Dynamic 13C-tracer study of storage carbohydrate pools in aerobic glucose-limited Saccharomyces cerevisiae confirms a rapid steady-state turnover and fast mobilization during a modest stepup in the glucose uptake rate

In this research, two dynamic ¹³C-labeling experiments confirmed turnover and rapid mobilization of stored glycogen and trehalose in an aerobic glucose-limited chemostat ( D =0.05 h⁻¹) culture of Saccharomyces cerevisiae . In one experiment, the continuous feed to an aerobic glucose-limited chemostat culture of S. cerevisiae was instantaneously switched from naturally labeled to fully ¹³C labeled while maintaining the same feed rate before and after the switch. The dynamic replacements of naturally labeled intracellular glycolytic intermediates and CO₂ (in the off-gas) with their ¹³C-labeled equivalents were measured. The data of this experiment suggest that the continuous turnover of glycogen and trehalose is substantial ( c . 1/3 of the glycolytic flux). The second experiment combined the medium switch with a shiftup in the glucose feeding rate (dilution rate shiftup from 0.05 to 0.10 h⁻¹). This experiment triggered a strong but transient mobilization of storage carbon, that was channelled into glycolysis, causing a significant disruption in the dynamic labeling profile of glycolytic intermediates. The off-gas measurements in the shiftup experiment confirmed a considerable transient influx of ¹²C-carbon into glycolysis after the combined medium switch and dilution rate shiftup. This study shows that for accurate in vivo kinetic interpretation of rapid pulse experiments, glycogen and trehalose metabolism must be taken into account.     

Contribution of 16S rRNA nucleotides forming the 30S subunit A and P sites to translation in Escherichia coli

Many contacts between the ribosome and its principal substrates, tRNA and mRNA, involve universally conserved rRNA nucleotides, implying their functional importance in translation. Here, we measure the in vivo translation activity conferred by substitution of each 16S rRNA base believed to contribute to the A or P site. We find that the 30S P site is generally more tolerant of mutation than the 30S A site. In the A site, A1493C or any substitution of G530 or A1492 results in complete loss of translation activity, while A1493U and A1493G decrease translation activity by >20-fold. Among the P-site nucleotides, A1339 is most critical; any mutation of A1339 confers a >18-fold decrease in translation activity. Regarding all other P-site bases, ribosomes harboring at least one substitution retain considerable activity, >10% that of control ribosomes. Moreover, several sets of multiple substitutions within the 30S P site fail to inactivate the ribosome. The robust nature of the 30S P site indicates that its interaction with the codon-anticodon helix is less stringent than that of the 30S A site. In addition, we show that G1338A suppresses phenotypes conferred by m(2)G966A and several multiple P-site substitutions, suggesting that adenine at position 1338 can stabilize tRNA interaction in the P site.     

Development of a program model to evaluate the potential for reuse of single-use medical devices: results of a pilot test study

Single-use medical devices (SUDs, or disposables) have become a major expense in hospital budgets. The need for cost reduction and the availability of sterilization technologies other than the autoclave have prompted hospitals worldwide to begin reusing disposables, in many cases without proper assessment of the true costs (time, personnel, etc) and ease/difficulty of implementation of an institutional reuse program. Our group has developed a rigorous program model to evaluate SUDs for reuse. The program comprises 3 sequential protocols: (1) device audit, (2) laboratory evaluation, and (3) clinical evaluation. Use of this model can produce scientific and financial data sufficient for any institution interested in reuse to reach an initial decision about its feasibility. In addition to the testing outcomes, regulatory requirements, the position of manufacturers and third-party reprocessors, and legal and ethical concerns must be considered. A successful reuse program must include ongoing evaluations to ensure that the safety levels and cost savings established during the initial audit and evaluation phases continue. Herein, we give the rationale and details of our program model and discuss results of our pilot application of the "ideal" protocol in a real-world context.     

ULTRASTRUCTURE AND ACID PHOSPHATASE IN PEDICEL ABSCISSION OF HIBISCUS

Pedicel abscission in Hibiscus rosa-sinensis was investigated by light and electron microscopy. During the pre-abscission period endoplasmic reticulum declined somewhat, dictyosomes increased in number and apparent activity, and mitochondria maintained their numbers. The observations suggested that dictyosomal vesicles were migrating to and fusing with the plasma membrane. The enzyme acid phosphatase was associated with dictyosomes and dictyosomal saccules, with small vacuoles and invaginations of the plasma membrane, and in the paramural region between the plasma membrane and the cell wall. Our interpretation is that acid phosphatase, (and probably also the enzymes involved in cell wall dissolution) are transported via an endoplasmic reticulum-dictyosome-vesicle carrier system to the paramural regions of the cell. In more general terms, our observations support the view that the enzymes involved in the cell wall hydrolysis of abscission are synthesized within a compartmentalized, lysosomal system prior to their release and action.     

Intraspecies evolution of enzymic mechanisms associated with the synthesis of α-1,4 storage glucans in two thermophilic-acidophilic algae of Cyanidium type

Cyanidium caldarium is an enigmatic eukaryotic alga which is both acidophilic and thermophilic. Its taxonomic position has been in doubt and, hence     

A Differential Stain for Rubber in Guayule

The following schedule, which combines an intense blue stain for rubber with sharply contrasting red counterstains, has been found satisfactory for use in an anatomical study of rubber deposition in guayule: Cut fresh or fixed sections about 50 to 100 % thick, transfer to 50% ethanol. Extract with acetone 5 minutes, treat with 1% NaOCl 5 minutes, saponify with 10% KOH in 95% ethanol 15 minutes, rinse 3 times with 50% ethanol, stain in oil blue NA (Calco) with safranin and Congo red 30 minutes at 55° C. Rinse in 50% ethanol 2 (or more) times to remove excess stain and mount in Karo syrup.