Different time course recovery of muscle edema within the quadriceps femoris and functional performance after single- vs multi-joint exercises.

This study aimed to verify the time course recovery of muscle edema within the quadriceps femoris and functional performance after lower-body single- and multi-joint exercises. For this within-participant unilateral and contralateral experimental design, fourteen untrained young males performed a unilateral knee extension exercise (KE), and a unilateral leg press (LP) exercise in a counterbalanced order. At pre-, post-, 24 h, 48 h, 72 h, and 96 h after exercise, the peak torque (PT), unilateral countermovement jump (uCMJ) performance, and rectus femoris (RF) and vastus lateralis (VL) muscle thicknesses were recorded in both legs. The PT decreased immediately after (p = 0.01) both exercises (KE and LP) and was fully recovered 24 h after KE (p = 0.38) and 48 h after LP (p = 0.68). Jump height and power, in the uCMJ, followed the same PT recovery pattern after both exercises. However, vertical stiffness (Kvert) was not affected at any time point after both protocols. The RF thickness increased after both exercises (p = 0.01) and was fully restored 48 h after KE (p = 0.86) and 96 h after LP (p = 1.00). The VL thickness increased after both exercises (p = 0.01) and was fully restored 24 h after LP (p = 1.00) and 48 h after KE (p = 1.00). The LP exercise, compared to KE, induced more prolonged impairment of functional performance and delayed recovery of RF muscle edema. However, the VL edema-induced muscle swelling recovery was delayed after the KE exercise. The different recovery kinetics between functional performance and muscle damage should be taken into consideration depending on the objectives of the next training sessions.

What Can Reinforcement Learning Models of Dopamine and Serotonin Tell Us about the Action of Antidepressants?

Although evidence suggests that antidepressants are effective at treating depression, the mechanisms behind antidepressant action remain unclear, especially at the cognitive/computational level. In recent years, reinforcement learning (RL) models have increasingly been used to characterise the roles of neurotransmitters and to probe the computations that might be altered in psychiatric disorders like depression. Hence, RL models might present an opportunity for us to better understand the computational mechanisms underlying antidepressant effects. Moreover, RL models may also help us shed light on how these computations may be implemented in the brain (e.g., in midbrain, striatal, and prefrontal regions) and how these neural mechanisms may be altered in depression and remediated by antidepressant treatments. In this paper, we evaluate the ability of RL models to help us understand the processes underlying antidepressant action. To do this, we review the preclinical literature on the roles of dopamine and serotonin in RL, draw links between these findings and clinical work investigating computations altered in depression, and appraise the evidence linking modification of RL processes to antidepressant function. Overall, while there is no shortage of promising ideas about the computational mechanisms underlying antidepressant effects, there is insufficient evidence directly implicating these mechanisms in the response of depressed patients to antidepressant treatment. Consequently, future studies should investigate these mechanisms in samples of depressed patients and assess whether modifications in RL processes mediate the clinical effect of antidepressant treatments.

High Supervised Resistance Training in Elderly Women: The Role of Supervision Ratio.

The objective of this study was to compare the effects of very high supervision (VHS-RT) versus high supervision (HS-RT) ratio resistance training (RT) on irisin, brain-derived neurotrophic factor (BNDF), muscle strength, functional capacity, and body composition in elderly women. Participants performed daily undulating periodized RT over 16 weeks with two different supervision ratios: VHS-RT at 1:2 (supervisor/subject) or HS-RT at 1:5. Serum was used to analyze brain derived neurotropic factor (BDNF) and irisin by enzyme-linked immunosorbent assay (ELISA). Body composition was evaluated by dual-energy X-ray absorptiometry, while functional capacity was evaluated using the Six-minute walk test, and Timed Up and Go (TUG). One- repetition maximum (1RM) was determined for bench press and 45° leg press exercises. For both groups, no differences between baseline and post-training were identified for irisin and lean mass (p > 0.05). Both groups improved bench press 1-RM, 45° leg press 1-RM, and TUG (p < 0.05). The VHS-RT group displayed higher effect sizes for 1-RM tests. Moreover, only VHS-RT group reduced body fat and body fat percentage (p < 0.05). In contrast, the HS-RT increased BDNF (p < 0.01). In this sense, RT enhances muscle strength and functional capacity in elderly women independent of supervision ratio. A greater supervision ratio during RT may induce more improvements in muscle strength, and body composition than lower supervision ratio during RT.

