Structure of a marine bacteriophage as revealed by the negative-staining technique

Valentine, Artrice F. (Georgetown University, Washington, D.C.), Peter K. Chen, Rita R. Colwell, and George B. Chapman. Structure of a marine bacteriophage as revealed by the negative-staining technique. J. Bacteriol. 91:819-822. 1966.-The morphology of a marine bacteriophage has been determined by negative-staining techniques and electron microscopy. The virus possesses a head, 600 A in diameter, and a tail which may be from 860 to 1,000 A in lenght. No tail sheath is seen. The appearance of the terminal tail structure is discussed.     

Comparative antioxidant-capacity and -content of leaves, bulbs, roots, flowers and fruit of Gethyllis multifolia L. Bolus and G. villosa Thunb. species

The total polyphenol, oxygen radical absorbance capacity (ORAC), ferric reducing antioxidant power (FRAP), radical cation scavenging ability, flavonol and flavanone contents were measured in the leaves, bulbs, roots, flowers and fruit (dry weight) of two natural populations of Gethyllis multifolia (Kukumakranka) and Gethyllis villosa. The flowers and fruit of G. multifolia and G. villosa showed higher, and in some cases significantly (P < 0.05) higher antioxidant activities when compared to the leaves, bulbs and roots. This, however, was not true for the flavanone content in both species. The total polyphenol content in the fruits of G. multifolia (21.54 mg GAE/g) and G. villosa (27.64 mg GAE/g) were found to be in agreement with those of raisins (28.30 mg GAE/g), blueberries (24 mg GAE/g) and strawberries (15.40 mg GAE/g). The FRAP values of G. multifolia flowers (76.66 μmole AAE/g) and fruit (91.51 μmole AAE/g) were found to be significantly (P < 0.05) higher than those of the other plant parts (16.76 to 39.08 μmol AAE/g). On the other hand, the flowers (590.23 μmol TE/g) and fruit (741.16 μmol TE/g) of G. villosa revealed a significantly (P < 0.05) higher ORAC when compared to the other plant parts (251.25 to 410.60 μmol TE/g). A strong correlation was evident in the fruit of both species between the total polyphenols and FRAP (r = 0.95), ORAC (r = 0.95) and flavonol content (r = 0.79).     

Insights into SOD1-linked amyotrophic lateral sclerosis from NMR studies of Ni2+- and other metal-ion-substituted wild-type copper–zinc superoxide dismutases

The dimeric Cu–Zn superoxide dismutase (SOD1) is a particularly interesting system for biological inorganic chemical studies because substitutions of the native Cu and/or Zn ions by a nonnative metal ion cause minimal structural changes and result in high enzymatic activity for those derivatives with Cu remaining in the Cu site. The pioneering NMR studies of the magnetically coupled derivative Cu₂Co₂SOD1 by Ivano Bertini and coworkers are of particular importance in this regard. In addition to Co²⁺, Ni²⁺ is a versatile metal ion for substitution into SOD1, showing very little disturbance of the structure in Cu₂Ni₂SOD1 and acting as a very good mimic of the native Cu ion in Ni₂Zn₂SOD1. The NMR studies presented here were inspired by and are indebted to Ivano Bertini’s paramagnetic NMR pursuits of metalloproteins. We report Ni²⁺ binding to apo wild-type SOD1 and a time-dependent Ni²⁺ migration from the Zn site to the Cu site, and the preparation and characterization of Ni₂Ni₂SOD1, which shows coordination properties similar to those of Cu₂Cu₂SOD1, namely, an anion-binding property different from that of the wild type and a possibly broken bridging His. Mutations in the human SOD1 gene can cause familial amyotrophic lateral sclerosis (ALS), and mutant SOD1 proteins with significantly altered metal-binding behaviors are implicated in causing the disease. We conclude by discussing the effects of the ALS mutations on the remarkable stabilities and metal-binding properties of wild-type SOD1 proteins and the implications concerning the causes of SOD1-linked ALS.     

Quantitative assessment of adverse events in clinical trials: Comparison of methods at an interim and the final analysis

In clinical study reports (CSRs), adverse events (AEs) are commonly summarized using the incidence proportion (IP). IPs can be calculated for all types of AEs and are often interpreted as the probability that a treated patient experiences specific AEs. Exposure time can be taken into account with time-to-event methods. Using one minus Kaplan–Meier (1-KM) is known to overestimate the AE probability in the presence of competing events (CEs). The use of a nonparametric estimator of the cumulative incidence function (CIF) has therefore been advocated as more appropriate. In this paper, we compare different methods to estimate the probability of one selected AE. In particular, we investigate whether the proposed methods provide a reasonable estimate of the AE probability at an interim analysis (IA). The characteristics of the methods in the presence of a CE are illustrated using data from a breast cancer study and we quantify the potential bias in a simulation study. At the final analysis performed for the CSR, 1-KM systematically overestimates and in most cases IP slightly underestimates the given AE probability. CIF has the lowest bias in most simulation scenarios. All methods might lead to biased estimates at the IA except for AEs with early onset. The magnitude of the bias varies with the time-to-AE and/or CE occurrence, the selection of event-specific hazards and the amount of censoring. In general, reporting AE probabilities for prespecified fixed time points is recommended.     

PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser^485/491 of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser^485/491 phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser^485/491 phosphorylation, which was associated with a ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser^485/491 phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser⁴⁹¹ in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser⁴⁹¹ that plays a negative role in insulin signaling in muscle cells.