A substantial number of children begin watching television at an earlier age and in greater amounts than the AAP recommends. Furthermore, these early viewing patterns persist into childhood. Preventive intervention research on television viewing should consider targeting infants and toddlers and their families.

The period of pregnancy and early parenthood is associated with worsening education-related disparities in smoking as well as substantial clustering of risk factors. These observations could influence the targeting and design of maternal smoking interventions.

We investigated the effect of human immunodeficiency virus type 1 (HIV-1) subtype on disease progression among 145 Kenyan women followed from the time of HIV-1 acquisition. Compared with those infected with subtype A, women infected with subtype D had higher mortality (hazard ratio, 2.3 [95% confidence interval, 1.0-5.6]) and a faster rate of CD4 cell count decline (P=.003). The mortality risk persisted after adjustment for plasma HIV-1 load. There were no differences in plasma viral load by HIV-1 subtype during follow-up. HIV-1 subtype D infection is associated with a >2-fold higher risk of death than subtype A infection, in spite of similar plasma HIV-1 loads.

We previously reported the design of a library of de novo proteins targeted to fold into 4-helix bundles. 1 The library was created using a "binary code" strategy in which the sequence locations of polar and nonpolar amino acids were specified explicitly, but the identities of these side chains were varied extensively. Combinatorial diversity was made possible by the organization of the genetic code: Positions designed to contain polar amino acids were encoded by the degenerate DNA codon NAN, which codes for Lys, His, Glu, Gln, Asp, or Asn. Positions designed to contain nonpolar amino acids were encoded by NTN, which codes for Met, Leu, Ile, Val, or Phe (N represents a mixture of DNA nucleotides). We subsequently reported that approximately half the sequences in our initial library 1 bind heme. 2 The enormous diversity of sequences within the binary code library presents an opportunity for the isolation of de novo heme-based enzymes. We now establish the catalytic potential of the binary code proteins by demonstrating that several of the de novo heme proteins function as peroxidases.Natural peroxidases such as horseradish peroxidase (HRP) catalyze the reduction of hydrogen peroxide or alkyl peroxides to water or alcohol, respectively. The peroxidase mechanism is described by the following steps: 3 E represents the ferric resting state of the heme enzyme. Compound I is an intermediate two oxidation states above the resting state, and compound II is one oxidation state above the resting state. AH 2 is the reducing agent. Two commonly used reducing agents are 2,2′,5,5′-tetramethyl-benzidine (TMB) and 2,2′-azino-di(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS). These reagents become colored upon oxidation, thereby allowing peroxidase activity to be monitored spectraphotometrically.To search for novel peroxidases in our library of heme proteins, we developed an activity screen that does not require purification of the de novo proteins from cellular contaminants. Samples were prepared by a rapid freeze/thaw protocol 4 shown previously to produce protein samples of sufficient quality for various studies including NMR spectroscopy 5a and H/D exchange kinetics. 5b Freeze/thaw samples from 37 binary code proteins 6 were screened by monitoring oxidation of TMB. The results (Figure 1) indicate that several of the de novo proteins display activity significantly above the controls.On the basis of this screen, we chose four proteins for detailed analysis. 9 Proteins were purified, reconstituted with heme, and analyzed for peroxidase activity using ABTS as the reducing agent. Experimental data for protein 86 are shown in Figure 2. Neither the apoprotein nor the heme alone 10 were active, thereby demonstrating the heme-protein complex is the active species.Maximal velocities (V max ) were determined by plotting the data according to the Michaelis-Menten model (Figure 3). Turnover numbers (k cat ) were calculated by dividing V max by the concentration of heme protein. More detailed kinetic constants (k 1 and k 3 ) were determine...