A first evaluation of the usefulness of Kudzu starch in cultural heritage restoration.

In recent times, the use of natural and harmless products for the environment and restorer is taking place in the field of Cultural Heritage restoration. In this sense, wheat, rice and corn starches as adhesives, have suitable characteristics without toxicity risks. A new starch in this field, is the Kudzu, an almost pure compound (99.5% starch) that is processed by a natural way from a plant called Pueraria lobata. This is a preliminary study of the potential use of Kudzu starch for the restoration of Cultural Heritage, focusing, firstly, in its capacity as adhesive through a comparative evaluation with common starches. The accelerated aging process carried out proved that Kudzu ensures optimal chromatic behaviour. On the other hand, the main problem in starch paste is the biological colonization. The daidzein, a natural antimicrobial compound implicit in Kudzu starch, confirmed the resistance to microorganism in this preliminary approach. The evaluation of the adhesive capacity, and the reversibility of the starches, suggest that Kudzu starch is a valid adhesive in the field of paper restoration. Thus, the potential of this starch in the conservation of Cultural Heritage is evidenced and its use as cleaner, resistance to biological colonization and consolidant is promising.

Melanin and pyomelanin in Aspergillus fumigatus: from its genetics to host interaction.

Aspergillus fumigatus is a worldwide-distributed saprophytic fungus and the major cause of invasive aspergillosis. This fungus can produce two types of melanin-dihydroxynaphthalene melanin (DHN-melanin) and pyomelanin. These pigments are considered important resistance mechanisms to stress, as well as virulence factors. The aim of this review is to present the current knowledge of the genetic basis and metabolic pathways of melanin production, their activation, function, and interaction with the host immune system. The DHN-melanin pathway is encoded in a cluster that includes six genes (abr1, abr2, ayg1, arp1, arp2, and pksP/alb1 genes) whose encoded proteins seem to be the origin of the pigment in endosomes. These vesicles are secreted and the pigment is subsequently located in the wall of the conidium beneath the rodlet layer. Unlike DHN-melanin, pyomelanin does not have its own biosynthetic pathway but is related to the activation of the L-tyrosine/L-phenylalanine degradation pathway that includes a cluster of six genes (hppD, hmgX, hmgA, fahA, maiA, and hmgR). Its production is due to the polymerization of homogentisic acid and is linked to conidial germination. Despite the knowledge gained in recent years, further studies will be necessary to confirm the pathways that produce these pigments and their role in the virulence mechanisms of A. fumigatus.

Rapid and specific detection of section Fumigati and Aspergillus fumigatus in human samples using a new multiplex real-time PCR.

Invasive aspergillosis is an opportunistic infection caused primarily by Aspergillus fumigatus. However, other common fungal pathogens belonging to section Fumigati are often misidentified as A. fumigatus. Thus, we have developed a multiplex real-time PCR (qPCR) assay with primers and specific TaqMan probes based on internal transcribed spacer regions or benA gene to discriminate, in less than 3 h, species of section Fumigati and, specifically, A. fumigatus. The multiplex qPCR showed a limit of detection of 20 and 50 fg of DNA for section Fumigati and A. fumigatus, respectively. Moreover, it enabled detection of a single germinated conidia. The inclusion of some PCR facilitators together with the dilution of samples makes it possible to completely avoid PCR inhibitions in all bronchoalveolar lavage (BAL) samples assayed. This technique may be a useful complementary tool in the diagnosis of invasive pulmonary aspergillosis caused by A. fumigatus using BAL fluid.

Characterization of a monoclonal antibody directed against Salmonella enterica serovar Typhimurium and serovar [4,5,12:i:-].