Corrigendum: Two Consecutive Days of Extreme Conditioning Program Training Affects Pro and Anti-inflammatory Cytokines and Osteoprotegerin without Impairments in Muscle Power.

[This corrects the article DOI: 10.3389/fphys.2016.00260.].

Two Consecutive Days of Crossfit Training Affects Pro and Anti-inflammatory Cytokines and Osteoprotegerin without Impairments in Muscle Power.

The aim of this study was to investigate the effects of two consecutive Crossfit® training sessions (24 h apart) designed to enhance work-capacity that involved both cardiovascular and muscular exercises on cytokines, muscle power, blood lactate and glucose. Nine male members of the CrossFit® community (age 26.7 ± 6.6 years; body mass 78.8 ± 13.2 kg; body fat 13.5 ± 6.2%; training experience 2.5 ± 1.2 years) completed two experimental protocols (24 h apart): (1) strength and power exercises, (2) gymnastic movements, and (3) metabolic conditioning as follows: 10 min of as many rounds as possible (AMRAP) of 30 double-unders and 15 power snatches (34 kg). The same sequence as repeated on session 2 with the following metabolic conditioning: 12 min AMRAP of: row 250 m and 25 target burpees. Serum interleukin-6 (IL-6), IL-10, and osteoprotegerin were measured before, immediately post and 24 h after workout of the day (WOD) 1, immediately post, 24 and 48 h after WOD 2. Peak and mean power were obtained for each repetition (back squat with 50% of 1 repetition maximum) using a linear position transducer measured before, immediately post and 24 h after WOD 1, immediately post and 24 h after WOD 2. Blood lactate and glucose were measured pre and immediately post WOD 1 and 2. Although both sessions of exercise elicited an significant increase in blood lactate (1.20 ± 0.41 to 11.84 ± 1.34 vs. 0.94 ± 0.34 to 9.05 ± 2.56 mmol/l) and glucose concentration (81.59 ± 10.27 to 114.99 ± 12.52 vs. 69.47 ± 6.97 to 89.95 ± 19.26 mg/dL), WOD 1 induced a significantly greater increase than WOD 2 (p ≤ 0.05). The training sessions elicited significant changes (p ≤ 0.05) in IL-6, IL-10 and osteoprotegerin concentration over time. IL-6 displayed an increase immediately after training WOD 1 [197 ± 109%] (p = 0.009) and 2 [99 ± 58%] (p = 0.045). IL-10 displayed an increase immediately after only WOD 1 [44 ± 52%] (p = 0.046), and decreased 24 and 48 h following WOD 2 (~40%; p = 0.018) as compared to pre-exercise values. Osteoprotegerin displayed a decrease 48 h following WOD 2 (~25%; p = 0.018) as compared with pre intervention. In conclusion, two consecutive Crossfit® training sessions increase pro/anti-inflammatory cytokines with no interference on muscle performance in the recovery period.

Salivary nitrite content, cognition and power in Mixed Martial Arts fighters after rapid weight loss: a case study.

