Water resource use and management by the United States forest products industry.

The connections between forest products operations and water resources in the United States is considered and, where possible, quantified. Manufacture of wood, pulp, and paper products and the influences of forest management and forest products manufacture on water quality are discussed. Most fresh water in the US originates in forested areas. Responsible harvesting strategies, best management practices, and forest re-growth combine to minimize or eliminate changes in water availability and degradation of water quality due to harvesting. Relative to alternative land uses and large-scale disturbance events, forested areas produce the highest quality of fresh water. Water inputs for the manufacture of forest products total about 5.8 billion m(3) per year, an amount equal about 0.4% of the surface and groundwater yield from timberland. Approximately 88% of water used in manufacturing is treated and returned directly to surface waters, about 11% is converted to water vapor and released during the manufacturing process, and 1% is imparted to products or solid residuals. Extensive study and continued monitoring of treated effluents suggest few or no concerns regarding the compatibility of current effluents with healthy aquatic systems.

Optical design of the atacama cosmology telescope and the millimeter bolometric array camera.

The Atacama Cosmology Telescope is a 6 m telescope designed to map the cosmic microwave background simultaneously at 145, 215, and 280 GHz with arcminute resolution. Each frequency will have a 32 by 32 element focal plane array of transition edge sensor bolometers. The telescope and the cold reimaging optics are optimized for millimeter-wave observations with these sensitive detectors. The design of each is described.

RNA triphosphatase is essential in Schizosaccharomyces pombe and Candida albicans.

BACKGROUND: The first two steps in the capping of cellular mRNAs are catalyzed by the enzymes RNA triphosphatase and RNA guanylyltransferase. Although structural and mechanistic differences between fungal and mammalian RNA triphosphatases recommend this enzyme as a potential antifungal target, it has not been determined if RNA triphosphatase is essential for the growth of fungal species that cause human disease. RESULTS: We show by classical genetic methods that the triphosphatase (Pct1) and guanylyltransferase (Pce1) components of the capping apparatus in the fission yeast Schizosaccharomyces pombe are essential for growth. We were unable to disrupt both alleles of the Candida albicans RNA triphosphatase gene CaCET1, implying that the RNA triphosphatase enzyme is also essential for growth of C. albicans, a human fungal pathogen. CONCLUSIONS: Our results provide the first genetic evidence that cap synthesis is essential for growth of an organism other than Saccharomyces cerevisiae and they validate RNA triphosphatase as a target for antifungal drug discovery.

T-loop phosphorylation stabilizes the CDK7-cyclin H-MAT1 complex in vivo and regulates its CTD kinase activity.

Cyclin-dependent kinase (CDK)7-cyclin H, the CDK-activating kinase (CAK) and TFIIH-associated kinase in metazoans can be activated in vitro through T-loop phosphorylation or binding to the RING finger protein MAT1. Although the two mechanisms can operate independently, we show that in a physiological setting, MAT1 binding and T-loop phosphorylation cooperate to stabilize the CAK complex of Drosophila. CDK7 forms a stable complex with cyclin H and MAT1 in vivo only when phosphorylated on either one of two residues (Ser164 or Thr170) in its T-loop. Mutation of both phosphorylation sites causes temperature-dependent dissociation of CDK7 complexes and lethality. Furthermore, phosphorylation of Thr170 greatly stimulates the activity of the CDK7- cyclin H-MAT1 complex towards the C-terminal domain of RNA polymerase II without significantly affecting activity towards CDK2. Remarkably, the substrate-specific increase in activity caused by T-loop phosphorylation is due entirely to accelerated enzyme turnover. Thus phosphorylation on Thr170 could provide a mechanism to augment CTD phosphorylation by TFIIH-associated CDK7, and thereby regulate transcription.

Reciprocal activation by cyclin-dependent kinases 2 and 7 is directed by substrate specificity determinants outside the T loop.

Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of the metazoan CDK-activating kinase (CAK), which activates CDKs, such as CDC2 and CDK2, through phosphorylation of a conserved threonine residue in the T loop. Full activation of CDK7 requires association with a positive regulatory subunit, cyclin H, and phosphorylation of a conserved threonine residue at position 170 in its own T loop. We show that threonine-170 of CDK7 is phosphorylated in vitro by its targets, CDC2 and CDK2, which also phosphorylate serine-164 in the CDK7 T loop, a site that perfectly matches their consensus phosphorylation site. In contrast, neither CDK4 nor CDK7 itself can phosphorylate the CDK7 T loop in vitro. The ability of CDC2 or CDK2 and CDK7 to phosphorylate each other but not themselves implies that each kinase can discriminate among closely related sequences and can recognize a substrate site that diverges from its usual preferred site. To understand the basis for this paradoxical substrate specificity, we constructed a chimeric CDK with the T loop of CDK7 grafted onto the body of CDK2. Surprisingly, the hybrid enzyme, CDK2-7, was efficiently activated in cyclin A-dependent fashion by CDK7 but not at all by CDK2. CDK2-7, moreover, phosphorylated wild-type CDK7 but not CDK2. Our results suggest that the primary amino acid sequence of the T loop plays only a minor role, if any, in determining the specificity of cyclin-dependent CAKs for their CDK substrates and that protein-protein interactions involving sequences outside the T loop can influence substrate specificity both positively and negatively.

From the lab to the police station. A successful application of eyewitness research.

The U.S. Department of Justice released the first national guide for collecting and preserving eyewitness evidence in October 1999. Scientific psychology played a large role in making a case for these procedural guidelines as well as in setting a scientific foundation for the guidelines, and eyewitness researchers directly participated in writing them. The authors describe how eyewitness researchers shaped understanding of eyewitness evidence issues over a long period of time through research and theory on system variables. Additional pressure for guidelines was applied by psychologists through expert testimony that focused on deficiencies in the procedures used to collect the eyewitness evidence. DNA exoneration cases were particularly important in leading U.S. Attorney General Janet Reno to notice the eyewitness literature in psychology and to order the National Institute of Justice to coordinate the development of national guidelines. The authors describe their experience as members of the working group, which included prosecutors, defense lawyers, and law enforcement officers from across the country.

Adapting the cognitive interview to enhance long-term (35 years) recall of physical activities.

The cognitive interview (CI) was modified for use in an epidemiological study in which respondents were asked to recall their daily physical activities from the distant past (35 years ago). In comparison to a traditional epidemiological interview, the CI elicited many more responses and also more precise responses. Several practical costs, however, were incurred by the CI: additional time to train interviewers and to conduct interviews and difficulties in coding the responses. The costs and benefits of conducting the CI are addressed, along with conceptual and methodological challenges. The article ends with an existential question: Is the CI a singular technique if it can be modified so radically for different settings?

Directed evolution to bypass cyclin requirements for the Cdc28p cyclin-dependent kinase.

To identify cyclin-dependent kinase mutants with relaxed cyclin requirements, CDC28 alleles were selected that could rescue a yeast strain expressing as its only CLN G1 cyclin a mutant Cln2p (K129A,E183A) that is defective for Cdc28p binding. Rescue of this strain by mutant CDC28 was dependent upon the mutant cln2-KAEA, but additional mutagenesis and DNA shuffling yielded multiply mutant CDC28-BYC alleles (bypass of CLNs) that could support highly efficient cell cycle initiation in the complete absence of CLN genes. By gel filtration chromatography, one of the mutant Cdc28 proteins exhibited kinase activity associated with cyclin-free monomer. Thus, the mutants' CLN bypass activity might result from constitutive, cyclin-independent activity, suggesting that Cdk targeting by cyclins is not required for cell cycle initiation.

Inhibition of transcription by the trimeric cyclin-dependent kinase 7 complex.

Cyclin-dependent kinase 7 (CDK7) can be isolated as a subunit of a trimeric kinase complex functional in activation of the mitotic promoting factor. In this study, we demonstrate that the trimeric cdk-activating kinase (CAK) acts as a transcriptional repressor of class II promoters and show that repression results from CAK impeding the entry of RNA polymerase II and basal transcription factor IIF into a competent preinitiation complex. This repression is independent of CDK7 kinase activity. We find that the p36/MAT1 subunit of CAK is required for transcriptional repression and the repression is independent of the promoter used. Our results demonstrate a central role for CAK in regulation of messenger RNA synthesis by either inhibition of RNA polymerase II-catalyzed transcription or stimulation of transcription through association with basal transcription repair factor IIH.