Flagellar extracts of Salmonella enterica serovars expressing phase 2 H1 antigenic complex (H:1,2, H:1,5, H:1,6, and H:1,7) and a mutant flagellin obtained by site-directed mutagenesis of the fljB gene from serovar Typhimurium at codon 218, transforming threonine to alanine, expressed in Escherichia coli (fljB218(A)) were used to analyze the H1 antigenic complex. Cross-reactions were detected by Western blotting and dot blotting using commercial polyclonal antibodies against the different wild-type extracts and mutant FljB218(A). Therefore, we produced a monoclonal antibody (MAb), 23D4, isotyped as immunoglobulin M, against H:1,2 S. enterica serovar Typhimurium flagellin. The mutant flagellin was not recognized by this MAb. When a large number of phase 1 and phase 2 flagellin antigens of different serovars were used to characterize the 23D4 MAb, only extracts of serovars Typhimurium and [4,5,12:i:-] reacted. The protein composition of phase 1 and phase 2 extracts and highly purified H:1,2 flagellin from serovar Typhimurium strain LT2 and extract of strain 286 (serovar [4,5,12:i:-]), which reacted with the MAb, was studied. Phase 2 flagellin (FljB(H:1,2)) was detected in phase 1 and phase 2 flagellar heat extracts of serovar Typhimurium and was the single protein identified in all spots of purified H:1,2 flagellin. FliC, FlgK, and other proteins were detected in some immunoreactive spots and in the flagellar extract of serovar [4,5,12:i:-]. Immunoelectron microscopy of complete bacteria with 23D4 showed MAb attachment at the base of flagella, although the MAb failed to recognize the filament of flagella. Nevertheless, the results obtained by the other immunological tests (enzyme-linked immunosorbent assay, Western blotting, and dot blotting) indicate a reaction against flagellins. The epitopes could also be shared by other proteins on spots where FljB is not present, such as aminopeptidase B, isocitrate lyase, InvE, EF-TuA, enolase, DnaK, and others. In conclusion, MAb 23D4 can be useful for detection and diagnostic purposes of S. enterica serovar Typhimurium and serovar [4,5,12:i:-] and could be also helpful for epitope characterization of flagellum-associated antigens.

Yeast associated with spontaneous fermentations of white wines from the "Txakoli de Bizkaia" region (Basque Country, North Spain).

The microbiota of eight spontaneous fermentation of white wine from different grape varieties and different wineries from the "Txakoli de Bizkaia" region (Basque country, North Spain), in 1996 and 1997 campaigns was studied. The yeast population was higher in grapes harvested in 1997, in which late summer and early autumn was warmer and drier. Eight species belonging to five genera were identified in total. The most frequent genera in grapes were Rhodotorula in 1996 and Kloeckera in 1997. Saccharomyces bayanus was the most frequent species during vigorous and final fermentation, and it was occasionally isolated from grapes and must. Only another Saccharomyces spp., i.e., S. kluyvery, was identified in some samples from 1997.

Differences in the Candida albicans antigenic expression after heat shock and infection.

Heat-shock and infection induce changes in protein expression in C. albicans. To investigate if these alterations induce changes in antigenicity, we have compared the reactivity mediated by IgA antibodies of protein extracts from a strain of C. albicans and the same strain recovered from an infected animal, both at 24 degrees C and 37 degrees C. The antigenic variability was detected mainly in antigens recognized by salivary IgA. Antigens of 223, 205, 180 and 140 kDa were over-expressed in both strains at 37 degrees C, indicating that variations due to heat shock were present before and after infection. The antigens were characterized as mannoproteins located at the outer side of the cell wall. An antigen of 61 kDa was also detected in which the expression decreased significantly after infection This was independent of heat shock.

Characterization of Candida albicans antigenic determinants by two-dimensional polyacrylamide gel electrophoresis and enhanced chemiluminescence.

The use of a two-dimensional polyacrylamide gel electrophoresis joined with Western blotting allowed us to investigate the reactivities of antibodies present in sera from mice and humans to antigens of Candida albicans blastoconidia. The analysis of the antibody response in the two models studied and the comparison between the antibody response in infected and noninfected individuals showed that the infection by C. albicans produces changes in the antibody response which may be of relevance in the serodiagnosis of invasive candidiasis. These changes include the induction of antibodies against new antigens, the disappearance of antibodies against a group of antigens and variations in the reactivity of antibodies directed to a different group of antigens. The technique used resolved the isoforms of several antigens including enolase. It is concluded that the antibody response in humans and mice with candidiasis is not homogeneously directed to all the isoforms of an antigen.

Re-expression by Candida albicans germ tubes of antigens lost during subculture of blastospores.

The effect of germ tube induction on the antigenic variability in C.albicans was studied in strains from blood cultures (Group I) and superficial candidiasis (Group II). When compared by immunoblotting with a rabbit antiserum, antigenic extracts from Group I strains grown as blastospores showed a higher reactivity than that of Group II strains. Major bands in Group I strains (45-47, 33, 30 kDa) were continuously expressed through the subcultures in vitro but, with the exception of the 45 kDa band, the reactivity of all of them decreased or disappeared after the tenth subculture in Group II strains. The induction of the germ tubes produced the re-expression of the antigens lost during subculture in the yeast form, the effect being very clear in Group II strains. The re-expression by C. albicans germ tubes of antigens lost during subculture of blastospores in vitro and the higher reactivity shown by Group I strains grown in mycelial phase should be taken into consideration when a test to detect anti-C. albicans antibodies is to be developed.

