A genotype-to-phenotype map of in vitro selected RNA-cleaving DNAzymes: implications for accessing the target phenotype

Herein, we describe a case study into the population dynamics of in vitro selection, using RNA-cleaving DNAzymes as a model system. We sought to understand how the composition of the population can change over time in response to different levels of selection pressure, and how well these changes are correlated with selection of the target phenotype. The model population is composed of 857 DNAzyme clones representing 215 discrete sequence classes, which had previously been identified from two parallel selection experiments, conducted under an increasingly stringent, or permissive and constant selection time pressure. In this report, we determined the principal phenotypic properties (i.e. k_obs, maximum cleavage yield and PCR efficiency) from a sample of 58 clones representing 46 different major and minor sequence classes from various rounds of each selection experiment. Interestingly, a positive correlation between the catalytic rate constant and the corresponding frequency and temporal position of a given DNAzyme was not consistently observed; however, the strength of the correlation was qualitatively higher under conditions of more stringent selection time pressure. These results suggest that the selective sampling paradigm on which in vitro selection is based, may underestimate the true functional capacity of any given random-sequence library.     

Proteins of the kidney microvillar membrane. Purification and properties of the phosphoramidon-insensitive endopeptidase (''endopeptidase-2'') from rat kidney.

A second endopeptidase is present in the renal microvillar membrane of rats that can be distinguished from endopeptidase-24.11 by its insensitivity to inhibition by phosphoramidon. The purification of this enzyme, referred to as endopeptidase-2, is described. The enzyme was efficiently released from the membrane by treatment with papain. The subsequent four steps depended on ion-exchange and gel-filtration chromatography. These steps were monitored by the hydrolysis of various substrates: 125I-insulin B chain (the normal assay substrate), benzoyl-L-tyrosyl-p-aminobenzoate (Bz-Tyr-pAB), azocasein and benzyloxycarbonyl-L-phenylalanyl-L-arginine 7-amino-4-methylcoumarylamide (Z-Phe-Arg-NMec). All four assays revealed comparable stepwise increases in activity in the main stages of the purification, although it was apparent that the last-named fluorogenic assay depended on traces of aminopeptidase activity present in the preparation. The Km for 125I-insulin B chain was 16 microM and that for Bz-Tyr-pAB was 4.7 mM. Several experimental approaches confirmed that both peptides were hydrolysed by the same enzyme. The pH optimum was 7.3. Phosphate buffers were inhibitory and shifted the optimum to above pH 9. Zinc was detected in the purified enzyme; EDTA and 1,10-phenanthroline were strongly inhibitory. SDS/polyacrylamide-gel electrophoresis revealed polypeptides of equal staining intensity of Mr 80,000 and 74,000 in reducing conditions. In non-reducing conditions a single band of apparent Mr 220,000 was seen. Gel filtration yielded an Mr of 436,000. These results are consistent with an oligomeric structure in which the alpha and beta chains are linked by disulphide bridges. Endopeptidase-2 hydrolysed a number of neuropeptides. Enkephalins resisted attack, only the heptapeptide [Met]enkephalin-Arg6-Phe7 being susceptible to slow hydrolysis. Luliberin (luteinizing-hormone-releasing hormone) and bradykinin were rapidly hydrolysed. Neurotensin was shown to be slowly attacked at the Tyr3-Glu4 bond. Thus the specificity appears to be limited to the hydrolysis of bonds involving the carboxy group of aromatic residues, provided that this P1 residue is extended by additional residues, at least to the P3' position. The relationship of this membrane metalloendopeptidase to mouse meprin and human 'PABA peptidase' is discussed.     

Lactate modeling and its application to endurance training

1. 1. In this short review, previous studies regarding the modeling of lactate (La) response to exercise and its application to endurance training have been summarized.

2. 2. Additionally the result of a recent study by the present authors are shown.

3. 3. Several models for La response to step and ramp exercise are already proposed and deductions derived from them are used for practical purposes such as the prediction of race performance in middle-and long-distance runners as well as for construction of their training regimens.

4. 4. Only a limited number of models however have tried to quantify whole body La kinetics to exercise in humans concomitantly with describing physiological mechanisms underlying the observed phenomenon.

5. 5. In a recent study described further in this paper a 2 compartment model was used for the purpose of clarifying the current “La production vs degradation” controversy during La adaptation to training.

6. 6. It was determined from this investigation that the La metabolic clearance rate during recovery is enhanced by the endurance training.

7. 7. This is in accordance with another recent observation of an increased La metabolic clearance rate at high absolute work rates and all relative work rates during exercise.

P38 Mitogen-activated Protein Kinase Activity Is Required during Mitosis for Timely Satisfaction of the Mitotic Checkpoint But Not for the Fidelity of Chromosome Segregation

Although p38 activity is reported to be required as cells enter mitosis for proper spindle assembly and checkpoint function, its role during the division process remains controversial in lieu of direct data. We therefore conducted live cell studies to determine the effect on mitosis of inhibiting or depleting p38. We found that in the absence of p38 activity the duration of mitosis is prolonged by ∼40% in nontransformed human RPE-1, ∼80% in PtK2 (rat kangaroo), and ∼25% in mouse cells, and this prolongation leads to an elevated mitotic index. However, under this condition chromatid segregation and cytokinesis are normal. Using Mad2/YFP-expressing cells, we show the prolongation of mitosis in the absence of p38 activity is directly due to a delay in satisfying the mitotic checkpoint. Inhibiting p38 did not affect the rate of chromosome motion; however, it did lead to the formation of significantly (10%) longer metaphase spindles. From these data we conclude that normal p38 activity is required for the timely stable attachment of all kinetochores to spindle microtubules, but not for the fidelity of the mitotic process. We speculate that p38 activity promotes timely checkpoint satisfaction by indirectly influencing those motor proteins (e.g., Klp10, Klp67A) involved in regulating the dynamics of kinetochore microtubule ends.     

Venom Alkaloid Chemistry of Australian Species of the Monomorium rothsteini Complex,with Particular Reference to Taxonomic Implications

A comparison of the morphology and of the venom alkaloids of the Australian Monomorium rothsteini complex was undertaken. These ants were collected in Australia from western New South Wales, northern Queensland, and northern Northern Territory. Additionally, samples from the M. sordidum complex and M. carinatum complex were examined. Thirteen previously described trans‐2,5‐dialkylpyrrolidines were detected in these ants, along with the novel trans‐2‐ethyl‐5‐[(Z)‐tridec‐4‐enyl]pyrrolidine ( 6 ), whose structure was confirmed by synthesis. The extent of variation and the correlation observed in the morphology and venom chemistry in M. rothsteini samples is very strongly indicative of multiple species in this complex. The presence and location of the C?C bond in 6 reinforces the remarkable structural similarity of the 2‐ethylpyrrolidines in these Monomorium species to the 2‐methylpiperidines in the venoms of many Solenopsis species, and may represent convergent evolution of biosynthetic processes in different genera of solenopsidine ants.