NMR study of the interaction between cytochrome b5 and cytochrome c. Observation of a ternary complex formed by the two proteins and [Cr(en)3]3+

The interaction between horse cytochrome c and the tryptic fragment of bovine liver microsomal cytochrome b5 in the absence and presence of [Cr(ethylenediamine)3]Cl3 was studied by 1H NMR spectroscopy. The protein-protein interaction region on cytochrome b5 was found to be different from the [Cr(en)3]3+-binding region. The solvent-exposed propionate-bearing edge of the haem of cytochrome b5 is accessible to [Cr(en)3]3+ in the interprotein complex.     

Pin1 inhibitors: Pitfalls,progress and cellular pharmacology

Compelling data supports the hypothesis that Pin1 inhibitors will be useful for the therapy of cancer: Pin1 deficient mice resist the induction of breast cancers normally evoked by expression of MMTV-driven Ras or Erb2 alleles. While Pin1 poses challenges for drug discovery, several groups have identified potent antagonists by structure based drug design, significant progress has been made designing peptidic inhibitors and a number of natural products have been found that blockade Pin1, notably epigallocatchechin gallate (EGCG), a major flavonoid in green tea. Here we critically discuss the modes of action and likely specificity of these compounds, concluding that a suitable chemical biology tool for probing the function of Pin1 has yet to be found. We conclude by outlining some open questions regarding the target validation of Pin1 and the prospects for identification of improved inhibitors in the future.     

A two-site kinetic mechanism for ATP binding and hydrolysis by E. coli Rep helicase dimer bound to a single-stranded oligodeoxynucleotide.

Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in reactions that are coupled to ATP binding and hydrolysis. We have investigated the kinetic mechanism of ATP binding and hydrolysis by a proposed intermediate in Rep-catalyzed DNA unwinding, the Rep "P2S" dimer (formed with the single-stranded (ss) oligodeoxynucleotide, (dT)16), in which only one subunit of a Rep homo-dimer is bound to ssDNA. Pre-steady-state quenched-flow studies under both single turnover and multiple turnover conditions as well as fluorescence stopped-flow studies were used (4 degrees C, pH 7.5, 6 mM NaCl, 5 mM MgCl2, 10 % (v/v) glycerol). Although steady-state studies indicate that a single ATPase site dominates the kinetics (kcat=17(+/-2) s-1; KM=3 microM), pre-steady-state studies provide evidence for a two-ATP site mechanism in which both sites of the dimer are catalytically active and communicate allosterically. Single turnover ATPase studies indicate that ATP hydrolysis does not require the simultaneous binding of two ATP molecules, and under these conditions release of product (ADP-Pi) is preceded by a slow rate-limiting isomerization ( approximately 0.2 s-1). However, product (ADP or Pi) release is not rate-limiting under multiple turnover conditions, indicating the involvement of a second ATP site under conditions of excess ATP. Stopped-flow fluorescence studies monitoring ATP-induced changes in Rep's tryptophan fluorescence displayed biphasic time courses. The binding of the first ATP occurs by a two-step mechanism in which binding (k+1=1.5(+/-0.2)x10(7) M-1 s-1, k-1=29(+/-2) s-1) is followed by a protein conformational change (k+2=23(+/-3) s-1), monitored by an enhancement of Trp fluorescence. The second Trp fluorescence quenching phase is associated with binding of a second ATP. The first ATP appears to bind to the DNA-free subunit and hydrolysis induces a global conformational change to form a high energy intermediate state with tightly bound (ADP-Pi). Binding of the second ATP then leads to the steady-state ATP cycle. As proposed previously, the role of steady-state ATP hydrolysis by the DNA-bound Rep subunit may be to maintain the DNA-free subunit in an activated state in preparation for binding a second fragment of DNA as needed for translocation and/or DNA unwinding. We propose that the roles of the two ATP sites may alternate upon binding DNA to the second subunit of the Rep dimer during unwinding and translocation using a subunit switching mechanism.     

