D. Lőrinczy

A simple model for the cooperative stabilisation of actin filaments by phalloidin and jasplakinolide

The stabilisation of magnesium actin filaments by phalloidin and jasplakinolide was studied using the method of differential scanning calorimetry. The results showed that actin could adapt three conformations in the presence of drugs. One conformation was adapted in direct interaction with the drug, while another conformation was identical to that observed in the absence of drugs. A third conformation was induced through allosteric inter‐protomer interactions. The effect of both drugs propagated cooperatively along the actin filaments. The number of the cooperative units determined by using a quantitative model was larger for jasplakinolide (15 actin protomers) than for phalloidin (7 protomers).

The effect of phalloidin and jasplakinolide on the flexibility and thermal stability of actin filaments

In this work the effect of phalloidin and jasplakinolide on the dynamic properties and thermal stability of actin filaments was studied. Temperature dependent fluorescence resonance energy transfer measurements showed that filaments of Ca‐actin became more rigid in the presence of phalloidin or jasplakinolide. Differential scanning calorimetric data implied that the stiffer filaments also had greater thermal stability in the presence of phalloidin or jasplakinolide. The fluorescence and calorimetric measurements provided evidences that the extent of stabilization by jasplakinolide was greater than that by phalloidin.

Differential scanning calorimetry (DSC) analysis of human plasma in melanoma patients with or without regional lymph node metastases

Melanoma malignum (MM) is a common type of skin cancer, and its incidence is increasing in the general population. We aimed to detect blood plasma components with differential scanning calorimetry (DSC) in 15 white adult MM patients, who had histopathologically diagnosed, operable cutaneous MM without any distant metastases. We observed that thermal changes (second Tm, calorimetric enthalpy) in blood plasma showed correlation with tumor thickness and the extent of regional invasion. Further studies are needed to elucidate these relationships, but our preliminary work has provided DSC should be a new tool for the early diagnosis and monitoring of MM patients.

Effect of oxygen free radicals on myosin in muscle fibres

Experiments were performed on glycerol‐extracted muscle fibres prepared from psoas muscle of rabbit in the presence of hydroxyl free radical generating system. Short irradiation of spin‐labelled muscle fibres by UV light showed the interaction of probe molecules with oxygen free radicals. The intensity of the EPR signal from maleimide or isothiocyanate spin labels attached to the essential thiol groups decreased following irradiation. Oxygen free radicals affected the rate constant of the transition AM.ADP.Vi→AM.ADP in the ATP hydrolysis cycle. It was found that the essential –SH groups of myosin were involved in the oxidation of sulphydryls by Ce(IV). Ce(IV) complexed to nitrilotriacetic acid in the presence of spin trap produced long‐lived free radicals located partly on SH‐1 sulphydryls.

DSC analysis of the abnormalities of human leg skeletal muscles: A preliminary study

Abstract The standard calorimetric properties of the healthy human skeletal muscle are presented and compared to the same of human skeletal muscles in primary peripheral leg deformities (congenital clubfeet) and secondary deformities caused by the malfunction of the central nervous system (cerebral palsy). To our best knowledge no calorimetric analysis of either the healthy or pathologic human skeletal muscles have been reported previously. Eleven muscle samples from the three groups of patients were analyzed and compared. We hope to add to the understanding of the primary and consecutive functional behavioral changes of the human skeletal muscle due to different etiologic reasons. It has been found that the human muscles have different DSC scans than the rabbit skeletal ones and they are very characteristic for the actual functional and structural state, they behave as a fingerprint. To find the precise biochemical and structural explanation of these alterations there is need for further detailed examinations.

Differential scanning calorimetry study of glycerinated rabbit psoas muscle fibres in intermediate state of ATP hydrolysis

BackgroundThermal denaturation experiments were extended to study the thermal behaviour of the main motor proteins (actin and myosin) in their native environment in striated muscle fibres. The interaction of actin with myosin in the highly organized muscle structure is affected by internal forces; therefore their altered conformation and interaction may differ from those obtained in solution. The energetics of long functioning intermediate states of ATP hydrolysis cycle was studied in muscle fibres by differential scanning calorimetry (DSC).ResultsSETARAM Micro DSC-II was used to monitor the thermal denaturation of the fibre system in rigor and in the presence of nucleotide and nucleotide analogues. The AM.ADP.Pi state of the ATP hydrolysis cycle has a very short lifetime therefore, we mimicked the different intermediate states with AMP.PNP and/or inorganic phosphate analogues Vi and AlF4 or BeFx. Studying glycerol-extracted muscle fibres from the rabbit psoas muscle by DSC, three characteristic thermal transitions were detected in rigor. The thermal transitions can be assigned to myosin heads, myosin rods and actin with transition temperatures (Tm) of 52.9 ± 0.7°C, 57.9 ± 0.7°C, 63.7 ± 1.0°C. In different intermediate states of the ATP hydrolysis mimicked by nucleotide analogues a fourth thermal transition was also detected which is very likely connected with nucleotide binding domain of myosin and/or actin filaments. This transition temperature Tm4 depended on the mimicked intermediate states, and varied in the range of 66°C – 77°C.ConclusionAccording to DSC measurements, strongly and weakly binding states of myosin to actin were significantly different. In the presence of ADP only a moderate change of the DSC pattern was detected in comparison with rigor, whereas in ADP.Pi state trapped by Vi, AlF4 or BeFx a remarkable stabilization was detected on the myosin head and actin filament which is reflected in a 3.0 – 10.0°C shift in Tm to higher temperature. A similar effect was observed in the case of the nonhydrolyzable AMP.PNP analogue. Differential DSC measurements suggest that stabilization actin structure in the intermediate states of ATP hydrolysis may play an additional role in actin-myosin interaction.

