The histidine residues in pig and horse colipases

The single histidine of horse colipase B was shown to be at position 29, thus definitely proving that His₈₆ present in the pig cofactor is not conserved in the horse. Carbethoxylation of histidines did not appreciably affect the capacity of the porcine cofactor to bind to hydrophobic interfaces and to anchor lipase in presence of bile salts, but it induced in the aromatic region of the molecule a significant transconformation detectable by spectrofluorimetry.     

Comparative biochemical and biophysical studies on rat brain synaptosomes

A teichoic acid degrading enzyme (teichoicase) was purified to apparent homogeneity from a water-soluble cell extract of sporulating Bacillus subtilis cells. A rapid test for the detection of teichoicase activity was developed. The purified teichoicase has an app. Mr = 310 000. It consists of 4 identical subunits of Mr = 78 000 each.     

β-amyrin-induced apoptosis in Candida albicans triggered by calcium

The emergence of drug-resistant pathogens has urged researchers to discover alternatives for traditional antibiotics. β-amyrin, which is included in the category of triterpenoids extracted from plants, is known for its antimicrobial activity, although the underlying mechanism has not yet been revealed. This study was conducted to elucidate the antifungal mode of action of β-amyrin against Candida albicans. Based on the relevance between triterpenoids and oxidative molecules, reactive oxygen species (ROS) concentrations were detected, which showed a noticeable increment. Disruption of Ca²⁺ homeostasis in the cytosol was additionally analyzed, which was supported by interactions between two. Subsequently, decrease in mitochondrial membrane potential, increment of mitochondrial Ca²⁺, and ROS concentration were monitored, which suggested mitochondrial dysfunction modulated by Ca²⁺. Further investigation confirmed oxidative damage through glutathione reduction and DNA fragmentation. Accumulation of lethal damages resulted in the activation of caspases and externalization of phosphatidylserine, indicating the induction of yeast apoptosis by β-amyrin in C. albicans.     

Immunological and receptor-binding properties of fragments resulting from cyanogen bromide cleavage of bovine growth hormone

Fragments obtained from bovine growth hormone (somatotropin) by cyanogen bromide cleavage were isolated and identified. Their activities were investigated in a radioimmunoassay for bovine growth hormone and in a radioreceptor assay for growth hormone which uses membrane-associated receptors from the liver of a pregnant rabbit. At least one antigenic determinant and the receptor-binding site could be located in the sequence comprising residues 1-124/6-124 plus 150–179 (disulfide-linked), although they appeared not to be identical. An apparent increase in affinity compared with unfractionated cyanogen bromide-cleaved hormone was observed in both assays for the fraction containing these fragments. Neither intactness of methionyl residues nor that of the hormone appeared to be absolutely required for antibody-binding and receptor-binding activity (although other antigenic determinants may have been lost as a result of cleavage). However, the activities of the disulfide-linked fragment were low, indicating that conformational or other changes had modified the antibody-binding and receptor-binding sites.     

Metabolic cofactors NAD(P)H and FAD as potential indicators of cancer cell response to chemotherapy with paclitaxel

Paclitaxel, a widely used antimicrotubular agent, predominantly eliminates rapidly proliferating cancer cells, while slowly proliferating and quiescent cells can survive the treatment, which is one of the main reasons for tumor recurrence and non-responsiveness to the drug. To improve the efficacy of chemotherapy, biomarkers need to be developed to enable monitoring of tumor responses. In this study we considered the auto-fluorescent metabolic cofactors NAD(P)H and FAD as possible indicators of cancer cell response to therapy with paclitaxel. It was found that, among the tested parameters (the fluorescence intensity-based redox ratio FAD/NAD(P)H, and the fluorescence lifetimes of NAD(P)H and FAD), the fluorescence lifetime of NAD(P)H is the most sensitive in tracking the drug response, and is capable of indicating heterogeneous cellular responses both in cell monolayers and in multicellular tumor spheroids. We observed that metabolic reorganization to a more oxidative state preceded the morphological manifestation of cell death and developed faster in cells that were more responsive to the drug. Our results suggest that noninvasive, label-free monitoring of the drug-induced metabolic changes by noting the NAD(P)H fluorescence lifetime is a valuable approach to characterize the responses of cancer cells to anti-cancer treatments and, therefore, to predict the effectiveness of chemotherapy.     

Effect of long-term injection of high doses of potassium iodide on iodine metabolism in rat thyroid gland

Concentration of thyroid hormones in the serum of the rats after 14-day injections of potassium iodide (1, 3, 10, 100, and 500 physiological daily doses) did not differ from the control values. Excessive administration of potassium iodide increased the total iodide content in the rat thyroid tissue by 60–121% (35–108% and 94–128% for the protein-bound and free iodide, respectively), indicating the activation of the uptake and organification of iodide. The long-term injection of both low and high doses of potassium iodide increased the activity of catalase by 8–18% and SOD by 33–50% and enhanced the level of toxic LPO products reacting with thiobarbituric acid by 15–38%. It is suggested that reactive oxygen species and the excessive iodination of proteins (particularly thyroglobulin) induced by the long-term administration of high doses of potassium iodide can play an important role in the development of thyroid dysfunctions and autoimmune diseases.     

