Identification and isolation of Lactobacillus fructivorans from wilted alfalfa silage with and without molasses.

AIM: To gain insights into the bacterial species associated with anaerobic storage and aerobic stability of alfalfa silage. METHODS AND RESULTS: Wilted alfalfa silage (498 g dry matter kg(-1) ) was prepared with and without the addition of molasses. Aerobic spoilage tests were conducted at 5, 10 and 60 days after ensiling. The composition of fermentation products and the bacterial communities of silage were determined at 1, 3, 5 and 7 days after silo opening. Silage without molasses had small amounts of lactic and acetic acids detectable at silo opening but resisted deterioration due to aerobic spoilage for at least 5 days after opening. Resistance to aerobic deterioration in silage increased with the addition of molasses. The predominant bacterial species in molasses-added silage was Lactobacillus fructivorans, which was detected by denaturing gradient gel electrophoresis analysis. Different bacterial growth media were used for Lact. fructivorans isolation from alfalfa silage with added molasses: isolation was successful using liver infusion sake medium, but was unsuccessful when de Man, Rogosa and Sharpe medium was used. CONCLUSION: A nonconventional lactic acid bacterium (LAB) species may be involved in the high aerobic stability of alfalfa silage. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings demonstrate that culture-independent microbiota analysis may be useful in the isolation and identification of nonconventional LAB species involved in fermentation and the aerobic stability of silage.

Lactic Acid Bacteria in Total Mixed Ration Silage Containing Soybean Curd Residue: Their Isolation, Identification and Ability to Inhibit Aerobic Deterioration.

We investigated the effects of the predominant lactic acid bacteria (LAB) on the fermentation characteristics and aerobic stability of total mixed ration (TMR) silage containing soybean curd residue (SC-TMR silage). The SC-TMR materials were ensiled in laboratory silos for 14 or 56 days. LAB predominant in SC-TMR silage were identified (Exp. 1). Lactobacillus fermentum (L. fermentum) and Streptococcus bovis (S. bovis) were found in the untreated materials, Leuconostoc pseudomesenteroides (L. pseudomesenteroides) in 14-day silage and Lactobacillus plantarum (L. plantarum) in all silages. Pediococcus acidilactici (P. acidilactici), Lactobacillus paracasei (L. paracasei), and Lactobacillus brevis (L. brevis) formed more than 90% of the isolates in 56-day silage. Italian ryegrass and whole crop maize were inoculated with P. acidilactici and L. brevis isolates and the fermentation and aerobic stability determined (Exp. 2). Inoculation with P. acidilactici and L. brevis alone or combined improved the fermentation products in ryegrass silage and markedly enhanced its aerobic stability. In maize silage, P. acidilactici and L. brevis inoculation caused no changes and suppressed deterioration when combined with increases in acetic acid content. The results indicate that P. acidilactici and L. brevis may produce a synergistic effect to inhibit SC-TMR silage deterioration. Further studies are needed to identify the inhibitory substances, which may be useful for developing potential antifungal agents.

Identification of lactic acid bacteria in the rumen and feces of dairy cows fed total mixed ration silage to assess the survival of silage bacteria in the gut.

The survival of silage lactic acid bacteria (LAB) in the gut of dairy cows was evaluated by examining the LAB communities of silage and gut contents. Samples were collected at 2 different research institutes (Mie and Okayama) that offered total mixed ration (TMR) silage throughout the year. Silage and feces were sampled in August, October, and November at the Mie institute, whereas silage, rumen fluid, and feces were sampled in June and August at the Okayama institute. Denaturing gradient gel electrophoresis using Lactobacillus-specific primers was performed to detect LAB species in the samples. The selected bands were purified for species identification and the band patterns were used for principal component analysis. Lactic acid was the predominant fermentation product in all the TMR silages analyzed, and the lactic acid level tended to be constant regardless of the sampling time and region. A total of 14 LAB species were detected in the TMR silage samples, of which 5 (Lactobacillus acetotolerans, Lactobacillus pontis, Lactobacillus casei, Lactobacillus suebicus, and Lactobacillus plantarum) were detected in the dairy cow feces. Most of the denaturing gradient gel electrophoresis bands for the feces samples were also detected in the rumen fluid, suggesting that any elimination of silage LAB occurred in the rumen and not in the postruminal gut segments. The principal component analysis indicated that the LAB communities in the silage, rumen fluid, and feces were separately grouped; hence, the survival of silage LAB in the cow rumen and lower gut was deemed difficult. It was concluded that, although the gut LAB community is robust and not easily affected by the silage conditions, several LAB species can inhabit both silage and feces, which suggests the potential of using silage as a vehicle for conveying probiotics.

