A fine structural modification of glycosaminoglycans is correlated with the progression of muscle regeneration after ischaemia: towards a matrix-based therapy?

Critical limb ischaemia often leads to amputation of the limb and potential mortality. Moreover, there are still significant problems with current therapeutic treatments, according to poor revascularisation of degenerated tissue probably due to modifications within the microenvironment. This study is focused on the changes of structure and bioactivity of glycosaminoglycans (GAGs), especially heparan sulphate (HS) and chondroitin sulphate (CS) in rat Extensor Digitorum Longus (EDL) muscle after ischaemia. Male Wistar rats were subjected to ischaemic-injury by ligation of the neurovascular trunk accompanying EDL-tendon. After 4, 8, 15, 21, 60 and 90 d, the rats were sacrificed and the muscles were collected and submitted to histological, biochemical and gene expression assays. We demonstrated that ischaemia induced modification of expression of enzymes involved in GAG biosynthesis which correlated with significant changes in HS and CS structural features such as size and sulphation pattern. These major structural changes are associated to modifications of GAG abilities to bind growth factors and to modulate cell activity. Moreover, a CS hallmark of injury is maintained as well after the regeneration process. Finally, we showed the relevance of the role of this glycanic matrix remodelling, since a GAG mimetic treatment accelerated muscle repair after ischaemia.

Arbuscular mycorrhizal fungi and rhizospheric bacteria diversity along an altitudinal gradient in South American Puna grassland.

Rhizospheric soil samples were taken from Puna native grasses along an altitudinal gradient. Biodiversity of arbuscular mycorrhizal fungi (AMF) and associated bacteria was analyzed considering altitude and grasses photosynthetic pathways (metabolic type C3, C4). Cultivation-dependent approaches were applied to obtain further information about the phylogeny of the dominating cultivable aerobic-heterotrophic bacteria communities present in rhizospheric soil samples. In average, the bacterial count ranged between 1.30 x 10(2) and 8.66 x 10(4) CFU g(-1) of dry weight of soil. Individual bacterial colonies of aerobic heterotrophic bacteria grown on R2A medium were morphologically grouped and identified as typical soil bacteria belonging to the genera Bacillus, Pseudomonas, and Arthrobacter. Ten AMF taxa were found: Acaulospora sp., A. laevis, A. spinosa, Gigaspora sp., Gi. ramisporophora, Glomus sp., Gl. aggregatum, Gl. ambisporum, Gl. sinuosum, and Scutellospora biornata. AMF diversity decreased with altitude.

Diverse responses to UV-B radiation and repair mechanisms of bacteria isolated from high-altitude aquatic environments.

Acinetobacter johnsonii A2 isolated from the natural community of Laguna Azul (Andean Mountains at 4,560 m above sea level), Serratia marcescens MF42, Pseudomonas sp. strain MF8 isolated from the planktonic community, and Cytophaga sp. strain MF7 isolated from the benthic community from Laguna Pozuelos (Andean Puna at 3,600 m above sea level) were subjected to UV-B (3,931 J m-2) irradiation. In addition, a marine Pseudomonas putida strain, 2IDINH, and a second Acinetobacter johnsonii strain, ATCC 17909, were used as external controls. Resistance to UV-B and kinetic rates of light-dependent (UV-A [315 to 400 nm] and cool white light [400 to 700 nm]) and -independent reactivation following exposure were determined by measuring the survival (expressed as CFU) and accumulation of cyclobutane pyrimidine dimers (CPD). Significant differences in survival after UV-B irradiation were observed: Acinetobacter johnsonii A2, 48%; Acinetobacter johnsonii ATCC 17909, 20%; Pseudomonas sp. strain MF8, 40%; marine Pseudomonas putida strain 2IDINH, 12%; Cytophaga sp. strain MF7, 20%; and Serratia marcescens, 21%. Most bacteria exhibited little DNA damage (between 40 and 80 CPD/Mb), except for the benthic isolate Cytophaga sp. strain MF7 (400 CPD/Mb) and Acinetobacter johnsonii ATCC 17909 (160 CPD/Mb). The recovery strategies through dark and light repair were different in all strains. The most efficient in recovering were both Acinetobacter johnsonii A2 and Cytophaga sp. strain MF7; Serratia marcescens MF42 showed intermediate recovery, and in both Pseudomonas strains, recovery was essentially zero. The UV-B responses and recovery abilities of the different bacteria were consistent with the irradiation levels in their native environment.