BACKGROUND AND AIM: Rapid weight loss (RWL) is extensively practiced by combat sports athletes, including Mixed Martial Arts (MMA), but its effects on performance are not well established with different magnitudes of RWL, including those higher than 5% of total body weight. The aim of the present study was to follow MMA athletes during RWL with subsequent weight regain to evaluate the responses of isometric strength, power, cognition and salivary nitrite ( NO 2 - ) content. METHODS: Two professional male MMA fighters, same age, competing in the same weight category underwent two magnitudes of RWL before a simulated competition period. Anthropometric measures, records of nutritional status, training, voluntary dehydration strategies, salivary samples, cognition response, isometric strength and muscular power were obtained: (I) 7 days before combat, (II) at the weigh-in moment, and (III) in the combat day. RESULTS AND CONCLUSIONS: Athlete 1 lost 7.2 kg (9.1% of total bodyweight) and Athlete 2 lost 4.0 kg (5.3% of total bodyweight). Athlete 1 had a lower and misbalanced caloric ingestion (708 ± 428 kcal), ingested 6 L of water during the first 5 days of RWL, underwent 2 days of fasting, water and sodium restriction before weigh-in. Athlete 2 was supervised by a nutritionist, had a balanced diet (1600 ± 0 kcal), ingested 2 L of water during the first 6 days of RWL, underwent only 1 day of fasting and water restriction, and did not restrict sodium. As expected, there was a negative effect of RWL in the evaluated parameters at the weigh-in moment, while in the combat day, salivary NO 2 - ) content. METHODS: Two professional male MMA fighters, same age, competing in the same weight category underwent two magnitudes of RWL before a simulated competition period. Anthropometric measures, records of nutritional status, training, voluntary dehydration strategies, salivary samples, cognition response, isometric strength and muscular power were obtained: (I) 7 days before combat, (II) at the weigh-in moment, and (III) in the combat day. RESULTS AND CONCLUSIONS: Athlete 1 lost 7.2 kg (9.1% of total bodyweight) and Athlete 2 lost 4.0 kg (5.3% of total bodyweight). Athlete 1 had a lower and misbalanced caloric ingestion (708 ± 428 kcal), ingested 6 L of water during the first 5 days of RWL, underwent 2 days of fasting, water and sodium restriction before weigh-in. Athlete 2 was supervised by a nutritionist, had a balanced diet (1600 ± 0 kcal), ingested 2 L of water during the first 6 days of RWL, underwent only 1 day of fasting and water restriction, and did not restrict sodium. As expected, there was a negative effect of RWL in the evaluated parameters at the weigh-in moment, while in the combat day, salivary NO 2 - was not completely reestablished at baseline levels (decreased by 35.9% in Athlete 1 and, 25.2% in Athlete 2, as compared with 7 days before). The athlete who underwent a lower weight loss (5.3%) presented better recovery of cognition and upper limbs power on the combat day as compared with the athlete who lost 9.1% of body weight. Although we cannot precisely conclude, this case report led us to believe that the recovery period between weigh-in and competition may be insufficient for total reestablishment of salivary NO 2 - after RWL, and higher amounts of RWLhave negative impacts on average power and cognition when compared with lower RWL.Relevance for patients: Scientific aspects related with performance in MMA athletes brought to light the absence of studies investigating the recovery of isometric strength, power, cognition and salivary NO 2 - during RWL with subsequent weight regain. This study revealed that athletes from the same categories can adopt different magnitudes of weight loss, and that this procedure impacts several important measures, for example, the reduction of salivary NO 2 - is associated with the lower O2 transport capacity, decreasing muscle performance.

Different acute cardiovascular stress in response to resistance exercise leading to failure versus not to failure in elderly women with and without hypertension--a pilot study.

OBJECTIVE: The purpose of the present study was to compare the effects of resistance exercise (RE) leading to failure versus not to failure on 24-h blood pressure (BP) and rate-pressure product (RPP) responses in normotensive and hypertensive trained elderly women. METHODOLOGY: Seven normotensive women and seven women with medically documented hypertension randomly performed three experimental sessions: (i) a non-exercise control session that involved 30 min of seated rest, (ii) whole body RE leading to failure that involved three sets with an eight repetitions maximum (8RM) load and (iii) whole body RE not to failure that involved three sets with 70% of an 8RM load. Systolic BP (SBP), diastolic BP (DBP) and mean BP (MBP) responses during each hour of sleep and awake states were measured. RESULTS: Results of all subjects revealed that the RPP was higher (P ≤ 0.05) during afternoon and night hours after the RE session leading to failure versus not to failure and the non-exercise control session. For the hypertensive group during the night hours, SBP remained higher after the RE session not to failure (P = 0.047) versus non-exercise control session. For the normotensive group, DBP remained higher after the RE session leading to failure over the 24-h period (approximately 8 mmHg h(-1), P = 0.044) and the period upon awaking (approximately 5 mmHg h(-1), P = 0.044) versus the hypertensive group. CONCLUSIONS: The normotensive elderly women of this pilot study presented a greater cardiovascular response to RE leading to failure, as a consequence of the higher training intensity.

