Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density [in colony forming units per gram of wood (CFU.g^-1) ] compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU.g^-1, while the threshold for the ST was greater than 0.1.10³ CFU^-1. No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU.g^-1, suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique.     

Ultrastruttura della cellula apicale di Halopteris scoparia (L.) Sauvageau (Phaeophyceae,Sphacelariales)

Abstract

Ultrastructure of the apical cell of Halopteris scoparia (L.) Sauvageau (Phaeophyceae, Sphacelariales). - The ultrastructure of resting apical cells of Halopteris scoparia (L.) Sauvageau from material collected in December is described. The cytoplasm is higly vacuolated with lipids, poliphenolic substances and polisaccharides occurring inside the vacuoles (the classic « physodes »).

Two cell organelles are prominently active at this stage: conspicuosly hypertrophic dictyosomes and the budding endoplasmic reticulum. Both light and electron microscope observations show that the cell wall has an outer stratification and inner discontinuous thickenings, the constituent material of which is uniformerly dispersed.

The above observations point out that the apical cell of Halopteris scoparia at this stage of its life cycle is engaged in an elaboration of vacuolar and parietal substances which will be therefore readly available at the outset of the growing season.     

Liver Accumulation of Plasmodium chabaudi-Infected Red Blood Cells and Modulation of Regulatory T Cell and Dendritic Cell Responses

It is postulated that accumulation of malaria-infected Red Blood Cells (iRBCs) in the liver could be a parasitic escape mechanism against full destruction by the host immune system. Therefore, we evaluated the in vivo mechanism of this accumulation and its potential immunological consequences. A massive liver accumulation of P. c. chabaudi AS-iRBCs (Pc-iRBCs) was observed by intravital microscopy along with an over expression of ICAM-1 on day 7 of the infection, as measured by qRT-PCR. Phenotypic changes were also observed in regulatory T cells (Tregs) and dendritic cells (DCs) that were isolated from infected livers, which indicate a functional role for Tregs in the regulation of the liver inflammatory immune response. In fact, the suppressive function of liver-Tregs was in vitro tested, which demonstrated the capacity of these cells to suppress naive T cell activation to the same extent as that observed for spleen-Tregs. On the other hand, it is already known that CD4+ T cells isolated from spleens of protozoan parasite-infected mice are refractory to proliferate in vivo. In our experiments, we observed a similar lack of in vitro proliferative capacity in liver CD4+ T cells that were isolated on day 7 of infection. It is also known that nitric oxide and IL-10 are partially involved in acute phase immunosuppression; we found high expression levels of IL-10 and iNOS mRNA in day 7-infected livers, which indicates a possible role for these molecules in the observed immune suppression. Taken together, these results indicate that malaria parasite accumulation within the liver could be an escape mechanism to avoid sterile immunity sponsored by a tolerogenic environment.     

Localization of Endo-Oligopeptidase (EC 3.4.22.19) in the Rat Nervous Tissue

The subcellular and regional distribution of endo-oligopeptidase (EC 3.4.22.19), an enzyme capable of generating enkephalin by single cleavage from enkephalin-containing peptides, was determined by an enzymatic assay using metorphamide and by immunochemical techniques in the CNS of the rat. The rat CNS contains a membrane-associated form of endo-oligopeptidase, an enzyme predominantly associated with the soluble fraction of brain homogenates. Subcellular fractionation showed that approximately 17% of the total activity of the enzyme is associated with membrane fractions including synaptosomes. Synaptosomal membranes were prepared from neocortex, striatum, hypothalamus, medulla, spinal cord, and cerebellum. The amount of EC 3.4.22.19 activity solubilized by 3-[( 3-cholamidopropyl]dimethylammonio)-1-propanesulfonate from synaptosomal membranes was similar in neocortex, striatum, and hypothalamus, being three- to 10-fold greater than in spinal cord, cerebellum, and medulla. A polyclonal antibody exhibiting high affinity for endo-oligopeptidase was raised in rabbits against the purified rat brain enzyme and used to localize endo-oligopeptidase by Western blotting and by immunoperoxidase techniques. A strong band corresponding to the Mr of EC 3.4.22.19 was found in solubilized proteins obtained from synaptosomal membranes prepared from hypothalamus, neocortex, and striatum when subjected to Western blotting. The immunohistochemical localization of endo-oligopeptidase indicated that the immunoreactivity was confined to gray matter in regions known to be rich in peptide-containing neurons such as the striatum. In the cerebellum, a region poor in peptides, no staining could be detected. The nonuniform distribution of endo-oligopeptidase in rat brain suggests a role in neurotransmitter processing in the CNS.     

Multiple Bacteriocin Production by Leuconostoc mesenteroidesTA33a and Other Leuconostoc/Weissella Strains

Leuconostoc (Lc.) mesenteroides TA33a produced three bacteriocins with different inhibitory activity spectra. Bacteriocins were purified by adsorption/desorption from producer cells and reverse phase high-performance liquid chromatography. Leucocin C-TA33a, a novel bacteriocin with a predicted molecular mass of 4598 Da, inhibited Listeria and other lactic acid bacteria (LAB). Leucocin B-TA33a has a predicted molecular mass of 3466 Da, with activity against Leuconostoc/Weissella (W.) strains, and appears similar to mesenterocin 52B and dextranicin 24, while leucocin A-TA33a, which also inhibited Listeria and other LAB strains, is identical to leucocin A-UAL 187. A survey of other known bacteriocin-producing Leuconostoc/Weissella strains for the presence of the three different bacteriocins revealed that production of leucocin A-, B- and C-type bacteriocins was widespread. Lc. carnosum LA54a, W. paramesenteroides LA7a, and Lc. gelidum UAL 187-22 produced all three bacteriocins, whereas W. paramesenteroides OX and Lc. carnosum TA11a produced only leucocin A- and B-type bacteriocins. Received: 11 April 1997 / Accepted: 10 June 1997     