Examination of the cyclophosphamide-induced polyneuropathy on guinea pig sciatic nerve and gastrocnemius muscle with differential scanning calorimetry

Polyneuropathy is defined as a simultaneous malfunction of several peripheral nerves, which could be a side effect of cancer therapy; however, this is reported to occur rarely and difficult to prove. The purpose of the study was to introduce for the first time the calorimetry in the diagnosis of neuropathy in an experimental animal model. The study was inspired by the forensic investigation of a 53-year-old cancer survival female patient, in whose case the development of polyneuropathy could have been caused by cyclophosphamide therapy. Adult guinea pigs were injected intraperitoneally with the dose of cyclophosphamide that comparable to the human dosage. Animals were euthanized; nerve and muscle samples were analyzed by a SETARAM Micro calorimeter. The denaturation temperatures were measured and the calorimetric enthalpies were calculated based on the areas under thermal absorption curves. The thermal denaturation of the samples decreased and the calorimetric enthalpy increased, depending on the therapeutic cyclophosphamide doses. The nerves were more sensitive to chemotherapy, compared to the muscles. The toxic effects of cyclophosphamide on peripheral nerves and muscles can be measured and analyzed by calorimetry, which effects were found dose dependent.

Nucleotide dependent differences between the α-skeletal and α-cardiac actin isoforms

The thermodynamic properties of the actin filaments prepared from cardiomyocytes were investigated with differential scanning calorimetry. This method could distinguish between the alpha-cardiac and alpha-skeletal components of the actin filaments polymerised from ADP-actin monomers by their different melting temperatures (T(m)). Similar separation was not possible with filaments polymerised from ATP-actin monomers. Further analyses revealed that the activation energy (E(act)) was greater for filaments of alpha-skeletal actin than for alpha-cardiac actin monomers when the filaments were polymerised from ADP-actin monomers. These results showed that the alpha-cardiac actin filaments were thermodynamically less stable than the filaments of alpha-skeletal actin and their difference was nucleotide dependent. Based on these results and considering previous observations it was concluded that the existence of two actin isoforms and their nucleotide dependent conformational differences are part of the tuning regulatory mechanism by which the cardiac muscle cells can maintain their biological function under pathological conditions.

MELTING PROPERTIES OF BUTTER FAT AND THE CONSISTENCY OF BUTTER Effect of modification of cream ripening and fatty acid composition

The cold unspreadable consistency of butter after taking it out of the refrigerator is a rightful objection on behalf of consumers. The possibilities to improve the cold spreadability of butter are: the enrichment with low melting point triglycerides and the application of a good cream-ripening method. In our investigations milk fat fractions of different low melting points and plant oils of low melting points obtained by cold pressing and extraction have been used to change the original fatty acid composition of milk fat. The cream-ripening, the traditional method and the heat-step ripening method, which seemed to be the most effective to our earlier research, have been applied. The consistency of butter was examined by penetration measurements and its thermal characteristics by differential scanning calorimetric (DSC) method. The cold unspreadable consistency of butter can only be improved by the combination of the heat-step cream ripening and enrichment with low melting point triglycerides to get stable consistency at room temperature. The milk fat fraction of melting point below 5°C made the spreadability better but the spreadable consistency of margarine still cannot be attained. Plant oils with melting point below 0°C improved the cold spreadability of butter to a significantly higher degree than the former did. In the case of the same melting point the plant oil obtained by a cold method (pressing) was more effective. There is a close relationship between the consistency of butter and its product characteristics. From DSC curves the cold spreadability and room temperature stability of butter can be directly concluded.

DSC and EPR study on AMP.PNP, BeFx and AlF4 containing myosin nucleotide complexes

Differential scanning calorimetry and electron paramagnetic resonance experiments were performed on glycerinated skeletal muscle fibres to study the effect of the binding of nucleotides and nucleotide analogues to myosin. The thermal unfolding of muscle fibres in rigor showed three discrete domain regions with thermal stability of 52.2, 58.8 and 67.8°C. AMP.PNP and ATP plus AlF3 or BeF2 affected markedly the transitions, which implies the strong interaction between AMP.PNP or nucleotide analogues and catalytic domain of myosin, and a partial dissociation of heads from actin. ADP.BeFx and states model the transition states of the ATP hydrolysis cycle which precede the powerstroke of the muscle fibres. Spectrum deconvolution on isothiocyanate-labelled fibres in AMP.PNP-state resulted in two populations; 50% of labels was highly ordered with respect to fibre axis, whereas the other 50% of labels was randomly oriented. The myosin heads which showed high degree of order were in the strongly binding ADP-state. The spectra in - and ADP.BeFx state reflected random orientation of labels with increased rotational mobility in comparison with rigor. The results suggest that myosin in muscle fibres in ADP.BeFx state exists in two forms.