Aerobic respiratory activity of the yeastlike phase of Sporotrichum schenckii as affected by high hydrogen ion concentrations

Summary Six strains ofSporotrichum schenckii were studied in regard to the effect of hydrogen ion concentration on certain aspects of the aerobic respiratory activity of the yeastlike phase.Optimal oxygen uptake of endogenous respiration occurred at pH 2.0, although no effects were observed on the oxygen-carbon dioxide exchange ratio (respiratory quotient) at this pH value when compared to the R.Q. obtained at pH 7.0. Endogenous respiratory activity at both pH 2 and 7 was markedly sensitive to the presence of certain respiratory inhibitors.Optimal respiratory activity using glucose as substrate occurred at pH 7.0 .On the other hand, oxidation of pyruvate as substrate proceeded at significant rates only at pH values below pH 4.0. With decreasing hydrogen ion concentration, accumulation of this organic acid occurred when glucose was employed as substrate. With the exception of acetate, none of the organic acid respiratory intermediates were found to stimulate respiration.The results reported herein suggest that the respiratory activity of the yeastlike phase ofS. schenckii differs in several respects from that observed for the yeastlike phases ofHistoplasma capsulatum andBlastomyces dermatitidis.From the Research Laboratory, Veterans Administration Hospital, Kansas City, Missouri, and The Department of Microbiology, Kansas University Medical Center, Kansas City, Kansas.Supported in part by USPHS Grant AI 03485.     

Amoeboid locomotion of Naegleria gruberi: the effects of cytochalasin B on cell-substratum interactions and motile behavior

The major manifestations of amoeboid locomotion in Naegleria-cytoplasmic streaming, pseudopod production, cell polarity and focal contact production-require that the actin-based cytoskeleton be extremely dynamic. Whether these features are causally linked is unclear. In an attempt to answer this question we have used the fungal product cytochalasin B (cyt B) to dissect the motility process. This drug can perturb the organisation of actin filaments both in vivo and in vitro. Essentially cyt B acts as a molecule which can cap the barbed ends of actin filaments. Not surprisingly, therefore cyt B has an effect on rates of actin polymerization and the dynamic state of actin in the cytoplasm. We have found that cyt B has a profound effect on focal contact production and breakdown. Within minutes of addition of cyt B focal contact production ceases, existing focal contacts are stabilised but cytoplasmic streaming and pseudopod production are not blocked. In conclusion it is now clear that the state of actin required for focal contact production is different from that required for pseudopod extension and cytoplasmic streaming.     

Changes in diadenosine tetraphosphate levels in Physarum polycephalum with different oxygen concentrations.

Cellular levels of diadenosine tetraphosphate (Ap4A) were measured, by a specific high-pressure liquid chromatography method, in microplasmodia of Physarum polycephalum subjected to different degrees of hypoxia, hyperoxia, and treatment with H2O2. Ap4A levels increased three- to sevenfold under anaerobic conditions, and the microplasmodia remained viable after such treatment. Elevated levels of Ap4A returned to the basal level within 5 to 10 min upon reoxygenation of the microplasmodia. The increases in Ap4A levels were larger in stationary-phase or starved microplasmodia than in fed, log-phase microplasmodia. The maximal increase measured in log-phase microplasmodia was twofold. No significant changes in Ap4A levels occurred in microplasmodia subjected to mild hypoxia, hyperoxia, or treatment with 1 mM H2O2. These results indicate that in P. polycephalum, Ap4A may function in the metabolic response to anaerobic conditions rather than in the response to oxidative stress.     

Herpes simplex virus type 1 immediate-early protein Vmw110 reactivates latent herpes simplex virus type 2 in an in vitro latency system.

Reactivation of latent herpes simplex virus type 2 (HSV-2) by the immediate-early protein Vmw110 was studied by using an in vitro latency system. Adenovirus recombinants that express Vmw110 reactivated latent HSV-2. An HSV-1 mutant possessing a deletion in a carboxy-terminal region of Vmw110 reactivated latent HSV-2, whereas mutant FXE, which has a deletion in the second exon, did not. Therefore, Vmw110 alone is required to reactivate latent HSV-2 in vitro, and the region of Vmw110 defined by the deletion in FXE is important for this process.     

The extracellular processing and catabolism of hyaluronan in cultured adult articular cartilage explants.

