Effect of microwave irradiation on the molecular and structural properties of hyaluronan

Hyaluronan (Na⁺ salt of hyaluronic acid, HA) was extensively depolymerised by HCl-catalyzed hydrolysis at pH 3 for up to 500 min under temperature-controlled microwave irradiation. The effects of microwave heating on the hydrodynamic properties of the polysaccharide were determined by SEC-MALLS and viscometry. The weight-average molecular mass (M_w) of HA decreased from 1.44 × 10⁶ to 5000, reaching the region of higher oligosaccharides. The scission of HA chains was found to proceed randomly during the whole degradation process. Treatment of the M_w and intrinsic viscosity data according to the Mark–Houwink equation, [η] = k × M_w^α suggested three relationships with α₁ = 0.46 for M_w > 500,000, α₂ = 0.84 for M_w between 500,000 and 50,000, and α₃ = 1.13 for M_w < 50,000. The results revealed that HA with M_w > 10,000 adopts a stiffish coil conformation in solution. As monitored by FT-IR and NMR spectroscopic techniques, the primary structure of the HA chains was maintained during the microwave-assisted hydrolysis at pH 3 at 105 °C. At reaction times larger than 240 min, uv spectroscopy suggested the depolymerisation of HA was accompanied by formation of by-products produced by side reaction.     

The application of microwave heating to the synthesis of 6-amino-6-deoxycellulose

Microwave heating was applied to the reactions involved in the synthesis of 6-amino-6-deoxycellulose, 4. These included, cellulose solubilization, bromination at C-6, displacement of bromine with azide ion, and reduction of the azido group to an amino group. Compared to conventional heating, this approach had the advantages of shortening reaction times and retaining the degree of polymerization of 4.     

Stability and structural changes of horseradish peroxidase: Microwave versus conventional heating treatment

Effects of conventional heating (CH) and microwave (MW) on the structure and activity of horseradish peroxidase (HRP) in buffer solution were studied. CH incubation between 30 and 45 °C increased activity of HRP, reaching 170% of residual activity (RA) after 4–6 h at 45 °C. CH treatment at 50 and 60 °C caused HRP inactivation: RA was 5.7 and 16.7% after 12 h, respectively. Secondary and tertiary HRP structural changes were analyzed by circular dichroism (CD) and intrinsic fluorescence emission, respectively. Under CH, activation of the enzyme was attributed to conformational changes in secondary and tertiary structures. MW treatment had significant effects on the residual activity of HRP. MW treatment at 45 °C/30 W followed by CH treatment 45 °C regenerated the enzyme activity. The greatest loss in activity occurred at 60 °C/60 W/30 min (RA 16.9%); without recovery of the original activity. The inactivation of MW-treated HRP was related to the loss of tertiary structure, indicating changes around the tryptophan environment.     

Improved synthetic routes to rhodium bipyridine complexes: Comparison of microwave vs. conventional synthesis

We report here two new, high purity, high yield, one-step syntheses of cis-[Rh(bpy)₂X₂][PF₆] {X = Cl, Br, I, bpy = 2,2′-bipyridine} directly from the RhX₃ · nH₂O starting materials - one conventional and one microwave. The key to obtaining the pure complexes appears to be maintaining a 2:1 ratio of bipyridine to rhodium in solution; thus all reactants must be completely dissolved prior to the start of the reaction. Comparison of the two methods is also discussed. These complexes are pure enough for emission spectroscopy after minimal work-up. The complete characterization of all the halide complexes and the first cyclic voltammetry data on the cis-[Rh(bpy)₂I₂][PF₆] complex are reported. The irreversible Rh(III)-Rh(I) redox potential becomes more positive from Cl to I. All three complexes show two reversible redox potentials corresponding to the bipyridine reductions. These data are consistent with the loss of the two halide ligands and formation of [Rh(bpy)₂]⁺ upon reduction of Rh(III) to Rh(I).     