Effects of rest interval length on Smith machine bench press performance and perceived exertion in trained men.

This study compared two different rest intervals (RI) between sets of resistance exercise. Ten resistance-trained men (M age = 24.3, SD = 3.5 yr.; M weigh t= 80.0 kg, SD = 15.3; M height = 1.75 m, SD = 0.04) performed five sets of Smith machine bench presses at 60% of one repetition maximum, either with 1.5 min. or 3 min. RI between sets. Their repetition performance, total training volume, velocity, fatigue, rating of perceived exertion, and muscular power were measured. All of these measures indicated that performance was significantly better and fatigue was significantly lower in the 3 min. RI as compared with the 1.5 min. RI, except the rating of perceived exertion which did not show a significant difference. A longer RI between sets promotes superior performance for the bench press.

Effect of bicarbonate on fluoride reactivity with enamel / Efeito do bicarbonato na reatividade do fluoreto com esmalte

Purpose: Bicarbonate (HCO-3) is an alkaline and buffering substance found in dentifrices, which could improve the anti-caries effect of fluoride (F). However, HCO-3 could reduce the formation of calcium fluoride-like (CaF2), the most important product of F reactivity with enamel, whose formation is higher in low pH. The aim of this in vitro study was to evaluate if HCO-3 interferes with the reactivity of F with human enamel. Methods: Five dentifrice formulations were evaluated: placebo of F and HCO-3 (pH 7.0); HCO-3 (pH 9.0); F (pH 7.0); F (pH 9.0) and F+HCO-3 (pH 9.0). F dentifrices contained NaF and all dentifrices were silica-based. The concentrations of total F (TF), CaF2 and firmly bound F (FA, fluorapatite-like) formed in enamel after 1-min reaction with dentifrice slurries (1:3) were determined. Results: The formation of TF, CaF2 and FA was reduced in 22.1 %, 47.9 % and 4.8 %, respectively, by the presence of HCO-3 in the dentifrice formulation. Conclusion: This in vitro data suggest that addition of HCO-3 to a dentifrice may interfere with the reactivity of F with enamel, reducing mainly the concentration of CaF2 formed.

Objetivo: O bicarbonato (HCO-3 ) é uma substância alcalina e tamponante encontrada em dentifrícios, que poderia melhorar o efeito anticárie do fluoreto (F). No entanto, HCO-3 poderia reduzir a formação de fluoreto de cálcio (CaF2), o mais importante produto da reatividade do F com esmalte, cuja formação é maior em baixo pH. O objetivo deste estudo foi avaliar in vitro se o HCO-3 interfere na reatividade do F com o esmalte humano. Metodologia: Cinco formulações de dentifrícios foram avaliadas: placebo de F e HCO-3(pH 7,0); HCO-3 (pH 9,0); F (pH 7,0); F (pH 9,0) e F+HCO-3 (pH 9,0). Os dentifrícios fluoretados continham NaF e todos continham sílica como abrasivo. As concentrações de F total (FT), CaF2 e F firmemente ligado (FA, tipo flúorapatita) formadas no esmalte após 1 minuto de reação com as suspensões dos dentifrícios (1:3) foram determinadas. Resultados: A formação de FT, CaF2 e FA foram reduzidas em 22,1 %; 47,9 % e 4,8 %, respectivamente, pela presença de HCO-3 na formulação do dentifrício. Conclusão: Os resultados in vitro sugerem que a adição de HCO-3 a um dentifrício pode interferir com a reatividade do F com o esmalte, principalmente reduzindo a concentração de CaF2 formado no esmalte.

Purification and characterization of the 1.0 MDa CCR4-NOT complex identifies two novel components of the complex.

