Synthesis and NMR characterization of new hyaluronan-based NO donors

Nitric oxide (NO) and hyaluronic acid (HA), two species widely different in terms of molecular complexity and biological competence, are both known to play an important role in the wound healing process. To combine the properties of HA and NO, we synthesized new NO-donors based on hyaluronic acid derivatives exhibiting a controlled NO-release under physiological conditions (in vitro tests). Since two molecules of NO can form a covalent bond with secondary amines to yield structures, named NONO-ates, able to release NO in solution, we used spermidine bound to HA as the NO-linker. The HA-spermidine derivative was obtained by controlled HA amidation in aqueous media, activating the biopolymer carboxylate groups with a water soluble carbodiimide. The resulting derivative, soluble in water, was fully characterized by high field 1H and 13C NMR spectroscopy. The amount of grafting of spermidine on HA was determined by integration of suitable 1H NMR signals. In addition, cross-linked derivatives of HA were synthesized by the Ugi's four-component reaction using formaldehyde, cyclohexylisocyanide, and spermidine. The HA-spermidine networks were characterized by 13C CP-MAS NMR spectroscopy. The degree of cross-linking of the networks was also determined. Finally, the release of NO from the swollen hydrogels freshly saturated with NO, in contact with aqueous media, was monitored by means of UV spectrophotometric measurements.     

Dispersal of a plasmid-borne chloramphenicol resistance gene in streptococcal and enterococcal plasmids

Plasmids coding for chloramphenicol resistance, five isolated from streptococci of groups A, B, and G, ten from enterococci (Enterococcus faecalis, Enterococcus faecium), and two from staphylococci, were tested for sequence homology with the chloramphenicol resistance gene of pIP501, a 30-kb plasmid originally isolated from a group B Streptococcus. The 6.3-kb HindIII fragment of pIP501, known to carry the chloramphenicol resistance gene, was cloned into pBR322. A 1.6-kb portion of the cloned fragment, which included most of the chloramphenicol resistance gene, was used as probe in DNA-DNA hybridization experiments. Sequence homology was detected between the probe and four of the streptococcal, seven of the enterococcal, and one of the staphylococcal plasmids. The absence of hybridization between this probe and one plasmid isolated from a group B Streptococcus, as well as three isolated from E. faecalis, indicated that there are at least two different plasmid-borne chloramphenicol resistance determinants in the streptococci and in the enterococci.     

Characterization of mixing in a novel wavy-walled bioreactor for tissue engineering

The wavy-walled bioreactor (WWB) possesses a novel geometry comprised of walls with sinusoidal waves that mimic baffles in an effort to promote mixing. This geometry provides a unique hydrodynamic environment suitable for the cultivation of mammalian cells and tissues and the investigation of fluid mechanical effects on cell and tissue growth and development. In the present study, mixing in WWB was characterized and compared to that in a conventional spinner flask (SF). The key parameters included in this characterization were mixing time, residence time distribution (RTD), and dissolved oxygen concentration during engineered cartilage tissue cultivation. Factors that influenced mixing in WWB included wave amplitude, agitation rate, and the ratio of the impeller diameter to the tank diameter (D/T). Data obtained from RTD and acid base neutralization studies confirmed the presence of different mixing zones in WWB. A theoretical comparison of WWB to a baffled spinner flask (BSF) using computational fluid dynamics (CFD) modeling predicted that while enhanced mixing was achieved in wavy-walled and BSF bioreactors, the shear stresses applied on tissue constructs were 15% lower in WWB. Improved mixing was achieved in WWB compared to the SF at similar D/T ratios, verified by improved oxygen transport and increased dispersion. However, for lower D/T ratios mixing in WWB was not necessarily improved. This study demonstrated the importance of characterization of mixing by showing the impact of even minor changes in bioreactor geometry and operating conditions.     

Differential distribution of ammonia- and nitrite-oxidising bacteria in flocs and granules from a nitrifying/denitrifying sequencing batch reactor

A lab-scale sequencing batch reactor was operated with alternating anoxic/aerobic conditions for nitrogen removal. Flocs and granules co-existed in the same reactor, with distinct aggregate structure and size, for over 180 days of reactor operation. Process data showed complete nitrogen removal, with temporary nitrite accumulation before full depletion of ammonia in the aerobic phase. Microbial quantification of the biomass by fluorescence in situ hybridisation showed that granules contained most of the nitrite-oxidising bacteria (NOB) whereas the ammonium-oxidising bacteria (AOB) seemed to be more abundant in the flocs. This was supported by microsensor measurements, which showed a higher potential of NO₂⁻ uptake than NH₄ uptake in the granules. The segregation is possibly linked to the different growth rates of the two types of nitrifiers and the reactor operational conditions, which produced different sludge retention time for flocs and granules. The apparent physical separation of AOB and NOB in two growth forms could potentially affect mass transfer of NO₂⁻ from AOB to NOB, but the data presented here shows that it did not impact negatively on the overall nitrogen removal.     

