Interaction of serum amyloid A with human cystatin C—assessment of amino acid residues crucial for hCC–SAA formation (part II)

Secondary amyloid A (AA) amyloidosis is an important complication of some chronic inflammatory diseases, primarily rheumatoid arthritis (RA). It is a serious, potentially life‐threatening disorder caused by the deposition of AA fibrils, which are derived from the circulatory, acute‐phase‐reactant, serum amyloid A protein (SAA). Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been proved. Using a combination of selective proteolytic excision and high‐resolution mass spectrometry, the binding sites in the SAA and hCC sequences were assessed as SAA(86–104) and hCC(96–102), respectively. Here, we report further details concerning the hCC–SAA interaction. With the use of affinity tests and florescent ELISA‐like assays, the amino acid residues crucial for the protein interaction were determined. It was shown that all amino acid residues in the SAA sequence, essential for the formation of the protein complex, are basic ones, which suggests an electrostatic interaction character. The idea is corroborated by the fact that the most important residues in the hCC sequence are Ser‐98 and Tyr‐102; these residues are able to form hydrogen bonds via their hydroxyl groups. The molecular details of hCC–SAA complex formation might be helpful for the design of new compounds modulating the biological role of both proteins. Copyright © 2013 John Wiley & Sons, Ltd.     

Temporal and Pharmacological Characterization of Angiostatin Release and Generation by Human Platelets: Implications for Endothelial Cell Migration

Platelets play an important role in thrombosis and in neo-vascularisation as they release and produce factors that both promote and suppress angiogenesis. Amongst these factors is the angiogenesis inhibitor angiostatin, which is released during thrombus formation. The impact of anti-thrombotic agents and the kinetics of platelet angiostatin release are unknown. Hence, our objectives were to characterize platelet angiostatin release temporally and pharmacologically and to determine how angiostatin release influences endothelial cell migration, an early stage of angiogenesis. We hypothesized anti-platelet agents would suppress angiostatin release but not generation by platelets. Human platelets were aggregated and temporal angiostatin release was compared to vascular endothelial growth factor (VEGF). Immuno-gold electron microscopy and immunofluorescence microscopy identified α-granules as storage organelles of platelet angiostatin. Acetylsalicylic acid, MRS2395, GPIIb/IIIa blocking peptide, and aprotinin were used to characterize platelet angiostatin release and generation. An endothelial cell migration assay was performed under hypoxic conditions to determine the effects of pharmacological platelet and angiostatin inhibition. Compared to VEGF, angiostatin generation and release from α-granules occurred later temporally during platelet aggregation. Consequently, collagen-activated platelet releasates stimulated endothelial cell migration more potently than maximally-aggregated platelets. Platelet inhibitors prostacyclin, S-nitroso-glutathione, acetylsalicylic acid, and GPIIb/IIIa blocking peptide, but not a P2Y12 inhibitor, suppressed angiostatin release but not generation. Suppression of angiostatin generation in the presence of acetylsalicylic acid enhanced platelet-stimulated endothelial migration. Hence, the temporal and pharmacological modulation of platelet angiostatin release may have significant consequences for neo-vascularization following thrombus formation.     

Heterogeneity of neural crest-derived melanocytes

The majority of melanocytes originate from the neural crest cells (NCC) that migrate, spread on the whole embryo’s body to form elements of the nervous system and skeleton, endocrinal glands, muscles and melanocytes. Human melanocytes differentiate mainly from the cranial and trunk NCC. Although melanocyte development has traditionally been associated with the dorsally migrating trunk NCC, there is evidence that a part of melanocytes arise from cells migrating ventrally. The ventral NCC differentiate into neurons and glia of the ganglia or Schwann cells. It has been suggested that the precursors for Schwann cells differentiate into melanocytes. As melanoblasts travel through the dermis, they multiply, follow the process of differentiation and invade the forming human fetal epidermis up to third month. After birth, melanocytes lose the ability to proliferate, except the hair melanocytes that renew during the hair cycle. The localization of neural crest-derived melanocytes in non-cutaneous places e.g. eye (the choroid and stroma of the iris and the ciliary body), ear (cells of the vestibular organ, cochlear stria vascularis), meninges of the brain, heart seems to indicate that repertoire of melanocyte functions is much wider than we expected e.g. the protection of tissues from potentially harmful factors (e.g. free radicals, binding toxins), storage ions, and anti-inflammatory action.     