Targeting deletion (homoeologous chromosome pairing locus) or addition line single copy sequences from cereal genomes.

We describe here a protocol for obtaining clones containing sequences present in low copy-number from genomic DNA where moderately and highly repeated sequences predominate. Specific chromosomal regions can be targeted by using deletion or addition line material. We have used this protocol to identify a sequence which has been deleted in both the tetraploid and hexaploid wheat mutants for the homoeologous chromosome pairing locus.     

Identification of a novel gene, DZIP (DAZ-interacting protein), that encodes a protein that interacts with DAZ (deleted in azoospermia) and is expressed in embryonic stem cells and germ cells

Evidence from diverse organisms, including humans, suggests that the DAZ (Deleted in Azoospermia) gene and a closely related homolog, DAZL (DAZ-like), are required early in germ cell development to maintain initial germ cell populations. Here we report the identification and characterization of the DZIP (DAZ-Interacting Protein) gene, which encodes at least three different protein isoforms that contain a C2H2 zinc-finger domain. The DZIP gene is expressed predominantly in human embryonic stem cells and fetal and adult germ cells; moreover, two DZIP protein isoforms colocalize with DAZ and/or DAZL proteins in these tissues. Finally, we provide evidence indicating that DZIP may associate with DAZ and its other cofactors in an RNA-binding protein complex that functions in both ES cells and germ cells.     

Seasonal dynamics of shallow-hyporheic-zone microbial community structure along a heavy-metal contamination gradient

Heavy metals contaminate numerous freshwater streams and rivers worldwide. Previous work by this group demonstrated a relationship between the structure of hyporheic microbial communities and the fluvial deposition of heavy metals along a contamination gradient during the fall season. Seasonal variation has been documented in microbial communities in numerous terrestrial and aquatic environments, including the hyporheic zone. The current study was designed to assess whether relationships between hyporheic microbial community structure and heavy-metal contamination vary seasonally by monitoring community structure along a heavy-metal contamination gradient for more than a year. No relationship between total bacterial abundance and heavy metals was observed (R(2) = 0.02, P = 0.83). However, denaturing gradient gel electrophoresis pattern analysis indicated a strong and consistent linear relationship between the difference in microbial community composition (populations present) and the difference in the heavy metal content of hyporheic sediments throughout the year (R(2) = 0.58, P < 0.001). Correlations between heavy-metal contamination and the abundance of four specific phylogenetic groups (most closely related to the alpha, beta, and gamma-proteobacteria and cyanobacteria) were apparent only during the fall and early winter, when the majority of organic matter is deposited into regional streams. These seasonal data suggest that the abundance of susceptible populations responds to heavy metals primarily during seasons when the potential for growth is highest.     

The receptors responsible for heat production in brown adipose tissue in the young rabbit

It is known that adrenergic agonists stimulate thermogenesis in the brown fat of the young rabbit but the receptors responsible for mediating the response have not been identified. The infusion of either noradrenaline or isoproterenol (1-2 micrograms . kg-1 X min-1) produced an increase in subcutaneous temperature (0.93 +/- 0.15 and 1.22 +/- 0.10 degrees C, respectively over the interscapular brown fat. At low doses (0.4 microgram . kg-1 X min-1) only isoproterenol was effective. The thermogenic response to isoproterenol was blocked by atenolol, a beta 1-adrenergic antagonist. Neither salbutamol or terbutaline, both beta 2-agonists, produced a temperature increase. Collectively, these data suggest that stimulation of beta 1-adrenoceptor is primarily responsible for the thermogenic activity of brown fat in the rabbit. However, it was found that 53% of the increase in temperature could be blocked by prazosin, an alpha 1-antagonist. Phentolamine was not effective as a blocker. Although a maximal brown fat thermogenic response can be achieved by stimulating the beta-adrenoceptors, the alpha-adrenoceptors appears to play at least an auxiliary role in young rabbit.