The CCR4-NOT complex is an evolutionarily conserved, transcriptional regulatory complex that is involved in controlling mRNA initiation, elongation and degradation. The CCR4-NOT proteins from Saccharomyces cerevisiae exist in two complexes, 1.9x10(6) Da and 1.0x10(6) Da (1.0 MDa) in size, and individual components of these complexes display such disparate functions as binding to and restricting TFIID functions, contacting SAGA and contributing to mRNA deadenylation. As a first step in characterizing the functional roles of the 1.0 MDa complex, we have purified it to near homogeneity. Mass spectrometric analysis was subsequently used to identify all the components of the complex. The 1.0 MDa complex was found to contain CCR4, CAF1, NOT1-5 and two new proteins, CAF40 and CAF130. CAF130 and CAF40 are two unique yeast proteins, with CAF40 displaying extensive homology to proteins from other eukaryotes. Immunoprecipitation and gel filtration experiments confirmed that CAF130 and CAF40 are components of both of the 1.9 MDa and 1.0 MDa CCR4-NOT complexes. Biochemical analysis indicated that the CAF40 and CAF130 proteins bind to the NOT1 protein and exist in a location separate from the two other subsets of proteins in the complex: the CCR4 and CAF1 proteins, and the NOT2, NOT4 and NOT5 proteins. Moreover, CAF40 was able to interact with human NOT1, suggesting that human CAF40 would also be a component of the recently identified human CCR4-NOT complex. Analysis of caf40 and caf130 deletions indicated that they elicited phenotypes shared by defects in other CCR4-NOT genes. The distinct location of CAF40 and CAF130 and the evolutionary conservation of CAF40 implicate them in novel roles in the function of the CCR4-NOT complex.

Genetic evidence supports a role for the yeast CCR4-NOT complex in transcriptional elongation.

The CCR4-NOT complex is involved in the regulation of gene expression both positively and negatively. The repressive effects of the complex appear to result in part from restricting TBP access to noncanonical TATAA binding sites presumably through interaction with multiple TAF proteins. We provide here genetic evidence that the CCR4-NOT complex also plays a role in transcriptional elongation. First, defects in CCR4-NOT components as well as overexpression of the NOT4 gene elicited 6-azauracil (6AU) and mycophenolic acid sensitivities, hallmarks of transcriptional elongation defects. A number of other transcription initiation factors known to interact with the CCR4-NOT complex did not elicit these phenotypes nor did defects in factors that reduced mRNA degradation and hence the recycling of NTPs. Second, deletion of ccr4 resulted in severe synthetic effects with mutations or deletions in the known elongation factors RPB2, TFIIS, and SPT16. Third, the ccr4 deletion displayed allele-specific interactions with rpb1 alleles that are thought to be important in the control of elongation. Finally, we found that a ccr4 deletion as well as overexpression of the NOT1 gene specifically suppressed the cold-sensitive phenotype associated with the spt5-242 allele. The only other known suppressors of this spt5-242 allele are factors involved in slowing transcriptional elongation. These genetic results are consistent with the model that the CCR4-NOT complex, in addition to its known effects on initiation, plays a role in aiding the elongation process.

The transcription factor associated Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae.

The major pathways of mRNA turnover in eukaryotes initiate with shortening of the poly(A) tail. We demonstrate by several criteria that CCR4 and CAF1 encode critical components of the major cytoplasmic deadenylase in yeast. First, both Ccr4p and Caf1p are required for normal mRNA deadenylation in vivo. Second, both proteins localize to the cytoplasm. Third, purification of Caf1p copurifies with a Ccr4p-dependent poly(A)-specific exonuclease activity. We also provide evidence that the Pan2p/Pan3p nuclease complex encodes the predominant alternative deadenylase. These results, and previous work on Pan2p/Pan3p, define the mRNA deadenylases in yeast. The strong conservation of Ccr4p, Caf1p, Pan2p, and Pan3p indicates that they will function as deadenylases in other eukaryotes. Interestingly, because Ccr4p and Caf1p interact with transcription factors, these results suggest an unexpected link between mRNA synthesis and turnover.

Characterization of CAF4 and CAF16 reveals a functional connection between the CCR4-NOT complex and a subset of SRB proteins of the RNA polymerase II holoenzyme.