Effects of storage temperature and ensiling period on fermentation products, aerobic stability and microbial communities of total mixed ration silage.

AIMS: To examine how storage temperatures influence ensiling fermentation, aerobic stability and microbial communities of total mixed ration (TMR) silage. METHODS AND RESULTS: Laboratory-scale silos were stored at 5, 15, 25 and 35°C for 10, 30 and 90 days. If silage was stored at 5°C, fermentation was weak until day 30, but acceptable lactic acid production was observed on day 90. The ethanol content was higher than the acetic acid content when stored at 15 and 25°C, whereas the ethanol content was lower when stored at 35 than at 25°C. Aerobic deterioration did not occur when silage was exposed to air at the same temperature at which it was stored. Although 10-day silages stored at 5 and 15°C deteriorated when the aerobic stability test was conducted at 25°C, heating was not observed in silages stored at 25 or 35°C or in any 90-day silages regardless of storage temperature. Denaturing gradient gel electrophoresis demonstrated that bands indicative of Lactobacillus plantarum and Lactobacillus delbrueckii were less prominent, while bands indicative of Lactobacillus panis became more distinct in silages stored at high temperatures. Bands of Kluyveromyces marxianus were seen exclusively in silages that were spoiled at 25°C. CONCLUSION: High ambient temperature enhances acetic acid production in TMR silage. Lactobacillus panis may be associated with changes in the fermentation products due to differences in storage temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: The role of Lacto. panis in ensiling fermentation and aerobic stability is worth examining.

Effects of wilting and molasses addition on fermentation and bacterial community in guinea grass silage.

AIMS: Acetic acid is considered an important preservative in tropical grass ensiling. The objective of the current experiments was to follow the ensiling fermentation of low dry matter (DM) tropical grass as a model to study changes in bacterial communities during acetic acid fermentation. METHODS AND RESULTS: Direct-cut and wilted guinea grass silage was prepared with and without molasses. A high acetic acid level was observed during the fermentation of direct-cut silage, and long storage increased the butyric acid and ethanol content if molasses was not added. The lactic acid production in wilted silage was greater than the acetic acid production, but prolonged ensiling decreased the lactic to acetic acid ratio regardless of molasses addition. Adding molasses enhanced the lactic acid content in both direct-cut and wilted silage. The bacterial community, identified by denaturing gradient gel electrophoresis, was affected by wilting and molasses addition. Bands for Pantoea sp. and Morganella sp. became faint when acetic acid fermentation was suppressed, and those for Pediococcus pentosaceus and Lactococcus garvieae were detected when lactic acid fermentation was enhanced by wilting and molasses addition. Lactobacillus plantarum and Lactococcus lactis were found throughout the ensiling process in all silage types. CONCLUSION: Distinct changes occurred in the bacterial community in guinea grass silage because of wilting and molasses addition. These changes could explain how lactic acid fermentation was enhanced but could not help determine which bacteria were associated with enhanced acetic acid fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The study reveals the effects of wilting and molasses during ensiling of low DM tropical grasses and the associated bacteria.

Survival of silage lactic acid bacteria in the goat gastrointestinal tract as determined by denaturing gradient gel electrophoresis.

AIMS: To determine the survival rate of silage lactic acid bacteria (LAB) in the ruminant gastrointestinal tract. METHODS AND RESULTS: Wilted Italian ryegrass (Lolium multiflorum Lam.) silage (containing 1·9×10(6) CFU LAB g(-1)) was fed ad libitum to three goats equipped with rumen cannulae. Silage was given alone or with concentrates at a 1:1 ratio on a dry matter basis. Rumen fluid was then obtained 2, 4 and 8h after the morning feeding. Denaturing gradient gel electrophoresis was performed to compare LAB communities in silage, rumen fluid and faeces. The LAB detected in the wilted silage included Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus murinus and Lactobacillus sakei. Bands indicative of Lact. murinus were detected in either the rumen fluid or faeces, whereas the bands indicative of Lact. plantarum, Lact. brevis and Lact. sakei were not. Although the rumen fluid LAB counts and volatile fatty acid concentrations were higher in goats fed silage plus concentrates compared with those fed silage alone, the LAB communities themselves remained unaffected. Sampling times and goat-to-goat variations did not affect the LAB communities found in the rumen fluid. CONCLUSION: LAB communities found in the gut are not remarkably affected by the consumption of silage LAB, even when the silage is accompanied by concentrates that facilitate gut fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Although silage can improve probiotic function, it may be difficult for silage LAB to survive the digestive process in the ruminant gastrointestinal tract.