Cdc2 activation in fission yeast depends on Mcs6 and Csk1, two partially redundant Cdk-activating kinases (CAKs).

Cyclin-dependent kinases (Cdks) are fully active only when phosphorylated by a Cdk-activating kinase (CAK) [1]. Metazoan CAK is itself a Cdk, Cdk7, whereas the CAK of Saccharomyces cerevisiae is a distinct enzyme unrelated to Cdks [1]. The Mcs6-Mcs2 complex of Schizosaccharomyces pombe is a putative CAK related to the metazoan enzyme [2] [3]. Although the loss of Mcs6 is lethal, it results in a phenotype that is inconsistent with a failure to activate Cdc2, the major Cdk in S. pombe [3]. We therefore tested the ability of Csk1, a putative regulator of Mcs6 [4], to activate Cdk-cyclin complexes in vitro. Csk1 activated both the monomeric and the Mcs2-bound forms of Mcs6. Surprisingly, Csk1 also activated Cdc2 in complexes with either Cdc13 or Cig2 cyclins. When a double mutant carrying a csk1 deletion and a temperature-sensitive mcs6 allele was incubated at the restrictive temperature, Cdc2 was not activated and the cells underwent a cell division arrest prior to mitosis. Cdc2-cyclin complexes isolated from the arrested cells could be activated in vitro by recombinant CAK, whereas complexes from wild-type cells or either of the single mutants were refractory to activation. Thus, fission yeast contains two partially redundant CAKs: the Mcs6-Mcs2 complex and Csk1. Inactivation of both CAKs is necessary and sufficient to prevent Cdc2 activation and cause a cell-cycle arrest. Mcs6, which is essential, may therefore have required functions other than Cdk activation.

The relation of output order and commission errors in free recall and eyewitness accounts.

We explored the relation between output order and the likelihood of a commission error in free recall under both laboratory and eyewitness conditions. In Experiment 1, participants studied a list of 20 unrelated words and, after a five-minute distractor task, were asked to recall those words. Whereas the items that participants recalled were mostly correct, commission errors were more likely to occur at the end of a participant's output. In Experiment 2, participants viewed a police film depicting an armed robbery. Participants described the perpetrators, their truck, and the sequence of events during the robbery. When describing the perpetrators or the truck, commission errors were more likely to occur at the end of the output. However, when describing the sequence of events, commission errors were more likely to occur in the middle of the output. In Experiment 3, we replicated the finding that commission errors are likely to occur at the end of the output order when participants are describing people. We speculate on the potential application of this finding and its theoretical underpinnings.

Cdk7 is essential for mitosis and for in vivo Cdk-activating kinase activity.

Cdk7 has been shown previously to be able to phosphorylate and activate many different Cdks in vitro. However, conclusive evidence that Cdk7 acts as a Cdk-activating kinase (CAK) in vivo has remained elusive. Adding to the controversy is the fact that in the budding yeast Saccharomyces cerevisiae, CAK activity is provided by the CAK1/Civ1 protein, which is unrelated to Cdk7. Furthermore Kin28, the budding yeast Cdk7 homolog, functions not as a CAK but as the catalytic subunit of TFIIH. Vertebrate Cdk7 is also known to be part of TFIIH. Therefore, in the absence of better genetic evidence, it was proposed that the CAK activity of Cdk7 may be an in vitro artifact. In an attempt to resolve this issue, we cloned the Drosophila cdk7 homolog and created null and temperature-sensitive mutations. Here we demonstrate that cdk7 is necessary for CAK activity in vivo in a multicellular organism. We show that cdk7 activity is required for the activation of both Cdc2/Cyclin A and Cdc2/Cyclin B complexes, and for cell division. These results suggest that there may be a fundamental difference in the way metazoans and budding yeast effect a key modification of Cdks.

