Calcium compartmentation in isolated renal tubules in suspension

Substantial increases of total cell Ca2+ have been observed in suspensions of isolated rabbit proximal tubules subjected to hypoxic injury or treated with exogenous ATP followed by apparent recovery with reoxygenation of the hypoxic tubules or continued incubation of ATP-treated tubules. Ca2+ compartmentation studies using digitonin and metabolic inhibitors were done to clarify the basis for these changes. Digitonin, 40-90 micrograms/mg tubule protein, rapidly permeabilized the tubule cells and did not impair mitochondrial Ca2+ sequestration. Most of the increases of tubule cell Ca2+ produced by hypoxia and ATP were accounted for by pools which could be rapidly removed by exposure of tubules to EGTA and the uncoupler carbonyl cyanide m-chlorophenyl hydrazone without concomitant use of digitonin, suggesting that the changes of Ca2+ predominantly reflect sequestration by mitochondria in severely damaged cells or mitochondria already released to the medium from them. The time course of uptake followed by spontaneous release of mitochondrial Ca2+ from tubule cells deliberately permeabilized with digitonin, then incubated for prolonged periods, indicated that the decreases of tubule cell Ca2+ during reoxygenation of hypoxic suspensions and prolonged incubation of ATP-treated tubules were likely to be attributable to loss of Ca2+ from free mitochondria and those in damaged cells rather than to extrusion by intact cells.     

Monitoring of denitrification byPseudomonas aeruginosa using on-line fluorescence technique

Summary The NAD(P)H fluorescence ofPseudomonas aeruginosa dropped sharply upon addition of nitrate to an anaerobic culture, indicating that denitrification is not limited by mass transfer of nitrate through cell membrane to reach nitrate reductase. The effect of added nitrate concentration on fluorescence drop followed a typical saturation kinetics. The maximum specific denitrification rate under the studied condition was found to be 0.26±0.05 g NO ₃ ⁻ -N/g cells-hr.     

Trimethyloxonium modification of single batrachotoxin-activated sodium channels in planar bilayers. Changes in unit conductance and in block by saxitoxin and calcium

Single batrachotoxin-activated sodium channels from rat brain were modified by trimethyloxonium (TMO) after incorporation in planar lipid bilayers. TMO modification eliminated saxitoxin (STX) sensitivity, reduced the single channel conductance by 37%, and reduced calcium block of inward sodium currents. These effects always occurred concomitantly, in an all-or-none fashion. Calcium and STX protected sodium channels from TMO modification with potencies similar to their affinities for block. Calcium inhibited STX binding to rat brain membrane vesicles and relieved toxin block of channels in bilayers, apparently by competing with STX for the toxin binding site. These results suggest that toxins, permeant cations, and blocking cations can interact with a common site on the sodium channel near the extracellular surface. It is likely that permeant cations transiently bind to this superficial site, as the first of several steps in passing inward through the channel.     

DNA replication initiates non-randomly at multiple sites near the c-myc gene in HeLa cells.

The origin of replication of the c-myc gene in HeLa cells was previously identified at low resolution within 3.5 kb 5' to the P1 promoter, based on replication fork polarity and the location of DNA nascent strands. To define the initiation events in the c-myc origin at higher resolution the template bias of nascent DNAs in a 12 kb c-myc domain has been analyzed by hybridization to strand specific probes. Strong switches in the asymmetry of nascent strand template preference confirm that replication initiates non-randomly at multiple sites within 2.4 kb 5' to the c-myc P1 promoter, and at other sites over a region of 12 kb or more. The strongest template biases occur in the 2.4 kb region 5' of the P1 promoter, shown earlier to contain sequences which allow the autonomous semiconservative replication of c-myc plasmids. An asymmetric pyrimidine heptanucleotide consensus sequence has been identified which occurs 12 times in the c-myc origin zone, and whose polarity exactly correlates with the polarity of nascent strand synthesis.     

Pathogenesis of SIVmac251 after atraumatic inoculation of the rectal mucosa in rhesus monkeys

Intrarectal inoculation of rhesus monkeys with low doses of SIV_mac led to a prolonged clinical and virological latency that was not observed for high intrarectal doses or for intravenous inoculation. Animals infected intrarectally with low virus doses remained negative for serum antibody responses to SIV for at least one year even though they readily transferred SIV to naive recipients via transfusion of whole blood.     

Determination of Gardnerella vaginalis Genome Size by Pulsed-Field Gel Electrophoresis

The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size.     

Litter and soil biodiversity jointly drive ecosystem functions

The decomposition of litter and the supply of nutrients into and from the soil are two fundamental processes through which the above- and belowground world interact. Microbial biodiversity, and especially that of decomposers, plays a key role in these processes by helping litter decomposition. Yet the relative contribution of litter diversity and soil biodiversity in supporting multiple ecosystem services remains virtually unknown. Here we conducted a mesocosm experiment where leaf litter and soil biodiversity were manipulated to investigate their influence on plant productivity, litter decomposition, soil respiration, and enzymatic activity in the littersphere. We showed that both leaf litter diversity and soil microbial diversity (richness and community composition) independently contributed to explain multiple ecosystem functions. Fungal saprobes community composition was especially important for supporting ecosystem multifunctionality (EMF), plant production, litter decomposition, and activity of soil phosphatase when compared with bacteria or other fungal functional groups and litter species richness. Moreover, leaf litter diversity and soil microbial diversity exerted previously undescribed and significantly interactive effects on EMF and multiple individual ecosystem functions, such as litter decomposition and plant production. Together, our work provides experimental evidence supporting the independent and interactive roles of litter and belowground soil biodiversity to maintain ecosystem functions and multiple services.