Guaraná (Paullinia cupana) as a commercial crop in Brazilian Amazonia

Guaraná (Paullinia cupana) is a woody vine or sprawling shrub native to the central Amazon Basin. The seeds are commercially produced on some 6,000 ha in the state of Amazonas near Manaus. The principal article of commerce is an amber-colored, carbonated soft drink. It is also widely used as a high caffeine stimulant and in local medicines. The plant is monoecious and is damaged by a number of diseases, the most severe being anthracnose. Prospects are excellent for greatly expanded international markets.     

Suppression of the rotifer Polyarthra remata by the omnivorous copepod Tropocyclops extensus: predation or competition

Indirect evidence from observations in the field suggested thata common and often abundant cyclopoid copepod, Tropocyclopsextensus, despite its small size (0.5 mm) and largely algaldiet, is an important predator of the rotifer Polyarthra remata.Laboratory experiments showed that copepodids and adults, butnot nauplii, markedly suppressed the population growth of thisrotifer. The suppression could be attributed entirely to predation,rather than to exploitative competition for shared food resourcesor to interference. Mortality rates predicted from a separatefeeding-rate experiment weresufficient to account for the declinein population size observed in the Tropocyclops treatment ofculture experiments. Also, monitoring the availability of cryptomonadfood in treatments with and without Tropocyclops throughoutthese experiments showed that cryptomonad densities always remainedhigh and were just as high or significantly higher in the copepodtreatments. Furthermore, direct videographic observations ofP. remata from cultures with and without Tropocyclops demonstratedno significant differences in swimming velocity, tendency todeviate from a straight-line path, or frequency and length ofspontaneous escape responses. Per capita ingestion rates ofT. extensus on P. remata varied from 2 to 8 day^-1, were significantlyhigher for adult females than adult males or copepodids V, andincreased significantly with rotifer density. A tendency ofcopepods to have higher ingestion rates on young than adultrotifers was not significant. Copepods cultured since birthwith cryptomonads and P. remata ate significantly fewer rotifersthan those cultured only with cryptomonads; this may be explainedby a more sated condition of the former and provided no evidencefor the idea that previous experience with this rotifer mightincrease predation efficiency. The results show that T. extensusin natural communities has the potential to deplete naturalpopulations of susceptible rotifer prey. Accordingly, it mayshift the species structure of rotifer assemblages in favourof resistant species, and provide selection pressure for avoidanceresponses.     

CORRESPONDENCE BETWEEN PHYLOGENY AND MORPHOLOGY OF SNOWELLA SPP. AND WORONICHINIA NAEGELIANA,CYANOBACTERIA COMMONLY OCCURRING IN LAKES1

In this study, the first reported isolates of the genera Snowella and Woronichinia were characterized by 16S rRNA gene sequencing and morphological analysis. Phylogenetic studies and sequences for these genera were not available previously. By botanical criteria, the five isolated strains were identified as Snowella litoralis (Häyrén) Komárek et Hindák Snowella rosea (Snow) Elenkin and Woronichinia naegeliana (Unger) Elenkin. This study underlines the identification of freshly isolated cultures, since the Snowella strains lost the colony structure and were not identifiable after extended laboratory cultivation. In the 16S rRNA gene analysis, the Snowella strains formed a monophyletic cluster, which was most closely related to the Woronichinia strain. Thus, our results show that the morphology of the genera Snowella and Woronichinia was in congruence with their phylogeny, and their phylogeny seems to support the traditional botanical classification of these genera. Furthermore, the genera Snowella and Woronichinia occurred commonly and might occasionally be the most abundant cyanobacterial taxa in mainly oligotrophic and mesotrophic Finnish lakes. Woronichinia occurred frequently and also formed blooms in eutrophic Czech reservoirs.     

The presence of chlorophyll b and the estimation of phaeopigments in marine phytoplankton

A reverse-phase h.p.l.c. technique was used to estimate theconcentration of chlorophyll b in phytoplankton cultures, fecalpellets of Calanus pacificus, and suspended paniculate matterfrom the Central North Pacific, Oregon coastal waters, and DabobBay (a temperate fjord in Puget Sound, WA, USA). The purposewas to assess the distribution of this pigment in the euphoticzone and its effect on the fluorometnc estimation of phaeopigments.Analyses of natural waters confirm high chlorophyll b concentrations(median mass ratio of b:a > 0.3) at the depth of the chlorophylla maximum in tropical waters while values for temperate planktonare relatively low (median mass ratio of chl b:a = 0.05) andpatchy. Zooplankton fecal pellets showed a significant enrichmentin chlorophyll b, suggesting grazing as a mechanism to explainhigh concentrations of this pigment at the bottom of the euphoticzone. It is estimated that the presence of chlorophyll b couldcause an average overestimation of phaeopigment concentrationby the fluorometnc technique of 38% between 0 and 200 m in theCentral North Pacific. This effect is more pronounced at thelayer of chlorophyll b maximum (120–140 m). ¹Present address: Marine Biology Research Division, A-002, ScrippsInstitution of Oceanography, La Jolla, CA 92093, USA