Critical role of the C-terminal domains of factor H in regulating complement activation at cell surfaces

The plasma protein factor H primarily controls the activation of the alternative pathway of complement. The C-terminal of factor H is known to be involved in protection of host cells from complement attack. In the present study, we show that domains 19-20 alone are capable of discriminating between host-like and complement-activating cells. Furthermore, although factor H possesses three binding sites for C3b, binding to cell-bound C3b can be almost completely inhibited by the single site located in domains 19-20. All of the regulatory activities of factor H are expressed by the N-terminal four domains, but these activities toward cell-bound C3b are inhibited by isolated recombinant domains 19-20 (rH 19-20). Direct competition with the N-terminal site is unlikely to explain this because regulation of fluid phase C3b is unaffected by domains 19-20. Finally, we show that addition of isolated rH 19-20 to normal human serum leads to aggressive complement-mediated lysis of normally nonactivating sheep erythrocytes and moderate lysis of human erythrocytes, which possess membrane-bound regulators of complement. Taken together, the results highlight the importance of the cell surface protective functions exhibited by factor H compared with other complement regulatory proteins. The results may also explain why atypical hemolytic uremic syndrome patients with mutations affecting domains 19-20 can maintain complement homeostasis in plasma while their complement system attacks erythrocytes, platelets, endothelial cells, and kidney tissue.     

Photodynamic activity of a new sensitizer derived from porphyrin-C60 dyad and its biological consequences in a human carcinoma cell line

The photokilling activity of a porphyrin-C₆₀ (P-C₆₀) dyad was evaluated on a Hep-2 human larynx-carcinoma cell line. This study represents the first evaluation of a dyad, with high capacity to form a photoinduced charge-separated state, to act as agent to inactivate cells by photodynamic therapy (PDT). Cell treatment was carried out with 1 μM P-C₆₀ incorporated into liposomal vesicles. No dark cytotoxicity was observed using 1 μM P-C₆₀ concentration and during long incubation time (24 h). The uptake of sensitizer into Hep-2 was studied at different times of incubation. Under these conditions, a value of 1.5 nmol/10⁶ cells was found after 4 h of incubation showing practically no change even after 24 h. The cell survival after irradiation of the cells with visible light was dependent upon light exposure level. A high photocytotoxic effect was observed for P-C₆₀, which inactivated 80% of the cells after 54 J/cm² of irradiation. Moreover, the dyad kept a high photoactivity even under argon atmosphere. Thus, depending on the microenviroment where the sensitizer is localized, this compound could produce a biological photodamage through either a ¹O₂-mediated photoreaction process or a free radical mechanism under low oxygen concentration.

The mechanism of cell death was analyzed by Hoechst-33258, toluidine blue staining, TUNEL and DNA fragmentation. Cell cultures treated for 24 h with P-C₆₀ and irradiated with a dose of 54 J/cm² showed a great amount of apoptotic cells (58%). Moreover, changes in cell morphology were analyzed using fluorescence microscopy with Hoechst-33258 under low oxygen concentration. Under this anaerobic condition, necrotic cellular death predominated on apoptotic pathway. There were more apoptotic cells under air irradiation condition than under argon irradiation condition. To determine the apoptotic pathway, caspase-3 activation was studied by caspase-3 activity detection kits. The last results showed that P-C₆₀ induced apoptosis by caspase-3-dependent pathway. These results indicated that molecular dyad, which can form a photoinduced charge-separated state, is a promising model for phototherapeutic agents and they have potential application in cell inactivation by PDT.     

CpG islands are the second main factor shaping codon usage in human genes

A correspondence analysis of codon usage in human genes revealed, as expected, that the first axis is strongly correlated with the base composition at synonymous third codon positions. At one extreme of the second axis were localized genes with a high frequency of NCG and CGN codons. The great majority of these sequences were embedded in CpG islands, while the opposite is true for the genes placed at the other extreme. The two main conclusions of this paper are: (1) the influence of CpG islands on codon usage, and (2) since the second axis is orthogonal (and therefore independent) of the first, GC3-rich genes are not necessarily associated with CpG islands.     

Complicaciones de la otitis media con parálisis del sexto par craneal contralateral en pediatría

Otitis media is a frequent infection during childhood. Complications may be present in up to 4 of 100 children including serious neurological complications, particularly in developing countries.We report the case of a 9-year-old girl with no disease history who presented with otitis media, otorrhea, intracranial hypertension syndrome, and paralysis of the VI cranial nerve contralateral to the lesion. A computed tomography scan of the skull and a brain magnetic resonance imaging revealed chronic otomastoiditis, petrous apicitis, and thrombosis of the transverse and sigmoid sinus, the jugular bulb, and the right internal jugular vein. She received antibiotics and surgical treatment.This case shows the spectrum of intra and extracranial complications associated with acute otitis media in the antibiotic era. The physical examination allows early identification of intracranial hypertension with signs such as papilledema and sixth contralateral nerve palsy as an unusual finding.     

Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in pathogenic fungi Magnaporthe grisea and Botryotinia fuckeliana

There is increasing evidence that in several fungi, rhamnose-containing glycans are involved in processes that affect host-pathogen interactions, including adhesion, recognition, virulence, and biofilm formation. Nevertheless, little is known about the pathways for the synthesis of these glycans. We show that rhamnose is present in glycans isolated from the rice pathogen Magnaporthe grisea and from the plant pathogen Botryotinia fuckeliana. We also provide evidence that these fungi produce UDP-rhamnose. This is in contrast to bacteria where dTDP-rhamnose is the activated form of this sugar. In bacteria, formation of dTDP-rhamnose requires three enzymes. Here, we demonstrate that in fungi only two genes are required for UDP-Rha synthesis. The first gene encodes a UDP-glucose-4,6-dehydratase that converts UDP-glucose to UDP-4-keto-6-deoxyglucose. The product was shown by time-resolved (1)H NMR spectroscopy to exist in solution predominantly as a hydrated form along with minor amounts of a keto form. The second gene encodes a bifunctional UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase that converts UDP-4-keto-6-deoxyglucose to UDP-rhamnose. Sugar composition analysis and gene expression studies at different stages of growth indicate that the synthesis of rhamnose-containing glycans is under tissue-specific regulation. Together, our results provide new insight into the formation of rhamnose-containing glycans during the fungal life cycle. The role of these glycans in the interactions between fungal pathogens and their hosts is discussed. Knowledge of the metabolic pathways involved in the formation of rhamnose-containing glycans may facilitate the development of drugs to combat fungal diseases in humans, as to the best of our knowledge mammals do not make these types of glycans.