Prevalence of fungi in carpeted floor environment: analysis of dust samples from living-rooms,bedrooms, offices and school classrooms

The results of 100 carpet dust analyses from atopic individuals' environment were compared according to the sampling period or the location. Dust samples were collected with a standard domestic vacuum cleaner, in locations with carpeted floor: in residences (living-room and/or bedroom), in school classrooms and in offices. The quantities of fungi vary from 5000 CFU/g to 66 000 000 CFU/g of dust. More than 100 species were isolated by dilution plating. The main species found in carpet dust wereEurotium repens, Penicillium chrysogenum, Alternaria alternata, Aureobasidium pullulans andPhoma herbarum. Strict xerophilic species were rather rare and detected in small quantities. Differences in the distribution of the CFU concentrations were examined for the four different sampling locations and were statistically significant (P=0.0174). In this study, schools were open spaces, and offices, mostly with air conditioning systems, were locations in which air is not confined. This, added to frequent professional carpet cleaning, probably explains the lowest levels of fungal concentration found in these locations. The majority of the homes had the largest fungal concentration in the living-room (median: 2×10⁵ CFU/g) while some bedrooms (median: 7×10⁴ CFU/g) had the highest concentrations. It is suggested that, when fungi are suspected to be the origin of respiratory allergy or irritating symptoms, the mycoflora of the bedroom, principally, should be investigated first.     

Subcellular distribution of carbonic anhydrase in Solanum tuberosum L. leaves

The intracellular compartmentation of carbonic anhydrase (CA; EC 4.2.1.1), an enzyme that catalyses the reversible hydration of CO₂ to bicarbonate, has been investigated in potato (Solanum tuberosum L.) leaves. Although enzyme activity was mainly located in chloroplasts (87% of total cellular activity), significant activity (13%) was also found in the cytosol. The corresponding CA isoforms were purified either from chloroplasts or crude leaf extracts, respectively. The cytosolic isoenzyme has a molecular mass of 255 000 and is composed of eight identical subunits with an estimated M _r of 30000. The chloroplastic isoenzyme (M _r 220000) is also an octamer composed of two different subunits with M _r estimated at 27 000 and 27 500, respectively. The N-terminal amino acid sequences of both chloroplastic CA subunits demonstrated that they were identical except that the M _r-27 000 subunit was three amino acids shorter than that of the M _r-27 500 subunit. Cytosolic and chloroplastic CA isoenzymes were found to be similarly inhibited by monovalent anions (Cl^–, I^–, N ₃ ^- and NO ₃ ^- ) and by sulfonamides (ethoxyzolamide and acetozolamide). Both CA isoforms were found to be dependent on a reducing agent such as cysteine or dithiothreitol in order to retain the catalytic activity, but 2-mercaptoethanol was found to be a potent inhibitor. A polyclonal antibody directed against a synthetic peptide corresponding to the N-terminal amino acid sequence of the chloroplastic CA monomers also recognized the cytosolic CA isoform. This antibody was used for immunocytolocalization experiments which confirmed the intracellular compartmentation of CA: within chloroplasts, CA is restricted to the stroma and appears randomly distributed in the cytosol.Abbreviations BSA bovine serum albumin - CA carbonic anhydrase - PMSF phenylmethylsulphonyl fluoride - BAM benzamidine - DTT dithiothreitol - 2-ME 2-mercaptoethanol - PVDF polyvinylidene difluoride The authors thanks P. Carrier and Dr. B. Dimon for technical assistance with the mass-spectrometry measurements.     

Pre-inoculation of Ri T-DNA-transformed pea roots with Pseudomonas fluorescens inhibits colonization by Pythium ultimum Trow: an ultrastructural and cytochemical study