Fourier transform infrared spectroscopic signature of blood plasma in the progression of breast cancer with simultaneous metastasis to lungs

Despite advanced diagnostic techniques used for detecting cancer, this disease still remains a leading cause of death in the developed world. What is more, the greatest danger for patients is not related with growing of tumor but rather with metastasis of cancer cells to the distant organs. In this study, Fourier transform infrared (FTIR) spectroscopy was used to track chemical changes in blood plasma to find spectral markers of metastatic breast cancer during the disease progression. Plasma samples were taken 1‐5 weeks after orthotropic inoculation of 4T1 metastatic breast cancer cells to mice. The earliest changes detected by FTIR spectroscopy in plasma were correlated with unsaturation of phospholipids and secondary structures of proteins that appeared 2 and 3 weeks, respectively, after 4T1 cells inoculation (micrometastatic phase). Significant alternations in the content and structure of lipids and carbohydrates were identified in plasma at the later stages (macrometastatic phase). When large primary tumors in breast and macrometastases in lung were developed, all bands in FTIR spectra significantly differed from those at earlier phases of the cancer progression. In conclusion, we showed that each phase of the breast cancer progression and its pulmonary metastasis can be characterized by a specific panel of spectral markers.     

Moderate severity heart failure does not involve a downregulation of myocardial fatty acid oxidation

Recent human and animal studies have demonstrated that in severe end-stage heart failure (HF), the cardiac muscle switches to a more fetal metabolic phenotype, characterized by downregulation of free fatty acid (FFA) oxidation and an enhancement of glucose oxidation. The goal of this study was to examine myocardial substrate metabolism in a model of moderate coronary microembolization-induced HF. We hypothesized that during well-compensated HF, FFA oxidation would predominate as opposed to a more fetal metabolic phenotype of greater glucose oxidation. Cardiac substrate uptake and oxidation were measured in normal dogs (n = 8) and in dogs with microembolization-induced HF (n = 18, ejection fraction = 28%) by infusing three isotopic tracers ([9,10-(3)H]oleate, [U-(14)C]glucose, and [1-(13)C]lactate) in anesthetized open-chest animals. There were no differences in myocardial substrate metabolism between the two groups. The total activity of pyruvate dehydrogenase, the key enzyme regulating myocardial pyruvate oxidation (and hence glucose and lactate oxidation) was not affected by HF. We did not observe any difference in the activity of carnitine palmitoyl transferase I (CPT-I) and its sensitivity to inhibition by malonyl-CoA between groups; however, malonyl-CoA content was decreased by 22% with HF, suggesting less in vivo inhibition of CPT-I activity. The differences in malonyl-CoA content cannot be explained by changes in the Michaelis-Menten constant and maximal velocity for malonyl-CoA decarboxylase because neither were affected by HF. These results support the concept that there is no decrease in fatty acid oxidation during compensated HF and that the downregulation of fatty acid oxidation enzymes and the switch to carbohydrate oxidation observed in end-stage HF is only a late-stage phenomenon.     

Correlation of HLA-Cw*06 allele frequency with some clinical features of psoriasis vulgaris in the population of northern Poland

Psoriasis is a common skin disease with a genetic background and significant human leukocyte antigen (HLA) associations. HLA-Cw6 is the most frequently described association, particularly with psoriasis of the early onset type. Few studies of its correlation with various psoriasis clinical phenotypes and severity of the disease have been published so far; none in the Polish population. In this study 78 patients with psoriasis vulgaris were evaluated clinically and subdivided according to the age of onset and the type of psoriasis. A system of disease severity evaluation was applied to each patient. All patients and the control group (70 unrelated persons) were typed for HLA-Cw*06. The results show that Cw*06 allele frequency was higher in psoriatics than in the control group. Our investigation confirms that the Cw*06 allele is positively associated with psoriasis vulgaris of the early onset type, with a positive family history and its more severe form.