The CCR4-NOT transcriptional regulatory complex affects transcription both positively and negatively and consists of the following two complexes: a core 1 x 10(6) dalton (1 MDa) complex consisting of CCR4, CAF1, and the five NOT proteins and a larger, less defined 1.9-MDa complex. We report here the identification of two new factors that associate with the CCR4-NOT proteins as follows: CAF4, a WD40-containing protein, and CAF16, a putative ABC ATPase. Whereas neither CAF4 nor CAF16 was part of the core CCR4-NOT complex, both CAF16 and CAF4 appeared to be present in the 1.9-MDa complex. CAF4 also displayed physical interactions with multiple CCR4-NOT components and with DBF2, a likely component of the 1.9-MDa complex. In addition, both CAF4 and CAF16 were found to interact in a CCR4-dependent manner with SRB9, a component of the SRB complex that is part of the yeast RNA polymerase II holoenzyme. The three related SRB proteins, SRB9, SRB10, and SRB11, were found to interact with and to coimmunoprecipitate DBF2, CAF4, CCR4, NOT2, and NOT1. Defects in SRB9 and SRB10 also affected processes at the ADH2 locus known to be controlled by components of the CCR4-NOT complex; an srb9 mutation was shown to reduce ADH2 derepression and either an srb9 or srb10 allele suppressed spt10-enhanced expression of ADH2. In addition, srb9 and srb10 alleles increased ADR1(c)-dependent ADH2 expression; not4 and not5 deletions are the only other known defects that elicit this phenotype. These results suggest a close physical and functional association between components of the CCR4-NOT complexes and the SRB9, -10, and -11 components of the holoenzyme.

Functional interaction of CCR4-NOT proteins with TATAA-binding protein (TBP) and its associated factors in yeast.

The CCR4-NOT transcriptional regulatory complex affects expression of a number of genes both positively and negatively. We report here that components of the CCR4-NOT complex functionally and physically interact with TBP and TBP-associated factors. First, mutations in CCR4-NOT components suppressed the his4-912delta insertion in a manner similar to that observed for the defective TBP allele spt15-122. Second, using modified HIS3 promoter derivatives containing specific mutations within the TATA sequence, we found that the NOT proteins were general repressors that disrupt TBP function irrespective of the DNA sequence. Third, increasing the dosage of NOT1 specifically inhibited the ability of spt15-122 to suppress the his4-912delta insertion but did not affect the Spt- phenotype of spt3 or spt10 at this locus. Fourth, spt3, spt8, and spt15-21 alleles (all involved in affecting interaction of SPT3 with TBP) suppressed ccr4 and caf1 defects. Finally, we show that NOT2 and NOT5 can be immunoprecipitated by TBP. NOT5 was subsequently shown to associate with TBP and TAFs and this association was dependent on the integrity of TFIID. These genetic and physical interactions indicate that one role of the CCR4-NOT proteins is to inhibit functional TBP-DNA interactions, perhaps by interacting with and modulating the function of TFIID.

The CCR4 and CAF1 proteins of the CCR4-NOT complex are physically and functionally separated from NOT2, NOT4, and NOT5.

The CCR4-NOT complex (1 mDa in size), consisting of the proteins CCR4, CAF1, and NOT1 to NOT5, regulates gene expression both positively and negatively and is distinct from other large transcriptional complexes in Saccharomyces cerevisiae such as SNF/SWI, TFIID, SAGA, and RNA polymerase II holoenzyme. The physical and genetic interactions between the components of the CCR4-NOT complex were investigated in order to gain insight into how this complex affects the expression of diverse genes and processes. The CAF1 protein was found to be absolutely required for CCR4 association with the NOT proteins, and CCR4 and CAF1, in turn, physically interacted with NOT1 through its central amino acid region from positions 667 to 1152. The NOT3, NOT4, and NOT5 proteins had no significant effect on the association of CCR4, CAF1, and NOT1 with each other. In contrast, the NOT2, NOT4, and NOT5 interacted with the C-terminal region (residues 1490 to 2108) of NOT1 in which NOT2 and NOT5 physically associated in the absence of CAF1, NOT3, and NOT4. These and other data indicate that the physical ordering of these proteins in the complex is CCR4-CAF1-NOT1-(NOT2, NOT5), with NOT4 and NOT3 more peripheral to NOT2 and NOT5. The physical separation of CCR4 and CAF1 from other components of the CCR4-NOT complex correlated with genetic analysis indicating partially separate functions for these two groups of proteins. ccr4 or caf1 deletion suppressed the increased 3-aminotriazole resistance phenotype conferred by not mutations, resulted in opposite effects on gene expression as compared to several not mutations, and resulted in a number of synthetic phenotypes in combination with not mutations. These results define the CCR4-NOT complex as consisting of at least two physically and functionally separated groups of proteins.