Candida albicans stress mannoproteins expression in superficial and systemic candidiasis. Stress mannoproteins in Candida albicans.

The presence of heat shock mannoproteins (HSMPs) reactive with sIgA was demonstrated in several C. albicans strains. The subculture of the C. albicans isolated from mucosal surfaces on Sabouraud's dextrose agar at 25 degrees C switched off the HSMP expression. A re-expression of the HSMPs was obtained in the same medium by shifting the temperature of incubation to 37 degrees C. However, expression of HSMPs in two strains isolated from deep infections was maintained during several subcultures on Sabouraud's dextrose agar at 25 degrees C. A glycoprotein of 200 kDa seemed to be the main HSMP reacting with vaginal sIgA. The data presented in this study suggest that factors other than temperature can influence the expression of C. albicans HSMPs and therefore these antigens should be referred as stress mannoproteins.

Heat-shock mannoproteins as targets of secretory IgA in Candida albicans.

The expression of Candida albicans antigenic determinants reacting with secretory IgA from patients with oral and vaginal candidiasis was investigated under different in vitro conditions. Reversible antigenic transitions were inducible in synthetic medium by temperature shifts, as the yeast cells were positive by an indirect immunofluorescence assay after being incubated at 37 degrees C but not at 25 degrees C. In vitro temperature-inducible C. albicans antigenic determinants reactive with secretory IgA were characterized by radioimmune Western blot as mannoproteins with molecular masses of 180-200, 130-150, 90-110, and 60-70 kDa. This is the first report on the expression of mannoproteins regulated by temperature involved in the secretory immune response during mucosal candidiasis.

Qualitative and quantitative differences in recognition patterns of Candida albicans protein and polysaccharide antigens by human sera.

Cytoplasmic and cell wall proteins and glycoproteins extracted from Candida albicans germ tubes were screened by Western blotting for their ability to differentiate between the serological responses of patients with candidosis and healthy individuals. Molecules of 114, 74 and 65 kDa were not recognized by any sera. Qualitative differences were observed for responses to proteins and glycoproteins from 29 to 60 kDa. Conversely, only quantitative differences were found to high molecular mass glycoproteins. Their recognition by control sera was invariably associated with reactivity against a 14-18 kDa antigen. However, despite a high level of antibodies against high molecular mass mannoproteins, some patients sera failed to react with the 14-18 kDa antigen, or lost this reactivity during the course of the disease.

Identification of Candida albicans cell wall antigens lost during subculture in synthetic media.

A high variability in reactivity was observed when Candida albicans strains freshly isolated from both patients with candidiasis and asymptomatic carriers were tested against different human sera. The highest reactivity was observed in C. albicans strains isolated from blood cultures. This high reactivity was observed when the isolates were tested against sera from patients with Candida oesophagitis, patients with vulvovaginal candidiasis, or asymptomatic carriers but not against sera from blood donors. The antigenic reactivity of the strongly reactive strains, but not that of the weakly reactive strains, decreased during subculture in synthetic media. Five major components of an apparent molecular mass of > 200, 67-70, 49-52, 33-35 and 29-31 kDa were observed in alpha-mannosidase extracts from C. albicans strains from both blood cultures (Group I) and patients with Candida oesophagitis (Group II) subcultured in synthetic media for different times. Changes in staining intensity through the different subcultures were observed for some bands. Group I strains showed a decrease in staining intensity for bands of > 200 and 67-70 kDa, an increase for bands of 33-35 and 29-31 kDa, but no changes were observed for the band of 49-52 kDa. Group II strains showed opposite changes in banding intensity. A decrease in staining intensity was observed for the proteins of 33-35 and 29-31 kDa, an increase for the protein of 49-52 kDa, and no change in intensity was observed for the band of 67-70 kDa. A component of > 200 kDa showed an irregular expression through the subcultures. The main antigen present in extracts from the first subculture of isolates from Group I and II had a molecular mass of 67-70 kDa. It could be related to the P antigens since it disappeared following subculture of the strains in synthetic media.