roots and shoots due to Ni treatment in situ. Under in vitro conditions, a gradual inhibition of RNase activity was observed with increasing concentrations of Ni²⁺ (0–2500 μM) in the assay medium. Artificial desiccation simulated by 40% PEG or the presence of 2.5 mM Ni²⁺ in the reaction medium resulted in about 52–53% loss of RNase activity. Such a loss could be partially restored by 1 M proline in the assay medium. The activity staining of RNase revealed seven and four isoforms of RNase in roots and shoots, respectively. The intensity of most of the bands decreased with increasing levels of Ni²⁺ treatment in situ. The results suggest that Ni toxicity in rice seedlings suppresses the hydrolysis of RNA and proteins by inhibiting the activity of RNase and protease, respectively. Proline appears to act as a protectant of the enzyme RNase against metal- and PEG-induced damages.     

A New Family of Highly Potent Inhibitors of Microbes: Synthesis and Conjugation of Elastin Based Peptides to Piperazine Derivative

Elastin protein-based polymers have their origin in repeating sequences of the mammalian elastic protein, elastin. The sequences of elastin peptides chosen are tetrapeptides, pentapeptides and tricosamers (30 amino acids) and also aromatic amino acids. These have been conjugated to 1-(2,3-dichlorophenyl)piperazine to study the effect of conjugation on the activity. The conjugates so obtained were characterized by physical and analytical techniques followed by the antimicrobial evaluation. The study revealed that all the conjugates have exhibited enhanced activity than the conventional drugs. Further, the conjugates of tricosamers have shown extraordinary activity against the fungal species with MIC value of 3–5 μg/ml which is five fold more potent than the antibiotic used.     

A comparative account of the microbiological characteristics of soils under natural forest,grassland and cropfield from Eastern India

Microbiological and physico-chemical characteristics of tropical forest, grassland and cropfield soils from India were investigated. The study revealed that the conversion of natural forest led to a reduction of soil organic C (26–36%), total N (26–35%), total P (33–44%), microfungal biomass (44–66%) and total microbial biomass C, N and P (25–60%) over a period of 30–50 years. Comparative analysis of microbial activity in terms of basal soil respiration revealed maximum activity in the forest and minimum in the cropfield soil. Analysis of microbial metabolic respiratory activity (qCO₂) indicated relatively greater respiratory loss of CO₂-C per unit microbial biomass in cropfield and grassland than in forest soil. Considering the importance of the microbial component in soil, we conclude that the conversion of the tropical forest to different land uses leads to the loss of biological stability of the soil.     

The metabolic burden of creatine synthesis

Creatine synthesis is required in adult animals to replace creatine that is spontaneously converted to creatinine and excreted in the urine. Additionally, in growing animals it is necessary to provide creatine to the expanding tissue mass. Creatine synthesis requires three amino acids: glycine, methionine and arginine, and three enzymes: l-arginine:glycine amidinotransferase (AGAT), methionine adenosyltransferase (MAT) and guanidinoacetate methyltransferase (GAMT). The entire glycine molecule is consumed in creatine synthesis but only the methyl and amidino groups, respectively, from methionine and arginine. Creatinine loss averages approximately 2 g (14.6 mmol) for 70 kg males in the 20- to 39-year age group. Creatinine loss is lower in females and in older age groups because of lower muscle mass. Approximately half of this creatine lost to creatinine can be replaced, in omnivorous individuals, by dietary creatine. However, since dietary creatine is only provided in animal products, principally in meat and fish, virtually all of the creatine loss in vegetarians must be replaced via endogenous synthesis. Creatine synthesis does not appear to place a major burden on glycine metabolism in adults since this amino acid is readily synthesized. However, creatine synthesis does account for approximately 40% of all of the labile methyl groups provided by S-adenosylmethionine (SAM) and, as such, places an appreciable burden on the provision of such methyl groups, either from the diet or via de novo methylneogenesis. Creatine synthesis consumes some 20–30% of arginine’s amidino groups, whether provided in the diet or synthesized within the body. Creatine synthesis is, therefore, a quantitatively major pathway in amino acid metabolism and imposes an appreciable burden on the metabolism of methionine and of arginine.     