Cellulose esters from waste cotton fabric via conventional and microwave heating

The esterification of cellulose from waste cotton fabric in a N,N-dimethylacetamide/lithium chloride solvent system was carried out using different types of fatty acid chloride including butyryl chloride, capryloyl chloride, and lauroyl chloride as esterifying agents, and N,N-dimethyl 1-4-aminopyridine as a catalyst under conventional and microwave activation. Microwave esterification was performed under 2.45 GHz with power varying from 90 to 450 W. The optimum conditions for esterification of cotton cellulose with various esterifying agents were investigated in terms of reaction time and temperature to attain appropriate %weight increase and degree of substitution of esterified-cellulose. The degree of substitution, functional group and chemical structure, and thermal stability of cellulose ester powder were characterized by ¹H NMR, FTIR, and TGA/SDTA analysis. Morphologies, crystallinity, and solubility of modified cellulose by two different heating methods were compared.     

Structure and properties of starch-based foams prepared by microwave heating from extruded pellets

The physical properties of microwave-foamed starch-based pellets, including density, porosity, cell structure, water absorption characteristics and mechanical properties were characterized. It was found that the physical properties of these starch-based foams produced by microwave heating are highly dependent on the raw materials and additives. Foam density decreased significantly after addition of 5.5–10.5% w/w salts, while foams containing nucleation agent (talc) were denser than the control with reduced cell size. A proprietary blowing agent did not affect the foam density markedly. Addition of salts also increased the water sorption of foams and plasticized cell walls. Mechanical behaviour of foamed pellets can be adjusted effectively by controlling the cell structure through using different additives. Mechanical properties of the foamed pellets in the elastic region as well as under large deformation (up to 40% strain) all follow a power–law relationship with foam density.     

Chain scission of hyaluronan by peroxynitrite

The reaction of peroxynitrite with the biopolymer hyaluronan has been studied using stopped-flow techniques combined with detection of molecular weight changes using the combination of gel permeation chromatography and multiangle laser light scattering. From the effect of peroxynitrite on the yield of hyaluronan chain breaks, it was concluded that the chain breaks were caused by hydroxyl radicals which escape a cage containing the *OH NO*(2) radical pair. The yield of free hydroxyl radicals was determined as 5+/-1% (as a proportion of the total peroxynitrite concentration). At high peroxynitrite concentrations, it was observed that the yield of chain breaks leveled out, an effect largely attributable to the scavenging of hydroxyl radicals by nitrite ions present in the peroxynitrite preparation. These experiments also provided some support for a previous proposal that the adduct formed between ONOOH and ONOO(-) might itself produce hydroxyl radicals. The rate of this reaction would have to be of the order of 0.05 s(-1) to produce hydroxyl radical yields that would account quantitatively for chain break yields at high peroxynitrite concentrations. By carrying out experiments at higher hyaluronan concentrations, it was also concluded that an additional yield of chain breaks was produced by the bimolecular reaction of the polymer with ONOOH at a rate constant of about 10 dm(3)mol(-1)s(-1). At 5.3 x 10(-3)mol dm(-3) hyaluronan, this amounted to 3.5% chain breaks (per peroxynitrite concentration). These conclusions support the proposal that the yield of hydroxyl radicals arising from the isomerization of ONOOH to nitrate ions is relatively low.     

Free amino acid concentrations in milk: Effects of microwaveversus conventional heating

Summary Microwave effects on free amino acid concentrations in milkversus a water bath heating were investigated in view of their importance for infant growth. Concentrations of few amino acids, such as aspartate, serine or lysine, are unchanged whatever the way and the temperature of heating. In contrast, tryptophan concentrations decreased similarly whatever the way of heating (110 ± 3µmol/l before heatingvs 84 ± 4µmol/l after 30°C microwave heating, p < 0.05). On the contrary, concentrations of glutamate and glycine increased more after water bath heating at 90°C (325 ± 4 and 101 ± 1µmol/l, respectively) than after microwave heating (312 ± 4 and 95 ± 1µmol/l, respectively, p < 0.05) suggesting milk proteolysis. Moreover, the accumulation of ammonia observed at 90°C with the water bath together with increase Glu levels might reflect a degradation of glutamine. An ornithine enrichment, more evident with microwave heating, was shown and could be of interest as it is a polyamine precursor. Also, considering few variations of free amino acid concentrations and the time saved, microwave heating appears to be an appropriate method to heat milk.     