Three-dimensional dynamics of fluctuations appearing during pellet ablation process around a pellet in a fusion plasma experiment.

Understanding pellet ablation physics is crucial to realizing efficient fueling into a high temperature plasma for the steady state operation of ITER and future fusion reactors. Here we report the first observation of the formation of fluctuation structures in the pellet plasmoid during the pellet ablation process by a fast camera in a medium-sized fusion device, Heliotron J. The fluctuation has a normalized fluctuation level of ~ 15% and propagates around the moving pellet across the magnetic field. By comparing the fluctuation structures with the shape of magnetic field lines calculated with the field line tracing code, we successfully reconstruct the spatio-temporal structure of the fluctuations during the pellet ablation process. The fluctuations are located at the locations displaced toroidally from the pellet and propagate in the cross-field direction around the pellet axis along the field line, indicating a three-dimensional behavior and structure of fluctuations. The fluctuation would be driven by a strong inhomogeneity formed around the pellet and invoke the relaxation of the gradient through a cross-field transport induced by the fluctuations, which could affect the pellet ablation and pellet fueling processes. Such fluctuations can be ubiquitously present at the inhomogeneity formed around a pellet in the pellet ablation process in fusion devices.

Monitoring the bacterial community of maize silage stored in a bunker silo inoculated with Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri.

AIMS: To monitor variations in the bacterial community and fermentation products of maize silage within and between bunker silos. METHODS AND RESULTS: Silage samples were collected in 2008 and 2009 from three dairy farms, wherein the farmers arranged for a contractor to produce maize silage using bunker silos. Silage was prepared using a lactic acid bacteria (LAB) inoculant consisting of Enterococcus faecium, Lactobacillus plantarum and Lactobacillus buchneri. Eight samples were collected from each bunker silo; 4 'outer' and 4 'inner' samples were collected from near the top and the bottom of the silo. The dry matter, lactic acid, acetic acid, ethanol, 1-propanol and 1,2-propanediol contents differed between bunker silos in both sampling years. Higher acetic acid, 1-propanol and 1,2-propanediol contents were found in the bottom than the top layers in the 2008 samples, and higher lactic acid content was found in the top than the bottom layers in the 2009 samples. The bacterial community varied more between bunker silos than within a bunker silo in the 2008 samples, whereas differences between the top and the bottom layers were seen across bunker silos in the 2009 samples. The inoculated LAB were uniformly distributed, while several nonconventional silage bacteria were also detected. Lactobacillus acetotolerans, Lactobacillus panis and Acetobacter pasteurianus were detected in both years. Stenotrophomonas maltophilia was detected in the 2008 samples, and Lactobacillus reuteri, Acinetobacter sp. and Rahnella sp. were detected in the 2009 samples. CONCLUSIONS: Although differences were seen within and between bunker silos, the bacterial community may indicate a different relationship between bunker silos and sampling locations within a bunker silo from that indicated by the fermentation products. SIGNIFICANCE AND IMPACT OF THE STUDY: Analysis of bacterial community can help understand how diverse non-LAB and LAB species are involved in the ensiling process of bunker-made maize silage.

Bacterial and fungal communities of wilted Italian ryegrass silage inoculated with and without Lactobacillus rhamnosus or Lactobacillus buchneri.

AIMS: To understand the effects of lactic acid bacteria (LAB) inoculation on fermentation products, aerobic stability and microbial communities of silage. METHODS AND RESULTS: Wilted Italian ryegrass was stored in laboratory silos with and without inoculation of Lactobacillus rhamnosus and Lactobacillus buchneri. The silos were opened after 14, 56 and 120 days and then subjected to aerobic deterioration for 7 days. Intensive alcoholic fermentation was found in untreated silage; the sum of ethanol and 2,3-butanediol content at day 14 was about 7 times higher than that of lactic and volatile fatty acids. Alcoholic fermentation was suppressed by L. rhamnosus and L. buchneri inoculation and lactic acid and acetic acid became the dominant fermentation products, respectively. Silages were deteriorated in untreated and L. rhamnosus-inoculated silages, whereas no spoilage was found in L. buchneri-inoculated silage. Enterobacteria such as Erwinia persicina, Pantoea agglomerans and Rahnella aquatilis were detected in untreated silage, whereas some of these bacteria disappeared or became faint with L. rhamnosus treatment. When silage was deteriorated, Lactobacillus brevis and Bacillus pumilus were observed in untreated and L. rhamnosus-inoculated communities, respectively. The inoculated LAB species was detectable in addition to untreated bacterial communities. Saccharomyces cerevisiae and Pichia anomala were the main fungi in untreated and L. rhamnosus-inoculated silages; however, P. anomala was not visibly seen in L. buchneri-inoculated silage either at silo opening or after exposure to air. CONCLUSION: Inoculation with L. rhamnosus can suppress alcoholic fermentation of wilted grass silage with elimination of enterobacteria at the beginning of fermentation. Addition of L. buchneri may improve aerobic stability, with distinct inhibitory effect observed on P. anomala after silo opening. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial and fungal community analyses help us to understand how inoculated LAB can function to improve the fermentation and aerobic stability of silage.