A complex containing RNA polymerase II, Paf1p, Cdc73p, Hpr1p, and Ccr4p plays a role in protein kinase C signaling.

Yeast contains at least two complex forms of RNA polymerase II (Pol II), one including the Srbps and a second biochemically distinct form defined by the presence of Paf1p and Cdc73p (X. Shi et al., Mol. Cell. Biol. 17:1160-1169, 1997). In this work we demonstrate that Ccr4p and Hpr1p are components of the Paf1p-Cdc73p-Pol II complex. We have found many synthetic genetic interactions between factors within the Paf1p-Cdc73p complex, including the lethality of paf1Delta ccr4Delta, paf1Delta hpr1Delta, ccr4Delta hpr1Delta, and ccr4Delta gal11Delta double mutants. In addition, paf1Delta and ccr4Delta are lethal in combination with srb5Delta, indicating that the factors within and between the two RNA polymerase II complexes have overlapping essential functions. We have used differential display to identify several genes whose expression is affected by mutations in components of the Paf1p-Cdc73p-Pol II complex. Additionally, as previously observed for hpr1Delta, deleting PAF1 or CDC73 leads to elevated recombination between direct repeats. The paf1Delta and ccr4Delta mutations, as well as gal11Delta, demonstrate sensitivity to cell wall-damaging agents, rescue of the temperature-sensitive phenotype by sorbitol, and reduced expression of genes involved in cell wall biosynthesis. This unusual combination of effects on recombination and cell wall integrity has also been observed for mutations in genes in the Pkc1p-Mpk1p kinase cascade. Consistent with a role for this novel form of RNA polymerase II in the Pkc1p-Mpk1p signaling pathway, we find that paf1Delta mpk1Delta and paf1Delta pkc1Delta double mutants do not demonstrate an enhanced phenotype relative to the single mutants. Our observation that the Mpk1p kinase is fully active in a paf1Delta strain indicates that the Paf1p-Cdc73p complex may function downstream of the Pkc1p-Mpk1p cascade to regulate the expression of a subset of yeast genes.

ADR1-mediated transcriptional activation requires the presence of an intact TFIID complex.

The yeast transcriptional activator ADR1, which is required for ADH2 and other genes' expression, contains four transactivation domains (TADs). While previous studies have shown that these TADs act through GCN5 and ADA2, and presumably TFIIB, other factors are likely to be involved in ADR1 function. In this study, we addressed the question of whether TFIID is also required for ADR1 action. In vitro binding studies indicated that TADI of ADR1 was able to retain TAFII90 from yeast extracts and TADII could retain TBP and TAFII130/145. TADIV, however, was capable of retaining multiple TAFIIs, suggesting that TADIV was binding TFIID from yeast whole-cell extracts. The ability of TADIV truncation derivatives to interact with TFIID correlated with their transcription activation potential in vivo. In addition, the ability of LexA-ADR1-TADIV to activate transcription in vivo was compromised by a mutation in TAFII130/145. ADR1 was found to associate in vivo with TFIID in that immunoprecipitation of either TAFII90 or TBP from yeast whole-cell extracts specifically coimmunoprecipitated ADR1. Most importantly, depletion of TAFII90 from yeast cells dramatically reduced ADH2 derepression. These results indicate that ADR1 physically associates with TFIID and that its ability to activate transcription requires an intact TFIID complex.

DBF2 protein kinase binds to and acts through the cell cycle-regulated MOB1 protein.