Applications of microwave dielectric heating in environment-related heterogeneous gas-phase catalytic systems

The application of microwave dielectric heating in a range of environment-related heterogeneous catalytic reaction systems has been reviewed. The reactions investigated include the decomposition of hydrogen sulfide, the reduction of sulfur dioxide with methane, the reformation of methane by carbon dioxide, the hydrodesulfurization of thiophene, and the oxidative coupling of methane. The interaction of microwave irradiation with heterogeneous catalytic systems and its consequence for the microwave heating behaviour of catalysts have been examined. The effect/mechanism of microwave dielectric heating on heterogeneous catalytic reaction systems has also been discussed.     

Degradation of high-molecular-weight hyaluronan by hydrogen peroxide in the presence of cupric ions

Dynamic viscosity (eta) of the high-molecular-weight hyaluronan (HA) solution was measured by a Brookfield rotational viscometer equipped with a Teflon cup and spindle of coaxial cylindrical geometry. The decrease of eta of the HA solution, indicating degradation of the biopolymer, was induced by a system containing H2O2 alone or H2O2 plus CuCl2. The reaction system H2O2 plus CuCl2 as investigated by EPR spin-trapping technique revealed the formation of a four-line EPR signal characteristic of a *DMPO-OH spin adduct. Thus, hydroxyl radicals are implicated in degradation of high-molecular-weight HA by the system containing H2O2 and CuCl2.     

Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.     

Comparative study on microwave and conventional methods for protein hydrolysis in food

Summary A rapid microwave hydrolysis procedure was developed for amino acid determination in food. The hydrolysis was performed with 6M HCl in sealed vessels using a microwave digestion system.Bovine Serum Albumin was chosen as a model protein to compare its theoric amino acid sequence with the experimental results obtained after hydrolysis by both the traditional oven heating and the microwave methods. Furthermore the selected microwave methods were carried out on different food matrices (cheese and durum wheat) and the obtained data were compared with the traditional method results.This comparative study shows that the rapid microwave hydrolysis is as accurate and precise as the traditional hydrolysis for determining amino acids in food.These results were presented at the Third International Congress on Amino Acids, Vienna, August 23–27th, 1993.     

Changes of amino acid gradients in brain tissues induced by microwave irradiation and other means

Focused microwave irradiation to the head (FMI) has been used extensively by neurochemists for rapid inactivation of enzymatic activity in brain tissues and the preservation, for in vitro analysis, of in vivo substrate concentrations. Periodically the suitability of this technique for regional studies has been questioned. Evidence has now been obtained, on the basis of altered concentration gradients for GABA and taurine from the Substantia Nigra (SN) to an Adjacent Dorsal Area (ADJ), that FMI not only inactivates enzymes, but also facilitates rapid diffusion of small molecules from areas of high concentrations to adjacent areas of lower concentration. To a lasser extent, the implantation of plastic injection cannulas also decreased these concentration gradients. These results offer clear evidence that FMI is ill suited and unreliable for studies designed to map and compare the in vivo regional concentrations of diffusible organic molecules (such as amino acids) in brain tissues. Any invasive technique that compromises membrane barriers is likely to produce smaller similar effects.     