Presence of sourdough lactic acid bacteria in commercial total mixed ration silage as revealed by denaturing gradient gel electrophoresis analysis.

AIMS: To characterize the bacterial communities in commercial total mixed ration (TMR) silage, which is known to have a long bunk life after silo opening. METHODS AND RESULTS: Samples were collected from four factories that produce TMR silage according to their own recipes. Three factories were sampled three times at 1-month intervals during the summer to characterize the differences between factories; one factory was sampled 12 times, three samples each during the summer, autumn, winter and spring, to determine seasonal changes. Bacterial communities were determined by culture-independent denaturing gradient gel electrophoresis. All silages contained lactic acid as the predominant acid, and the contents appeared stable regardless of factories and product seasons. Acetic acid and 1-propanol contents were different between factories and indicated seasonal changes, with increases in warm seasons compared to cool seasons. Both differences and similarities existed among the bacterial communities from each factory and product season. Lactobacillus parabuchneri was found in the products from three of four factories. Various sourdough lactic acid bacteria (LAB) were identified in commercial TMR silage; Lactobacillus panis, Lactobacillus hammesii, Lactobacillus mindensis, Lactobacillus pontis, Lactobacillus frumenti and Lactobacillus farciminis were detected in many products. Moreover, changes owing to product season were distinctive, and Lact. pontis and Lact. frumenti became detectable in summer products. CONCLUSION: Sourdough LAB are involved in the ensiling of commercial TMR silage. Silage bacterial communities vary more by season than by factory. The LAB species Lact. parabuchneri was detected in the TMR silage but may not be essential to the product's long bunk life after silo opening. SIGNIFICANCE AND IMPACT OF THE STUDY: Commercial TMR silage resembles sourdough with respect to bacterial communities and long shelf life. The roles of sourdough LAB in the ensiling process and aerobic stability are worth examining.

Bacterial community associated with ensilage process of wilted guinea grass.

AIMS: To determine the effects of wilting, storage period and bacterial inoculant on the bacterial community and ensiling fermentation of guinea grass silage. METHODS AND RESULTS: Fermentation products, colony counts and denaturing gradient gel electrophoresis (DGGE) profiles were determined. There was more lactic acid than acetic acid in all silages, but the lactic acid to acetic acid ratio decreased with storage time. This shift from lactic to acetic acid was not prevented even with a combination of wilting and bacterial inoculant. The DGGE analyses suggest that facultatively heterofermentative lactic acid bacteria (Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus pentosus) were involved in the shift to acetic acid fermentation. CONCLUSIONS: Lactic acid can dominate the fermentation in tropical grass silage with sufficient wilting prior to ensiling. Prolonged storage may lead to high levels of acetic acid without distinctive changes in the bacterial community. SIGNIFICANCE AND IMPACT OF THE STUDY: The bacterial community looks stable compared to fermentation products over the course of long storage periods in tropical grass silage. Acetic acid fermentation in tropical grass silage can be a result of the changes in bacterial metabolism rather than community structure.

Ensiling of soybean curd residue and wet brewers grains with or without other feeds as a total mixed ration.