Hyaluronan was shown to have the same turnover time as aggrecan in explant cultures of adult bovine articular cartilage. Inclusion of fetal calf serum in the culture medium resulted in a similar decrease in the rate of catabolism of both hyaluronan and proteoglycan. Less than 9% of the hyaluronan lost from the explants in the course of the experiment was recovered from the culture medium as hyaluronan, suggesting that the catabolism of hyaluronan involves the uptake of this glycosaminoglycan by the chondrocytes. Analysis of the molecular size of the newly synthesized hyaluronan in these cultures showed that the hyaluronan was initially synthesized as large macromolecules that were gradually depolymerized with time within the extracellular matrix. The resulting size distribution of newly synthesized hyaluronan molecules after 12 days in culture was similar to that determined for the endogenous hyaluronan. The kinetics of depolymerization of the newly synthesized hyaluronan was consistent with a random fragmentation of the macromolecule. The rate constants for the depolymerization of hyaluronan indicate that oxygen-derived radicals may be involved in the fragmentation of this macromolecule. Inclusion of either cycloheximide or proteinase inhibitors in the medium of the explant cultures resulted in a marked decrease in the rate of loss of hyaluronan from the tissue and in the inhibition of the depolymerization of the newly synthesized macromolecule. This suggests that both the catabolism and the depolymerization of hyaluronan are cell mediated and depend on metabolically active cells.     

Mitochondrial gene mutation: the ageing process and degenerative diseases

Polymerase chain reaction (PCR) amplification was carried out on total DNA from a range of autopsy tissues from deceased human subjects with no known mitochondrial disease, aged from birth (80 minutes) to 87 years. We report the finding of an age-related 5 kb deletion in the mitochondrial genomes of these subjects. The deletion occurs between nucleotide positions 8470 and 13459 of the mitochondrial genome, and is flanked by a 13 bp direct repeat. All tissues from adult subjects showed the presence of mitochondrial DNA molecules with the deletion after a 30 cycle PCR amplification; by contrast the deletion was not similarly detected in any of the infant tissues analysed. However, the occurrence of the deletion was detected in the infant tissues after 60 PCR cycles of MtDNA amplification. It is concluded that such deletions are not necessarily associated with particular mitochondrial diseases but occur naturally, and with increasing frequency with age. A consequence of the accumulation of this deletion could be a progressive decrease with age of bioenergetic capacity which in turn could influence the rate of ageing and predispose to age-associated degenerative diseases.     

Genetic variability in two hydrophilous species ofPotamogeton,P. pectinatus andP. filiformis (Potamogetonaceae)

Genetic variability inPotamogeton pectinatus andP. filiformis was studied by means of isozymes. The overall levels of variability were similar to some other well studied hydrophilous species, and were shown to be distributed more between than within populations. This partitioning of variability was attributed to three main factors. (1) Clonal growth (as measured by the frequency of multi-enzyme phenotypes) was shown to be a major factor in both species, although more important inP. pectinatus. (2) Low levels of sexual reproduction were shown to be a likely contributor to the partitioning of variability inP. pectinatus; such reproduction is probably limited by seedling recruitment rather than by infrequent flowering. (3) Geographical isolation was indicated as a factor regulating gene flow at distances of more than about 1000 km in both species, with the data suggesting that dispersal between populations is mainly by seed rather than by vegetative means.     

A bioassay of the availability of residual 15N fertilizer eight years after application to a forest soil in interior British Columbia

Although a high proportion of fertilizer N may be immobilized in organic forms in the soil, no studies have examined the long-term availability of residual fertilizer ¹⁵N in forestry situations. We investigated this by growing lodgepole pine (Pinus contorta) seedlings in surface (0–10 cm) soil sample eight years after application of ¹⁵N-urea, ¹⁵NH₄NO₃ and NH₄ ¹⁵NO₃ to lodgepole pine in interior British Columbia. After nine months of growth in the greenhouse, seedlings took up an average of 8.5% of the ¹⁵N and 4.6% of the native N per pot. Most of the mineral N in the pots without seedlings was in the form of nitrate, while pots with seedlings had very low levels of mineral N. In contrast to the greenhouse study, there was no significantuptake of ¹⁵N by trees in the field study after the first growing season, although half of the soil organic ¹⁵N was lost between one and eight years after fertilization. This indicates the need to understand the mechanisms which limit the uptake of mineral N by trees in the field, and the possible mismatch of tree demand and mineral N availability.