Enhancement of hydrocarbon waste biodegradation by addition of a biosurfactant from Bacillus subtilis O9.

A non-sterile biosurfactant preparation (surfactin) was obtained from a 24-h culture of Bacillus subtilis O9 grown on sucrose and used to study its effect on the biodegradation of hydrocarbon wastes by an indigenous microbial community at the Erlenmeyer-flask scale. Crude biosurfactant was added to the cultures to obtain concentrations above and below the critical micelle concentration (CMC). Lower concentration affected neither biodegradation nor microbial growth. Higher concentration gave higher cell concentrations. Biodegradation of aliphatic hydrocarbons increased from 20.9 to 35.5% and in the case of aromatic hydrocarbons from nil to 41%, compared to the culture without biosurfactant. The enhancement effect of biosurfactant addition was more noticeable in the case of long chain alkanes. Pristane and phytane isoprenoids were degraded to the same extent as n-C17 and n-C18 alkanes and, consequently, no decrease in the ratios n-C17/pri and n-C18/phy was observed. Rapid production of surfactin crude preparation could make it practical for bioremediation of ship bilge wastes.

Continuous production of L(+)-lactic acid by Lactobacillus casei in two-stage systems.

A two-stage two-stream chemostat system and a two-stage two-stream immobilized upflow packed-bed reactor system were used for the study of lactic acid production by Lactobacillus casei subsp casei. A mixing ratio of D12/D2 = 0.5 (D = dilution rate) resulted in optimum production, making it possible to generate continuously a broth with high lactic acid concentration (48 g l-1) and with a lowered overall content of initial yeast extract (5 g l-1), half the concentration supplied in the one-step process. In the two-stage chemostat system, with the first stage at pH 5.5 and 37 degrees C and a second stage at pH 6.0, a temperature change from 40 degrees C to 45 degrees C in the second stage resulted in a 100% substrate consumption at an overall dilution rate of 0.05 h-1. To increase the cell mass in the system, an adhesive strain of L. casei was used to inoculate two packed-bed reactors, which operated with two mixed feedstock streams at the optimal conditions found above. Lactic acid fermentation started after a lag period of cell growth over foam glass particles. No significant amount of free cells, compared with those adhering to the glass foam, was observed during continuous lactic acid production. The extreme values, 57.5 g l-1 for lactic acid concentration and 9.72 g l-1 h-1 for the volumetric productivity, in upflow packed-bed reactors were higher than those obtained for free cells (48 g l-1 and 2.42 g l-1 h-1) respectively and the highest overall L(+)-lactic acid purity (96.8%) was obtained in the two-chemostat system as compared with the immobilized-cell reactors (93%).

Chemostat production of plantaricin C by Lactobacillus plantarum LL441.

Plantaricin C, a bacteriocin synthesized by Lactobacillus plantarum LL441, was optimally produced in chemostats kept at pH 5.0, 30 degreesC, 150 rpm, and a dilution rate of 0.05 h-1 when glucose was used as carbon source and a dilution rate of 0.10 to 0.12 h-1 when sucrose or fructose was used instead. Production was abolished at high dilution rates, i.e., when the cells grew rapidly in all carbon sources.

Fed-batch and continuous culture ofPhaffia rhodozyma (Xanthophyllomyces dendrorhous).

The basidiomycetous yeastPhaffia rhodozyma was grown in batch, fed-batch and continuous culture, and some parameters governing growth and total carotenoid production were determined.

Effects of oxidative stress on the production of carotenoid pigments byPhaffia rhodozyma (Xanthophyllomyces dendrorhous).

The resistance to killing by free radicals of two mutants ofPhaffia rhodozyma was determined. Mutant 5-7 did not produce astaxanthin but produced beta-carotene, while mutant 3-4 did not produce any carotenoid pigments. The resistance of mutant 5-7 was the same as that of the wild type but mutant 3-4 was rapidly killed. Carotenoid pigments increased the resistance to killing by free radicals. We investigated the effects of free radicals, generated by H(2)O(2) and Fe(2+) added to the medium, on wild-type cells and mutants ofP. rhodozyma. Unpigmented mutants of basidiomycetous yeasts (Rhodotorula spp. and others) are more susceptible to killing by UV-irradiation than the pigmented, wild-type strains. Therefore, we investigated the effect of free radicals on a similar basidiomycetous yeast,P. rhodozyma, a species of economic importance, in the biological production of astaxanthin.