Wet brewers grains and soybean curd residue were stored in laboratory-scale silos without (BG and SC silages, respectively) or with other ingredients as total mixed rations (BGT and SCT silages, respectively). Silages were opened after 14 and 56 d, and microbial counts, fermentation products, and aerobic stability were determined. Denaturing gradient gel electrophoresis was carried out to examine bacterial communities, and several bacteria that appeared to be involved in fermentation were identified. Lactic acid content was greater in SCT than in BGT silage, but lower in SC than in BG silage. Ethanol content was greater in BG than in SC regardless of silage type. Aerobic deterioration occurred promptly in ensiling materials (nonensiled by-products and total mixed ration mixtures) and in silages stored alone; however, SCT and BGT silages resisted deterioration and no heating was found for more than 5.5 d regardless of storage period. Silages were stable even with high yeast populations at silo opening, whereas prolonged ensiling decreased yeast counts in the 2 total mixed ration silages. The denaturing gradient gel electrophoresis profiles appeared similar between SCT and BGT silages but not between SC and BG silages. Weissella spp. and Lactobacillus brevis were common in aerobically stable SCT and BGT silages, and Lactobacillus buchneri was detected only in BGT silage. Both L. brevis and L. buchneri were found in silage but not in ensiling materials. Several other lactic acid bacteria were also identified in SCT and BGT silages, but did not appear to be related to fermentation and aerobic stability.

Cyclic hydroxamic-acid-containing peptide 31, a potent synthetic histone deacetylase inhibitor with antitumor activity.

Cyclic hydroxamic-acid-containing peptide 1 (CHAP1), designed as a hybrid of trichostatin A and trapoxin, is a lead compound for the development of potent inhibitors of histone deacetylase (HDAC). In this study, we synthesized a series of CHAP derivatives and evaluated their biological activities by monitoring the potency of their inhibition of HDAC activity, their ability to augment the expression of MHC class-I molecules in B16/BL6 cells, and their effect on cell proliferation. A structure-activity relationship study using these three assay systems revealed several requirements of their structure for the strong inhibition of HDAC not only in the cell-free situation, but also in cells. When the structures of CHAP derivatives are represented as cyclo(-Asu(NHOH)-AA(2)-AA(3)-Pro or Pip-)(n), where Asu(NHOH) and Pip are zeta-hydroxamide-alpha-aminosuberic acid and pipecolic acid, respectively, (a) the tetrapeptide structure (n = 1) was better than the octapeptide one (n = 2); (b) AA(2) and AA(3) should be hydrophobic; and (c) the combination of amino acid chirality should be LDLD for the strongest inhibition of HDAC in cells (LDLD > LLLD, LDLL > LLDL). cyclo(-L-Asu(NHOH)-D-Tyr(Me)-L-Ile-D-Pro-) or CHAP31 was selected as one of the strongest CHAPs, and its biological activity was characterized further. CHAP31 was much more stable in the presence of cultured cells (t(1/2) > 3000 h) than trichostatin A (t(1/2) = 14.7 h) or trapoxin A (t(1/2) = 2.10 h). CHAP31 exhibited antitumor activity in C57BL x DBA/2 F(1) (BD2F(1)) mice bearing B16/BL6 tumor cells. Furthermore, CHAP31 inhibited the growth in four of five human tumor lines implanted into nude mice. These results suggest CHAP31 to be promising as a novel therapeutic agent for cancer treatment.

Activation of human plasma prekallikrein by Pseudomonas aeruginosa elastase in vitro.

Human plasma prekallikrein, precursor of the bradykinin-generating enzyme, was activated in a purified system under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase which is a tissue-destructive metalloproteinase. Compared with that, Pseudomonas aeruginosa alkaline proteinase poorly activated it with a rate as low as less than one-twentieth of that of elastase. The activation by elastase was blocked with a specific inhibitor of elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2 (10 microM). Generation of kallikrein-like amidolytic activity was also observed in plasma deficient in Hageman factor by treatment with elastase, but was not in plasma deficient in prekallikrein. The kallikrein-like activity generated in Hageman factor deficient plasma as well as the generation process itself was indeed inhibited by anti-human prekallikrein goat antibody. These results suggest that the pathological activation of the kallikrein-kinin system might occur under certain clinical conditions in pseudomonal infections.

Anti-chymotrypsin and anti-elastase activities of a synthetic bicyclic fragment containing a chymotrypsin-reactive site of soybean Bowman-Birk inhibitor.

A bicyclic hexadecapeptide, which corresponds to the sequence 36-51 and contains the chymotrypsin-reactive Leu-43-Ser-44 bond of soybean Bowman-Birk inhibitor, has been synthesized. This peptide consists of two loops formed by disulfide bridges between Cys-36 and Cys-51 and between Cys-41 and Cys-49. The bicyclic peptide showed a strong anti-chymotryptic activity with a Ki of 7.1.10(-7) M. Comparison of inhibitory activity and digestive stability against chymotrypsin with other hexadecapeptides having the same sequence but lacking one or both disulfide bridges suggested that the compact bicyclic structure increases the activity and protects the Leu-Ser bond from chymotryptic digestion. Interestingly, the bicyclic peptide was found to inhibit porcine pancreatic elastase with a Ki of 4.3.10(-5) M, indicating the broad specificity of this ring system.