The CDK7-cycH-p36 complex of transcription factor IIH phosphorylates p53, enhancing its sequence-specific DNA binding activity in vitro.

Phosphorylation is believed to be one of the mechanisms by which p53 becomes activated or stabilized in response to cellular stress. Previously, p53 was shown to interact with three components of transcription factor IIH (TFIIH): excision repair cross-complementing types 2 and 3 (ERCC2 and ERCC3) and p62. This communication demonstrates that p53 is phosphorylated by the TFIIH-associated kinase in vitro. The phosphorylation was found to be catalyzed by the highly purified kinase components of TFIIH, the CDK7-cycH-p36 trimeric complex. The phosphorylation sites were mapped to the C-terminal amino acids located between residues 311 and 393. Serines 371, 376, 378, and 392 may be the potential sites for this kinase. Phosphorylation of p53 by this kinase complex enhanced the ability of p53 to bind to the sequence-specific p53-responsive DNA element as shown by gel mobility shift assays. These results suggest that the CDK7-cycH-p36 trimeric complex of TFIIH may play a role in regulating p53 functions in cells.

CDKs and cyclins in transition(s).

Understanding of cyclin-dependent kinase (CDK) regulation in mammalian cells has deepened even as the functions ascribed to these enzymes have multiplied. We know from crystallographic studies how a prototypic CDK-cyclin complex is activated and inactivated; the challenge now is to extend this knowledge to other CDKs involved in cell cycle progression. At the same time, as CDKs turn up in some unexpected places, interest in CDK regulation has spread beyond the cell cycle field.

Alternative mechanisms of CAK assembly require an assembly factor or an activating kinase.

We have cloned a mouse cDNA that encodes p36, a novel subunit of the CDK-activating kinase (CAK). p36 contains a C3HC4 zinc-binding domain or RING factor and is associated both with a TFIIH-bound form of CAK and with a free trimeric form. p36 promotes the assembly of CDK7 and cyclin H in vitro, stabilizing the transient CDK7-cyclin H complex. Stabilization and activation of CAK by p36 is independent of the phosphorylation state of T170, the conserved activating residue of CDK7. Assembly of active CDK7-cyclin H dimers can also occur through an alternative p36-independent pathway that requires phosphorylation of T170 by a CAK-activating kinase, or CAKAK. Thus, CDK7-cyclin H complex formation can be achieved by multiple mechanisms.

Facilitating children's eyewitness recall with the revised cognitive interview.

Eighty-six 2nd-grade children participated in a Simon says game with an unfamiliar adult. The children were subsequently interviewed twice with either a standard interview or the revised cognitive interview (CI), once within 3 hr of the event and then 2 weeks later. On both the initial interview and the 2-week delayed interview, children receiving the revised CI recalled significantly more correct information than did children receiving a standard interview. In addition, children who were interviewed twice with the revised CI recalled more unique accurate facts (M = 25.44) than children who received 2 standard interviews (M = 16.75). The CI also elicited more inaccurate facts; however, the accuracy rate (proportion of reported facts that were accurate) for the 2 groups was equivalent. The research has implications for police and others who interview real child victims and witnesses.

Cdk-activating kinase complex is a component of human transcription factor TFIIH.

Transcription factor IIH (TFIIH) contains a kinase capable of phosphorylating the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII). Here we report the identification of the Cdk-activating kinase (Cak) complex (Cdk7 and cyclin H) as a component of TFIIH after extensive purification of TFIIH by chromatography. We find that affinity-purified antibodies directed against cyclin H inhibit TFIIH-dependent transcription and that both cyclin H and Cdk7 antibodies inhibit phosphorylation of the CTD of the largest subunit of the RNAPII in the preinitiation complex. Cak is present in at least two distinct complexes, TFIIH and a smaller complex that is unable to phosphorylate RNAPII in the preinitiation complex. Both Cak complexes, as well as recombinant Cak, phosphorylate a CTD peptide. Finally, TFIIH was shown to phosphorylate both Cdc2 and Cdk2, suggesting that there could be a link between transcription and the cell cycle machinery.