Profiling of the eye aqueous humor in exfoliation syndrome by high-performance liquid chromatographic analysis of hyaluronan and galactosaminoglycans

The concentrations of hyaluronan and galactosaminoglycans - i.e., chondroitin sulfate and dermatan sulfate - were measured in the aqueous humor of the eye from patients with exfoliation syndrome and from healthy persons. The glycosaminoglycans/proteoglycans were almost completely precipitated (>97%) with ethanol in the presence of dextran as carrier and, following enzymic digestion, hyaluronan and galactosaminoglycans, were quantitatively converted to Δ^4,5-disaccharides. Non-degraded heparan sulfate and proteins/glycoproteins were removed by ultrafiltration using a Centricon 3 membrane. Separation and determination of hyaluronan- and galactosaminoglycan-derived Δ-disaccharides were performed by ion-suppression HPLC. For an accurate analysis in triplicate, as little as 50 μl of aqueous humor is required. Application of this method to the analysis of samples from six patients with exfoliation syndrome and three healthy persons showed that hyaluronan levels in patients (6.65–16.15 μg ml⁻¹) were significantly higher (3–8 times) than in healthy persons (2.0–2.24 μg ml⁻¹). There was no significant alteration in the galactosaminoglycan concentration. The obtained data open a new area in the deeper understanding of the exfoliation syndrome pathophysiology and in establishing a highly sensitivity and accurate HPLC method for its diagnosis and patient's follow-up.     

Hydrolysis of plant seed gums by microwave irradiation

Under microwave irradiation (MW), the seed gums, guar and Ipomoea quamoclit were hydrolyzed to constituent monosaccharides and oligosaccharides in very mild conditions and short reaction time. Under MW both the seed gums could be completely hydrolyzed using very dilute acid (0.00625N H₂SO₄) within two minutes. Hydrolysis occurs in 2 min and 20 s even in absence of acid under the MW irradiation. Thus hydrolytic fragmentation under MW provides an efficient tool in structural elucidation of polysaccharides.     

The distribution of renal hyaluronan and the expression of hyaluronan synthases during water deprivation in the Spinifex hopping mouse, Notomys alexis

Hyaluronan (HA) is a glycosaminoglycan that is synthesized by a family of enzymes called hyaluronan synthases (HASs), of which there are three isoforms (HAS1, 2 and 3) in mammals. The HASs have different tissue expression patterns and function, indicating that synthesis of HA and formation of the HA matrix may be regulated by various factors. The HA matrix has an important role in renal water handling and the production of a concentrated urine. We investigated the distribution of HA and the expression of HAS1, HAS2 and HAS3 mRNAs in the kidney of the Spinifex hopping mouse, Notomys alexis, a native Australian desert rodent that is reported to produce the most concentrated urine of any mammal. After periods of three, seven and fourteen days of water deprivation, the distribution of renal HA changed considerably, and there was a general down-regulation of HAS mRNA expression. It is proposed that the regulation of HA synthesis by the different HAS isoforms during water deprivation in N. alexis, could be influenced by the molecular mass of the HA chains produced by each isoform, followed by the rate at which the individual HAS produces HA.     

Comparison of bacterial growth to high-intensity microwave exposure and conventional heating

Escherichia coli pol A⁺ and pol A^? strains were exposed to 8.8-GHz microwaves pulsed at 1,000 Hz (1-μs pulse width) and an SAR of 40 W/kg, which increased the temperature of the cell culture by 7 °C. Two-way analysis of variance showed no significant difference between the growth rates of microwave irradiated and thermally exposed cells.     

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed,paraffin embedded tissues

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.     

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed, paraffin embedded tissues

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.     

Facile synthesis of glycosylated Fmoc amino acid building blocks assisted by microwave irradiation

The synthesis of glycosylated Fmoc amino acids by reaction of mono- and disaccharide peracetates with Fmoc amino acids having free carboxyl groups was rapidly promoted by Lewis acids (SnCl₄, BF₃·Et₂O) under microwave irradiation. The products are useful building blocks for the synthesis of glycopeptides.