Design and characterization of novel hybrid peptides from LFB15(W4,10), HP(2-20), and cecropin A based on structure parameters by computer-aided method

The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. The pivotal assets of the antimicrobial peptide include potential for rapid bactericidal activity and low propensity for resistance. The four new antimicrobial hybrid peptides were designed based on peptides LFB15(W4,10), HP(2-20), and cecropin A according to the structure–activity relationship of the amphipathic and cationic antimicrobial peptides. Their structural parameters were accessed by bioinformatics tools, and then two hybrids with the most potential candidates were synthesized. The hybrid peptide LH28 caused an increase in antibiotic activity (MIC₅₀ = 1.56–3.13 μM) against given bacterial strains and did not cause obvious hemolysis of rabbit erythrocytes at concentration of 3.13 μM with effective antimicrobial activity. The results demonstrate that evaluating the structural parameters could be useful for designing novel antimicrobial peptides. Zi-gang Tian and Tian-tang Dong contributed equally to this paper     

Hydrogen exchange and proteolytic degradation of ribonuclease A. The local splitting of the native structure and the conformation of loop segments

The limited proteolytic sites or nicksites are present only in one of the five loops of the RNase A molecule. The splitted loop 15–23 connects two structural domains in the hinge region of the interdomain contacts of the V-shaped molecule. The other four loops are inside two domains, 64–71 and 112–115 in the domain I (1–19, 47–81, 102–106) and 36–42 and 88–95 in the domain II (20–46, 82–101). Because of enhanced chain flexibility of the splitted loop in the pH-dependent conformational isomerization, deformation of its structure is slighter under the influence of the intermolecular contacts in the crystal lattice and more significant changes occur in loop conformation at the formation of the 3D swapped dimer of the RNase A molecule. The proteolytic splitting of the 15–23 loop proceeds due to the local fluctuation of the native protein structure.     

Structural Modification and Expression of Attacin A from Glossina morsitans morsitans in E. coli and its Antibacterial Activities

Attacin A from Glossina morsitans morsitans belongs to the type of Gly-rich antimicrobial peptides, with mature peptide of 188 amino acids and molecular weight of 19.4 kDa. In this work, a truncated form of Attacin A was studied to evaluate its N-terminal and G1 domain for antibacterial activities with the help of genetic engineering technology. Genes coding for mature full length Attacin A together with its truncated form Attacin AP1 containing 1–99 amino acids of Attacin A were cloned, expressed and purified. The expression level for Attacin A and Attacin AP1 were about 10 and 20% of total cell protein, respectively. The purity of bioactive recombinant proteins, which showed virtually homogenous as determined by SDS–PAGE, was over 90%. Antibacterial testing shows that Attacin A could inhibit the growth of Gram-negative bacteria such as E. coli, Enterobacter cloacae and Klebsiella pneumoniae at relatively high concentration comparing with Ampicillin, but had little activity against Gram-positive Staphylococcus aureus. Attacin AP1 maintained the inhibitory activities against E. coli, E. cloacae and K. pneumoniae. This result makes it possible for better understanding of the structure–activity relationship of antimicrobial peptides.     

Inhibition of ribonuclease and protease activities in germinating rice seeds exposed to nickel

When the seeds of two rice cvs. Malviya-36 and Pant-12 were germinated up to 120 h in the presence of 200 and 400 μM NiSO₄, a significant reduction in the germination of seeds occurred. Seeds germinating in the presence of 400 μM NiSO₄ showed about 12–20% decline in germination percent, about 20–53% decline in lengths and about 8–34% decline in fresh weights of roots and shoots at 120 h of germination. Ni²⁺ exposure of germinating seeds resulted in apparent increased levels of RNA, soluble proteins, and free amino acids in endosperms as well as embryo axes. A 400 μM Ni²⁺ treatment led to about 58–101% increase in the level of soluble proteins and about 39–107% increase in the level of free amino acids in embryo axes at 96 h of germination. Activities of ribonuclease and protease declined significantly with increasing levels of Ni²⁺ treatment. Isoenzyme profile of RNase as revealed by activity staining indicated decline in the intensities of 3–4 preexisting enzyme isoforms in embryo axes of both the rice cultivars and disappearance of one of the two isoforms in endosperms of cv. Pant-12 due to 400 μM Ni²⁺ treatment. Results suggest that the presence of high level of Ni²⁺ in the medium of germinating rice seeds serves as a stress factor resulting in decreased hydrolysis as well as delayed mobilization of endospermic RNA and protein reserves and causing imbalance in the level of biomolecules like RNA, proteins, and amino acids in growing embryo axes. These events would ultimately contribute to decreased germination of rice seeds in high Ni²⁺ containing environment.