The DBF2 gene of the budding yeast Saccharomyces cerevisiae encodes a cell cycle-regulated protein kinase that plays an important role in the telophase/G1 transition. As a component of the multisubunit CCR4 transcriptional complex, DBF2 is also involved in the regulation of gene expression. We have found that MOB1, an essential protein required for a late mitotic event in the cell cycle, genetically and physically interacts with DBF2. DBF2 binds MOB1 in vivo and can bind it in vitro in the absence of other yeast proteins. We found that the expression of MOB1 is also cell cycle regulated, its expression peaking slightly before that of DBF2 at the G2/M boundary. While overexpression of DBF2 suppressed phenotypes associated with mob1 temperature-sensitive alleles, it could not suppress a mob1 deletion. In contrast, overexpression of MOB1 suppressed phenotypes associated with a dbf2-deleted strain and suppressed the lethality associated with a dbf2 dbf20 double deletion. A mob1 temperature-sensitive allele with a dbf2 disruption was also found to be synthetically lethal. These results are consistent with DBF2 acting through MOB1 and aiding in its function. Moreover, the ability of temperature-sensitive mutated versions of the MOB1 protein to interact with DBF2 was severely reduced, confirming that binding of DBF2 to MOB1 is required for a late mitotic event. While MOB1 and DBF2 were found to be capable of physically associating in a complex that did not include CCR4, MOB1 did interact with other components of the CCR4 transcriptional complex. We discuss models concerning the role of DBF2 and MOB1 in controlling the telophase/G1 transition.

The NOT proteins are part of the CCR4 transcriptional complex and affect gene expression both positively and negatively.

The CCR4 transcriptional regulatory complex consisting of CCR4, CAF1, DBF2 and other unidentified factors is one of several groups of proteins that affect gene expression. Using mass spectrometry, we have identified the 195, 185 and 116 kDa species which are part of the CCR4 complex. The 195 and 185 kDa proteins were found to be NOT1 and the 116 kDa species was identical to NOT3. NOT1, 2, 3 and 4 proteins are part of a regulatory complex that negatively affects transcription. All four NOT proteins were found to co-immunoprecipitate with CCR4 and CAF1, and NOT1 co-purified with CCR4 and CAF1 through three chromatographic steps in a complex estimated to be 1.2x10(6) Da in size. Mutations in the NOT genes affected many of the same genes and processes that are affected by defects in the CCR4 complex components, including reduction in ADH2 derepression, defective cell wall integrity and increased sensitivity to monoand divalent ions. Similarly, ccr4, caf1 and dbf2 alleles negatively regulated FUS1-lacZ expression, as do defects in the NOT genes. These results indicate that the NOT proteins are physically and functionally part of the CCR4 complex which forms a unique and novel complex that affects transcription both positively and negatively.

Dhh1p, a putative RNA helicase, associates with the general transcription factors Pop2p and Ccr4p from Saccharomyces cerevisiae.

The POP2 (Caf1) protein in Saccharomyces cerevisiae affects a variety of transcriptional processes and is a component of the Ccr4p complex. We have isolated five multicopy suppressor genes of a pop2 deletion mutation: CCR4, DHH1 (a putative RNA helicase), PKC1, STM1, and MPT5 (multicopy suppressor of pop two). Overexpression of either the CCR4 or DHH1 genes effectively suppressed phenotypes associated with pop2 mutant cells; overexpression of PKC1, STM1, or MPT5 genes produced only partial suppression. Disruption of the CCR4 or DHH1 genes resulted in phenotypes similar to those observed for pop2 cells. In addition, overexpression of the DHH1 gene also suppressed the ccr4 mutation, suggesting a close relationship between the POP2, CCR4, and DHH1 genes. Two-hybrid analysis and coimmunoprecipitation experiments revealed that Pop2p and Dhh1p interact physically, and these and other data suggest that Dhh1p is also a component of the Ccr4p complex. Finally, we investigated the genetic interaction between factors associated with POP2 and the PKC1 pathway. The temperature-sensitive growth defect of dhh1 or mpt5 cells was suppressed by overexpression of PKC1, and the defect of mpk1 cells was suppressed by overexpression of MPT5. These results and phenotypic analysis of double mutants from the POP2 and PKC1 pathways suggested that the POP2 and the PKC1 pathways are independent but have some overlapping functions.