Pseudomonas aeruginosa alkaline proteinase might share a biological function with plasmin.

Pseudomonas aeruginosa alkaline proteinase, which is a zinc-dependent bacterial endopeptidase, preferentially hydrolyzed Boc-Val-Leu-Lys-methylcoumarylamide (MCA) which was originally designed as a specific substrate of plasmin, a plasma serine proteinase. The hydrolytic capacity was resistant to tosyl-lysine chloromethylketone at a concentration as high as 1 mM, but was blocked by a treatment with metal chelator such as o-phenanthroline at the concentration of 5 mM. Kinetic parameters of the amidolytic reaction were Km = 21 microM, kcat = 0.067 s-1 and kcat/Km = 3190 M-1 s-1. A synthetic peptide inhibitor which bore a possible ligand for zinc atom at the carboxy terminal was designed. This inhibitor, Ac-Val-Leu-Lys-4-mercaptoanilide, blocked the amidolytic activity of the pseudomonal alkaline proteinase in a competitive manner with the dissociation constant (Ki) value of 24 microM. The results imply that P. aeruginosa alkaline proteinase must be an unusual zinc-dependent 'C (COOH)-type' endopeptidase, which hydrolyzes the peptide bond of certain amino acid residues at the carboxyl group side by specific recognition, like serine- and cysteine-proteinases. In comparison, P. aeruginosa elastase which is a typical 'N (NH2)-type' metalloproteinase did not hydrolyze all of the commercially available peptide-MCA substrates tested at the present study. P. aeruginosa alkaline proteinase also hydrolyzed natural substrates of plasmin, such as fibrin and fibrinogen, with similar specific activities to plasmin. The susceptible subunits of fibrinogen were the A-alpha and B-beta ones, in this order. P. aeruginosa alkaline proteinase also exhibited an anti-coagulant activity in human plasma attributed to the direct fibrinogenolytic function. Such potential anti-coagulant capacity of the P. aeruginosa alkaline proteinase might explain, at least partly, the most characteristic pathologic feature of the P. aeruginosa septicemia, hemorrhagic lesions with lacking thrombi (Fetzer, A.E. et al. (1967) Am. Rev. Respirat. Dis. 96, 1121-1130).

Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro.

Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2, at a concentration as low as 10 microM. In this activation Hagemen factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation were kcat = 5.8 x 10(-3) s-1, Km = 4.3 x 10(-7) M and kcat/Km = 1.4 x 10(4) M-1 x s-1. This Km value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 micrograms/ml), the activation rate by the elastase increased several fold, with the kinetic parameters of kcat = 13.9 x 10(-3) s-1, Km = 1.6 x 10(-7) M and kcat/Km = 8.5 x 10(4) M-1 x s-1. These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.

Conformational studies of amphipathic alpha-helical peptides containing an amino acid with a long alkyl chain and their anchoring to lipid bilayer liposomes.

In order to investigate the conformation and orientation of lipid-bound peptides and proteins in the lipid bilayer, basic amphipathic alpha-helical peptides with a long alkyl chain, palmitoyl-(Leu-Ala-Arg-Leu)3-NHCH3 (P-4(3)) and Ac-Leu-Ala-Arg-Leu-Trp-Amy-Arg-Leu-Leu-Ala-Arg-Leu-NHCH3 (Amy-4(3), Amy; alpha-aminomyristic acid) were designed and synthesized. The conformational features and spectroscopic behavior in a buffer solution and in neutral and acidic liposomes were studied by CD, dye-leakage, and fluorescence measurements. The CD data indicated that P-4(3) took an alpha-helical structure in aqueous solution and in neutral and acidic liposomes. On the other hand, Amy-4(3) took a beta-structure in aqueous solution and an alpha-helical structure in neutral and acidic liposomes. The conformational change of Amy-4(3) was confirmed by fluorescence study on lipid titration of the peptide. The dye-leakage experiment showed that both peptides interacted with acidic liposomes to perturb them, but less effectively than Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4(3)) which has no long alkyl group. Based on these results, a discussion is made concerning the conformation and orientation of peptides in aqueous solution and in the lipid bilayer.