The influence exerted by Pseudomonas fluorescens, strain 63-28R, in stimulating plant defense reactions was investigated using an in-vitro system in which Ri T-DNA-transformed pea (Pisum sativum L.) roots were subsequently infected with Pythium ultimum. Cytological investigations of samples from P. fluorescens-inoculated roots revealed that the bacteria multiplied abundantly at the root surface and colonized a small number of epidermal and cortical cells. Penetration of the epidermis occurred through the openings made by the disruption of the fibrillar network at the junction of adjacent epidermal cell walls. Direct cell wall penetration was never observed and bacterial ingress into the root tissues proceeded via an intercellular route. Striking differences in the extent of fungal colonization were observed between bacterized and non-bacterized pea roots following inoculation with P. ultimum. In non-bacterized roots, the pathogen multiplied abundantly through most of the tissues while in bacterized roots, pathogen growth was restricted to the epidermis and the outer cortex. At the root surface, the bacteria interacted with the pathogen, in a way similar to that observed in dual culture tests. Most Pythium cells were severely damaged but fungal penetration by the bacteria was never observed. Droplets of the amorphous material formed upon interaction between the bacteria and the host root were frequently found at the fungal cell surface. Incubation of sections with a -1,4-exoglucanase-gold complex revealed that the cell wall of markedly altered Pythium hyphae was structurally preserved. Successful penetration of the root epidermis was achieved by the few hyphae of P. ultimum that could escape the first defensive line in the rhizosphere. Most hyphae of the pathogen that penetrated the epidermis exhibited considerable changes. The unusual occurrence of polymorphic wall appositions along the host epidermal cells was an indication that the host plant was signalled to defend itself through the elaboration of physical barriers.Abbreviations AGL Aplysia gonad lectin - PGPR plant growth-promoting rhizobacteria The authors wish to thank Sylvain Noël for excellent technical assistance. This study was supported by grants from the Fonds Québécois pour la formation de chercheurs et l'Aide à la Recherche (FCAR), the Natural Sciences and Engineering Council of Canada (NSERC) and the Ministère de l'Industrie, du Commerce, de la Science et de la Technologie (SYNERGIE).     

Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase.

Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the alpha-decarboxylation of L-glutamic acid to yield gamma-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-beta-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65 degrees C but not at 68 degrees C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.     

Effects of external stimuli on environmental bacterial strains harboring analgD-lux bioluminescent reporter plasmid for the study of corrosive biofilms

An alginic acid biosynthesis bioluminescent reporter plasmid, pUTK50, was transconjugated into environmental strains ofPseudomonas putida, Pseudomonas fluorescens, andStenotrophomonas maltophilia. Bioluminescent transconjugates were selected from each strain for investigation of environmental stress factors that promote alginic acid exopolymer biosynthesis in developing biofilms. Environmental stimuli associated with increased levels of alginate synthesis, in a previously developed organism,P. aeruginosa FRD1, were applied to the environmental strains. Increased salt concentrations and higher ratios of nitrate vs ammonium ions as the limiting nitrogen source induced bioluminescence in FRD1 and the environmental strains. However, for environmental strains ofP. putida, P. fluorescens andS. maltophilia, polysaccharides were detected with low uronic acids content and different structural components. When tested within a biofilm,S. maltophilia O46 demonstrated exceptional adhesive and corrosive properties while alginic acid synthesis was not high. In most of the environmental strains, periods of increased bioluminescence were induced by external stimuli, but exopolysaccharides other than alginic acid were expressed. It is hypothesized that the environmental strains have homologous but nonidentical promoter sequences which are responsive to certain environmental stimuli and may control genes necessary for the production of alternative exopolysaccharides.     

Effect of vertebrate predation on the spatio-temporal distribution of cladocerans in a temperature eutrophic lake

We analysed the spatio-temporal distribution of zooplankton along a profile of 10 stations from the shore to the pelagic zone from April to September 1988, the period when the larvae and juveniles Rutilus rutilus, the most abundant species in the Lake, are in the littoral zone. The digestive tracts of the young roach were analysed. They fed essentially on rotifers and on cladocerans. For comparison, zooplankton was also analysed at one littoral area without fish fry. There was an increase of cladoceran density from the vegetated nearshore zone to the offshore zone. Considering the density of Bosmina longirostris, Daphnia longispina, Chydorus sphaericus and Ceriodaphnia quadrangula, we observed a different distribution pattern in the course of the year. In the nearshore zone, the relative abundance of small species, Bosmina and Chydorus, was much higher than that of the larger Daphnia. From April to September, predation pressure mainly affected the smallest species: in contrast to the inshore station without fish fry, the density of Bosmina decreased in May in the littoral with fish. Chydorus was concentrated in the littoral between February and April, then grew into the pelagic zone, where predation pressure obviously was low during the warm season. The number of Daphnia, which was eaten by the fish fry at any time, remained low in the nearshore zone, which suggests that the presence of fish may cause Daphnia to avoid this zone. Ceriodaphnia which was not affected by this predation, was scarce in the nearshore zone during mid-summer. The low density of the cladocerans in the nearshore zone is likely associated with vertebrate predation by roach fry and juveniles, the result of such a process being either a depletion in density of the prey, or an avoidance behaviour.     

Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing.

Cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the hepatitis C virus polyprotein requires a viral serine protease activity residing in the N-terminal one-third of the NS3 protein. Sequence comparison of the residues flanking these cleavage sites reveals conserved features including an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. In this study, we used site-directed mutagenesis to assess the importance of these and other residues for NS3 protease-dependent cleavages. Substitutions at the P7 to P2' positions of the 4A/4B site had varied effects on cleavage efficiency. Only Arg at the P1 position or Pro at P1' substantially blocked processing at this site. Leu was tolerated at the P1 position, whereas five other substitutions allowed various degrees of cleavage. Substitutions with positively charged or other hydrophilic residues at the P7, P3, P2, and P2' positions did not reduce cleavage efficiency. Five substitutions examined at the P6 position allowed complete cleavage, demonstrating that an acidic residue at this position is not essential. Parallel results were obtained with substrates containing an active NS3 protease domain in cis or when the protease domain was supplied in trans. Selected substitutions blocking or inhibiting cleavage at the 4A/4B site were also examined at the 3/4A, 4B/5A, and 5A/5B sites. For a given substitution, a site-dependent gradient in the degree of inhibition was observed, with a 3/4A site being least sensitive to mutagenesis, followed by the 4A/4B, 4B/5A, and 5A/5B sites. In most cases, mutations abolishing cleavage at one site did not affect processing at the other serine protease-dependent sites. However, mutations at the 3/4A site which inhibited cleavage also interfered with processing at the 4B/5A site. Finally, during the course of these studies an additional NS3 protease-dependent cleavage site has been identified in the NS4B region.     

The last 140 ka in the Afro-Asian arid/semi-arid transitional zone

During the last ten years, a great number of geological or other proxy records provided radiocarbon, U/Th or TL dated information on the past climatic oscillations in the arid/semi-arid zones extending between the Atlantic and the Pacific. Comparisons and a synthetic view of these results can now be attempted for the last 140 ka, and compared with global changes, as registered in oceanic or ice cores, and with palaeomonsson models: for this purpose, wide spatial and time scales have to be used. On the whole, arid/humid alternations roughly fit global changes, cold phases corresponding to an extension of the arid areas to the South, warm phases corresponding to the shrinking of the same to the North.

The last interglacial is associated with an increase of precipitation throughout the area considered. Isotopic stage 4 brings no evidence whatsoever of humid conditions. Two wetter episodes are registrated during stage 3. A major rainfall decrease is everywhere associated with the Last Glacial Maximum (21-15 ka in most regions), the arid or semi-arid zones extending several hundred kilometers southwards, relative to the present-day pattern. The two abrupt deglaciation steps and the Younger Dryas are recorded in all of the most sensitive regions, at the margins of the present-day monsoonal range. During the Holocene, the precipitation increases everywhere (by 100–400 mn, relative to the present-day values), the optimum being at 8.5-6.5 ka. A climatic deterioration follows with an irregular pattern of dry/wet episodes, according to the different geographic conditions. The humid phase terminates at 3.5-3 ka in the whole transitional zone.

The major southward shift of the monsoonal precipitation range since its optimum, some 8000 years ago, fits the astronomical neoglacial trend and may possibly be correlated with past stage 5d, although its rapidity and spatial importance may just reflect one of the sharp successive cold/warm variations registered by GRIP during the whole stage 5.     

Polygalacturonase-inhibiting protein accumulates in Phaseolus vulgaris L. in response to wounding, elicitors and fungal infection

Polygalacturonase-inhibiting protein (PGIP) is a cell wall-associated protein that specifically binds to and inhibits the activity of fungal endopolygalacturonases. The Phaseolus vulgaris gene encoding PGIP has been cloned and characterized. Using a fragment of the cloned pgip gene as a probe in Northern blot experiments, it is demonstrated that the pgip mRNA accumulates in suspension-cultured bean cells following addition of elicitor-active oligogalacturonides or fungal glucan to the medium. Rabbit polyclonal antibodies specific for PGIP were generated against a synthetic peptide designed from the N-terminal region of PGIP; the antigenicity of the peptide was enhanced by coupling to KLH. Using the antibodies and the cloned pgip gene fragment as probes in Western and Northern blot experiments, respectively, it is shown that the levels of PGIP and its mRNA are increased in P. vulgaris hypocotyls in response to wounding or treatment with salicylic acid. Using gold-labeled goat-anti-rabbit secondary antibodies in EM studies, it has also been demonstrated that, in bean hypocotyls infected with Colletotrichum lindemuthianum, the level of PGIP preferentially increases in those cells immediately surrounding the infection site. The data support the hypothesis that synthesis of PGIP constitutes an active defense mechanism of plants that is elicited by signal molecules known to induce plant defense genes.