Determination of radial growth rate of colonies of Sclerotium rolfsii F-6656 for the evaluation of culture medium, optimum incubation temperature, osmo- and halotolerance.

The measurement of the colony radial growth rate (Kr) on solid medium of colonies of Sclerotium rolfsii Proimi F-6656 for the evaluation of scleroglucan production medium and other different media, incubation temperature and tolerance to diverse concentrations of sucrose and NaCl were studied. The optimum growth temperature observed was 30 degrees C. The Kr value reached on the Production Medium used (0.66 mm.h-1) showed no differences compared with those of the other media tested, indicating that all the requirements for growth were provided. Poor growth was only observed on Soil Extract Agar. The fungus tolerated concentrations of sucrose from 0.15 to 1.17 M, on both Czapek and production medium. Growth was limited by the highest concentrations of sucrose tested (0.88 and 1.17 M), as indicated by a slower increase in colony size. Addition of 0.86 M NaCl to the production medium and YM agar did not inhibit growth completely, but decreased the radial growth rate considerably (80 and 70% respectively).

Selection of an adhesive phenotype of Streptococcus salivarius subsp. thermophilus for use in fixed-bed reactors.

Streptococcus salivarius subsp. thermophilus was cultivated in a chemostat in order to obtain an adhesive phenotype of this strain. When the system was operated at low dilution rates (D < 0.2 h-1) for about 4 weeks, the strain formed a visible film on the surface of the culture vessel. The biofilm cells were not washed out even when dilution rates were increased (D = 6.9 h-1), and this resulted in a high biomass productivity (P = 4.1 g1(-1) h-1). On the other hand, when the culture was grown at dilution rates faster than 0.2 h-1, only the free suspended cells were present in the culture broth, and were washed out at velocities of about 1.0 h-1. The biomass productivity was consequently lower (P = 1.33 g1(-1) h-1) than in the previous case. The selected adhesive phenotype was grown on different glass beads and the possibility of lactate fermentation in a continuous and semicontinuous mode was demonstrated.

Construction of an integrative shuttle vector for Zymomonas mobilis.

An integrative shuttle vector, pZMOCP1, was constructed by ligating EcoRV digests of the plasmid cloning vector pBluescript and pZMP1, a cryptic plasmid of Zymomonas mobilis PROIMI A1. The 7.2-kb plasmid pZMOCP1 replicated in Escherichia coli and could also be transferred from this host by electroporation to Z. mobilis ATCC 29191. The transformants were selected by ampicillin resistance. The integrative characteristic was detected by hybridization in situ. The vector was stably maintained in Z. mobilis after 200 generations without selective pressure.

[The biomass in upflow anaerobic filters]. / El contenido biológico de filtros anaeróbicos de flujo ascendente.

A cylindrical upflow filter packed with non-reticulated polyurethane foam, seeded with anaerobic sewage sludge and geared to biological treatment of dairy industrial wastewater, was used to determine the biomass content of the biofilm and suspended flora. This microflora is responsible for the conversion to methane and carbon dioxide of most of organic matter in wastewater. The methanogenic process reduces the COD of liquid wastes in more than 83% when operate at organic loading rate of 6 Kg COD/m3/d. Sequential sampling showed that biomass could be determined by measurement of volatile solids of each filter section. Those solids are related to filter geometry an produce accumulation of flocs (0.7g/l) in the bottom zone corresponding to liquid inlet.

El contenido biológico de filtros anaeróbicos de flujo ascendente / [The biomass in upflow anaerobic filters]

A cylindrical upflow filter packed with non-reticulated polyurethane foam, seeded with anaerobic sewage sludge and geared to biological treatment of dairy industrial wastewater, was used to determine the biomass content of the biofilm and suspended flora. This microflora is responsible for the conversion to methane and carbon dioxide of most of organic matter in wastewater. The methanogenic process reduces the COD of liquid wastes in more than 83

when operate at organic loading rate of 6 Kg COD/m3/d. Sequential sampling showed that biomass could be determined by measurement of volatile solids of each filter section. Those solids are related to filter geometry an produce accumulation of flocs (0.7g/l) in the bottom zone corresponding to liquid inlet.