Microalga biofertilizer improves potato growth and yield,stimulating amino acid metabolism

Journal of Applied Phycology - In the context of a growing demand for healthy food, nature-friendly alternatives such as biofertilizers and biostimulants, which are natural sources capable of...     

Changes in the Serum Urate Level Can Predict the Development of Parkinsonism in the 6-Hydroxydopamine Animal Model

Epidemiological studies indicate that a higher plasma level of uric acid (UA) associates with the reduced risk of Parkinson’s disease (PD). To confirm the role of UA as a biomarker for PD, we evaluated changes in the serum UA level in the 6-hydroxydopamine (6-OHDA)-induced hemiparkinsonism in rat. For this purpose, 6-OHDA was administered in the medial forebrain bundle by stereotaxic surgery. According to the apomorphine-induced rotational test, the increased intensity of behavioral symptoms as a function of time was associated with the further reduction of UA level. On the other hand, the level of UA increased in the midbrain of the injured hemisphere. The level of reduction in the serum UA level of rats with severe and moderate symptoms was significantly higher than that of rats with mild symptoms. The immunohistofluorescence and biochemical analyses showed that the serum UA level was also correlated with the death of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNc), reduced level of striatal dopamine, and severity of oxidative stress in the midbrain. The rats with mild symptoms also showed a significant decrease in TH-positive neurons and striatal dopamine level. These findings suggest a positive correlation between the level of reduction in the serum urate level and severity of 6-OHDA-induced Parkinsonism. In addition, our findings indicated that UA had no marked neuroprotective effects, at least at concentrations obtained in this study. On the other hand, UA was introduced as a biomarker for PD, as a significant decline was observed in the serum UA level of rats with mild behavioral symptoms but with significant dopaminergic cell death in the SNc.     

Characterization of the bioactive form and molecular determinants of recognition of cyclic enkephalin peptides at the δ-opioid receptor

An extensive and systematic search strategy to determine the conformational profile of 12 cyclic disulfide-bridged opioid peptides with varying affinities at the δ receptor has been carried out to identify the structure that is recognized by the δ receptor for each analogue. The methods and procedures used here for the conformational search have already been validated for [D -Pen2, D -Pen5] enkephalin (DPDPE), one member of this family. Use of these methods led to a low-energy solution conformation of DPDPE in excellent agreement with all the geometric properties deduced from its solution nmr spectra. Each of the analogue was subjected to the same procedure, involving a combination of molecular dynamics simulations at high and low temperature. The study was repeated in two environmental conditions, an apolar environment, simulated by using a distance-dependent dielectric constant, and a polar environment by embedding the peptides in a high constant dielectric ( ε = 80). An automated comparison of the different conformers based on their backbone rms and average distance between the key aromatic moieties was followed by graphic analysis using maximum structural overlap. The cross-comparison of the conformations for each analogue revealed a unique conformer that may be recognized by the δ receptor for each high-affinity analogue that permitted maintaining the critical elements required for recognition in a simple spatial orientation, while maximizing similarity in other regions. © 1993 John Wiley & Sons, Inc.     

A case study of airborne culturable microorganisms in a poultry slaughterhouse in Styria, Austria

According to Council Directive 90/679/EEC on the protection of workers from risks related to exposure to biological agents at work, nature, degree and duration of workers’ exposure to microorganisms must be determined. This directive has already been implemented in waste and wastewater management. The present case study investigates concentration and composition of microorganisms in a poultry slaughterhouse in the State of Styria, Austria. From June to November 2002, measurements were conducted at the sampling sites ‘moving rail’ and ‘gall bladder separation’ using the Andersen six stage viable cascade impactor and the SKC BioSampler. The results of this study were compared with other previous studies which were carried out using the same device (ACFM) and the same measurement methods. At the processing area of the ‘moving rail’, the median concentration of airborne mesophilic bacteria was 1.7×10⁶ CFU/m³ which is 8000 times higher than the background concentration of residential areas (approx. 210 CFU/m³). The airborne microorganisms concentration was 1.7×10⁴ CFU/m³ at composting plants which is 100 times lower than at a workplace of a poultry slaughterhouse. The study shows that poultry slaughterhouse employees are exposed to high concentrations of airborne microorganisms throughout the entire work time without using a respiratory protective device. For the protection of employees against airborne biological agents, relevant measures should be introduced to this field of work.