The antifungal plant defensin RsAFP2 from radish induces apoptosis in a metacaspase independent way in Candida albicans

We show that the antifungal plant defensin Raphanus sativus antifungal protein 2 (RsAFP2) from radish induces apoptosis and concomitantly triggers activation of caspases or caspase-like proteases in the human pathogen Candida albicans. Furthermore, we demonstrate that deletion of C. albicans metacaspase 1, encoding the only reported (putative) caspase in C. albicans, significantly affects caspase activation by the apoptotic stimulus acetic acid, but not by RsAFP2. To our knowledge, this is the first report on the induction of apoptosis with concomitant caspase activation by a defensin in this pathogen. Moreover, our data point to the existence of at least two different types of caspases or caspase-like proteases in C. albicans.     

Determination of iodide amounts in urine and water by isotope dilution analysis

Urinary iodide and iodine in drinking water were determined in 318 healthy children aged 0 to 18 yr living in Izmir and environmental rural and urban areas in the western part of Turkey. The method is based on substochiometric isotope dilution analysis. Iodide was precipitated by substoichiometric amounts of AgNO₃. Iodide-131 was used as a tracer. Electrophoresis was performed to separate Ag¹³¹I from excess¹³¹I-. The Ag¹³¹I zone was cut off the electrophoresis paper and counted with a Nal(Tl) scintillation counter. Count rates were plotted versus added KI concentrations. The unknown iodide amount was found by using these linear plots. Iodide concentration ranges were within 1.8 –100.45 Μg/L in the analyzed drinking water samples. The mean value was 44.14 ±17.33 Μg/L and the median was 58.08 Μg/L. Urinary iodide concentration ranges were 0.22 –142.22 Μg/L. The median of the distribution was 37.71 Μg/L and the mean was 40.30 ±24.05 Μg/L. The results show that the examined area suffers moderate iodine deficiency.     

Mutation in dystrophin-encoding gene affects energy metabolism in mouse myoblasts

Duchenne Muscular Dystrophy is characterized by severe defects in differentiated muscle fibers, including abnormal calcium homeostasis and impaired cellular energy metabolism. Here we demonstrate that myoblasts derived from dystrophic (mdx) mouse exhibit reduced oxygen consumption, increased mitochondrial membrane potential, enhanced reactive oxygen species formation, stimulated glycolysis but unaffected total cellular ATP content. Moreover, reduced amounts of specific subunits of the mitochondrial respiratory complexes and ATP-synthase as well as disorganized mitochondrial network were observed. Both the dystrophic and control myoblasts used were derived from a common inbred mouse strain and the only difference between them is a point mutation in the dystrophin-encoding gene, thus these data indicate that this mutation results in multiple phenotypic alterations demonstrating as early as in undifferentiated myoblasts. This finding sheds new light on the molecular mechanisms of Duchenne Muscular Dystrophy pathogenesis.     

Role of α-helical conformation of histatin-5 in candidacidal activity examined by proline variants

Human salivary histatin-5 (Hsn-5) is a potent in vitro anticandidal agent. The aim of this study was to investigate the importance of α-helical structure of Hsn-5 for its candidacidal activity. The following three Hsn-5 variants, where one or more functionally nonessential residues were replaced with proline (potent α-helix breaker), were produced by Escherichia coli expression system: H21P (1P), H19P/H21P (2P), and E16P/H19P/H21P (3P). The activities of purified proteins were determined by candidacidal assays, and the secondary structures by circular dichroism (CD) spectroscopy in trifluoroethanol (TFE) that is considered the helix-promoting solvent, and lysophosphatidyl-glycerol (LPG) micelles, the environment that more closely resembles the biological membranes. Our results indicated that 3P variant displayed a candidacidal activity which was similar to that of unaltered Hsn-5 (0P), while 1P and 2P variants showed lower cidal activity. The CD spectra in TFE indicated that 3P variant has less helical characteristics than the 0P, 1P and 2P. These results suggested that the α-helical content of Hsn-5 proline variants does not correlate with the candidacidal activity. Further, the CD spectral analysis of peptides in LPG micelles indicated the formation of β-turn structures in 0P and 3P variants. In conclusion, 3P variant which exhibited comparable candidacidal activity to 0P contains lower percentage of α-helical structure than 1P and 2P variants, which exhibited lower candidacidal activity. This suggests α-helix may not be important for anticandidal activity of Hsn-5.