Magnetic particle hyperthermia: Néel relaxation in magnetic nanoparticles under circularly polarized field.

The mechanism of magnetization reversal in single-domain ferromagnetic particles is of interest in many applications, in most of which losses must be minimized. In cancer therapy by hyperthermia the opposite requirement prevails: the specific loss power should be maximized. Of the mechanisms of dissipation, here we study the effect of Néel relaxation on magnetic nanoparticles unable to move or rotate and compare the losses in linearly and circularly polarized fields. We present exact analytical solutions of the Landau-Lifshitz equation as derived from the Gilbert equation and use the calculated time-dependent magnetizations to find the energy loss per cycle. In frequencies lower than the Larmor frequency, linear polarization is found to be the better source of heat power, at high frequencies (beyond the Larmor frequency) circular polarization is preferable.

Yield improvement of heterologous peptides expressed in yps1-disrupted Saccharomyces cerevisiae strains.

Heterologous protein expression levels in Saccharomyces cerevisiae fermentations are highly dependent on the susceptibility to endogenous yeast proteases. Small peptides, such as glucagon and glucagon-like-peptides (GLP-1 and GLP-2), featuring an open structure are particularly accessible for proteolytic degradation during fermentation. Therefore, homogeneous products cannot be obtained. The most sensitive residues are found at basic amino acid residues in the peptide sequence. These heterologous peptides are degraded mainly by the YPS1-encoded aspartic protease, yapsin1, when produced in the yeast. In this article, distinct degradation products were analyzed by HPLC and mass spectrometry, and high yield of the heterologous peptide production has been achieved by the disruption of the YPS1 gene (previously called YAP3). By this technique, high yield continuous fermentation of glucagon in S. cerevisiae is now possible.

A removable spacer peptide in an alpha-factor-leader/insulin precursor fusion protein improves processing and concomitant yield of the insulin precursor in Saccharomyces cerevisiae.

An alpha-factor leader/insulin precursor fusion protein was produced in Saccharomyces cerevisiae and metabolically labeled in order to analyse the efficiency of maturation and secretion. A substantial fraction of the secreted material was found in a hyperglycosylated unprocessed form, indicating incomplete Kex2p endopeptidase maturation. Introduction of a spacer peptide (EAEAEAK) after the dibasic Kex2p site, creating a N-terminal extension of the insulin precursor, greatly increased the Kex2p catalytic efficiency and the fermentation yield of insulin precursor. The N-terminal extension features a Lys to allow subsequent proteolytic removal by trypsin or the Achromobacter lyticus Lys-specific protease. Dipeptidyl aminopeptidase A (DPAPA) activity removing Glu-Ala dipeptides from the extension was inhibited by adding a Glu N-terminally to the extension. Unexpectedly, this modified N-terminal extension (EEAEAEAK) was partially cleaved after the Lys during fermentation. This monobasic proteolytic activity was demonstrated to be associated with Yap3p. Yap3p cleavage could be prevented by insertion of a Pro before the Lys (EEAEAEAPK).

Soluble, fatty acid acylated insulins bind to albumin and show protracted action in pigs.

We have synthesized insulins acylated by fatty acids in the epsilon-amino group of LysB29. Soluble preparations can be made in the usual concentration of 600 nmol/ml (100 IU/ml) at neutral pH. The time for 50% disappearance after subcutaneous injection of the corresponding TyrA14(125I)-labelled insulins in pigs correlated with the affinity for binding to albumin (r = 0.97), suggesting that the mechanism of prolonged disappearance is binding to albumin in subcutis. Most protracted was LysB29-tetradecanoyl des-(B30) insulin. The time for 50% disappearance was 14.3 +/- 2.2 h, significantly longer than that of Neutral Protamine Hagedorn (NPH) insulin, 10.5 +/- 4.3 h (p < 0.001), and with less inter-pig variation (p < 0.001). Intravenous bolus injections of LysB29-tetradecanoyl des-(B30) human insulin showed a protracted blood glucose lowering effect compared to that of human insulin. The relative affinity of LysB29-tetradecanoyl des-(B30) insulin to the insulin receptor is 46%. In a 24-h glucose clamp study in pigs the total glucose consumptions for LysB29-tetradecanoyl des-(B30) insulin and NPH were not significantly different (p = 0.88), whereas the times when 50% of the total glucose had been infused were significantly different, 7.9 +/- 1.0 h and 6.2 +/- 1.3 h, respectively (p < 0.04). The glucose disposal curve caused by LysB29-tetradecanoyl des-(B30) insulin was more steady than that caused by NPH, without the pronounced peak at 3 h. Unlike the crystalline insulins, the soluble LysB29-tetradecanoyl des-(B30) insulin does not elicit invasion of macrophages at the site of injection. Thus, LysB29-tetradecanoyl des-(B30) insulin might be suitable for providing basal insulin in the treatment of diabetes mellitus.

A pathogen-induced gene of barley encodes a protein showing high similarity to a protein kinase regulator.

A full length cDNA of a barley leaf messenger, found to increase in amount during infection attempts by the powdery mildew fungus (Erysiphe graminis), is characterized. The messenger encodes a polypeptide of 261 amino acid residues with a calculated mass of 29.2 kDa and a pI of 4.6. Sequence comparisons as well as serological studies demonstrate that the encoded protein is closely related to a family of mammalian proteins believed to have functions associated with the multifunctional Ca(2+)-dependent protein kinases.

Induction, purification and characterization of chitinase isolated from pea leaves inoculated with Ascochyta pisi.

Chitinase (EC 3.2.1.14.) activity increased in pea (Pisum sativum L.) leaves inoculated with both virulent and avirulent isolates of Ascochyta pisi Lib. Three basic chitinase isoenzymes were purified: two, A1 and A2, separated by high performance liquid chroma tography, had a relative molecular mass (Mr) of approx. 34 000, with an isoelectric point (pI) of 8.5, and one, B, had an Mr of approx. 25 000, with a pI of 9.0. Elevated levels of chitinase isoenzymes were found in the leaves: thus 150 h after inoculation, there was a ninefold increase for isoenzymes A1 and A2 combined, with a threefold increase for isoenzyme B in susceptible plants, and a sevenfold increase for isoenzymes A1 and A2 combined, with a twofold increase for isoenzyme B in hypersensitive-reacting plants. The N-terminal 15-23 amino-acid residues of the three chitinase isoenzymes were sequenced, and considerable sequence similarity was found to chitinase sequences from tobacco, potato and bean, as well as to hevein and wheat-germ agglutinin. Purified chitinase protein cross-reacted with specific antiserum raised against a chitinase isoenzyme from sugar beet (Beta vulgaris L.). Immunoblots prepared using leaf proteins isolated at different time points following inoculation revealed the accumulation of polypeptides corresponding to at least two of the chitinase isoenzymes.