El contenido biológico de filtros anaeróbicos de flujo ascendente. / [The biomass in upflow anaerobic filters]

A cylindrical upflow filter packed with non-reticulated polyurethane foam, seeded with anaerobic sewage sludge and geared to biological treatment of dairy industrial wastewater, was used to determine the biomass content of the biofilm and suspended flora. This microflora is responsible for the conversion to methane and carbon dioxide of most of organic matter in wastewater. The methanogenic process reduces the COD of liquid wastes in more than 83

when operate at organic loading rate of 6 Kg COD/m3/d. Sequential sampling showed that biomass could be determined by measurement of volatile solids of each filter section. Those solids are related to filter geometry an produce accumulation of flocs (0.7g/l) in the bottom zone corresponding to liquid inlet.

Characteristics of an inulinase produced by Bacillus subtilis 430A, a strain isolated from the rhizosphere of Vernonia herbacea (Vell Rusby).

Bacillus subtilis 430A, isolated from the Vernonia herbacea (Vell Rusby) rhizosphere, produced an exocellular inulinase that fits the requirements for the production of syrups on an industrial scale. The partially purified enzyme, obtained by acetone precipitation, displayed a higher specificity for inulin (Km, 8 mM) than for sucrose (56 mM) and a total invertase/total inulase ratio of 0.62. In addition, it is stable at an optimal temperature of 45 to 50 degrees C for at least 7 h and is inhibited by the end product, fructose, at 14 mM.

Presence of an L(+)-lactate dehydrogenase in cells of Lactobacillus delbrueckii ssp. bulgaricus.

Lactobacillus delbrueckii ssp. bulgaricus ATCC 11842 was cultured in a chemostat and growth conditions were varied as required. Synthesis of L(+)-lactate was observed in all cases as well as activity of L(+)-lactate dehydrogenase in cell-free extracts. This enzyme was responsible for the formation of the L(+) isomer of lactate, since a lactate racemase was not present.

Use of the UASB reactor for the anaerobic treatment of stillage from sugar cane molasses.

The feasibility of applying the UASB concept for the anaerobic treatment of stillage of distilleries in the sugar producing area of Argentina was subject to study. Results obtained in a 100-L UASB reactor treating stillages with COD values between 35 and 100 g COD/L are presented. Loading rates of up to 24 g COD/L/day were applied with an average COD removal of 75% and a biogas production of more than 9 L/L/day, with an average methane content of 58%. The settling velocity distribution of sludge particles would indicate a good formation of biomass pellets. System interruptions of months without feed and at ambient temperature (20-24 degrees C) were well tolerated.

Membrane-bound cooperative enzymes. Stokes' radii, Hill plots and membrane fluidity in the regulation of adenosinetriphosphatase from Escherichia coli.

The soluble Ca2+-ATPase from Escherichia coli had a distinctive behavior with respect to inhibition by Na+ measured at 36 degrees C and 19 degrees C. At the first temperature the Hill plots are linear and show a slope of 1.1 while at 19 degrees C the plots are biphasic, with slopes of 1.8 and 0.8 before and after the break, respectively. The break occurs at about 50 nM NaCl. Gel chromatography was performed in jacketed Sepharose 4B columns kept at 2 temperatures in the presence of different concentrations of NaCl. It was found that the Stokes' radius of the enzyme was dependent on the temperature and on the salt concentration. Equilibrium sucrose gradients run at 19 degrees C showed that the sedimentation constant of the enzyme remained constant irrespective of the NaCl concentration used. It is concluded that a "folding" of the enzyme takes place in the presence of NaCl, the process being complete at about 50 mM NaCl at 19 degrees C and at about 20 mM at 36 degrees C. The results are in excellent agreement with the kinetic data: the "folded" or "compact" configuration would show no cooperative response towards Na+ while the "expanded" conformer would present strong cooperativity. This is also in agreement with the results obtained with the enzyme embedded in the membrane: when the membrane is fluid a high n value (Hill coefficient) is found; when the membrane is more rigid the value of n falls. A model explaining all our results is proposed and discussed.