Drainage status of grassland peat soils in Ireland: Extent, efficacy and implications for GHG emissions and rewetting efforts.

Peatlands have been artificially drained and degraded over 100s of years and have released huge amounts of carbon dioxide (CO2) as a result. In organic grassland soils, raising the water table to prevent such emissions is being proposed to meet national greenhouse gas emission targets for the land use sector. At present, all of these soils (335,000 ha) are assumed to be drained (as no information has been available on their drainage status) within national emission inventory reporting and are therefore responsible for significant emissions (8-9 million tonnes CO2-equivalent annually). The objective of the present study was to collate studies relating to the drainage status of peat soils in Ireland to present alternative scenarios with regard to actual drainage status of organic soils and their estimated emissions. From a drainage design perspective, evidence suggests that relatively small proportions of the grassland peat area was drained effectively using optimal in-field drain spacings required to control the water table at 0.4-0.5 m. Open drains excavated on such soils have limited capacity to laterally control the water table depth beyond short distances. Furthermore, the lack of long-term routine maintenance post installation ensures the redundancy of many drainage systems over time. New drainage installations are therefore likely replacing existing infrastructure and not necessarily increasing the drained area at any given time. This evidence supports literature from the 1980s which state that relatively low proportions of the grassland peat area has been subjected to effective drainage. Scenario testing results showed that likely emissions from the most probable scenario (with total area drained equating to 90,000-120,000 ha) are 3.6-4.7 million tonnes CO2-equivalent, approximately 40-53% of current national emission inventory estimates. The incorporation of such a refinement into the national inventory could offer a significant reduction in estimated GHG emissions from the grassland land use sector in national emission inventory reporting.

Targeting mitochondrial cardiolipin and the cytochrome c/cardiolipin complex to promote electron transport and optimize mitochondrial ATP synthesis.

BACKGROUND AND PURPOSE: Cardiolipin plays an important role in mitochondrial respiration and cardiolipin peroxidation is associated with age-related diseases. Hydrophobic interactions between cytochrome c and cardiolipin converts cytochrome c from an electron carrier to a peroxidase. In addition to cardiolipin peroxidation, this impedes electron flux and inhibits mitochondrial ATP synthesis. SS-31 (D-Arg-dimethylTyr-Lys-Phe-NH2 ) selectively binds to cardiolipin and inhibits cytochrome c peroxidase activity. Here, we examined whether SS-31 also protected the electron carrier function of cytochrome c. EXPERIMENTAL APPROACH: Interactions of SS-31 with cardiolipin were studied using liposomes and bicelles containing phosphatidylcholine alone or with cardiolipin. Structural interactions were assessed by fluorescence spectroscopy, turbidity and nuclear magnetic resonance. Effects of cardiolipin on electron transfer kinetics of cytochrome c were determined by cytochrome c reduction in vitro and oxygen consumption using mitoplasts, frozen and fresh mitochondria. KEY RESULTS: SS-31 interacted only with liposomes and bicelles containing cardiolipin in about 1:1 ratio. NMR studies demonstrated that the aromatic residues of SS-31 penetrated deep into cardiolipin-containing bilayers. SS-31 restored cytochrome c reduction and mitochondrial oxygen consumption in the presence of added cardiolipin. In fresh mitochondria, SS-31 increased state 3 respiration and efficiency of ATP synthesis. CONCLUSIONS AND IMPLICATIONS: SS-31 selectively targeted cardiolipin and modulated its interaction with cytochrome c. SS-31 inhibited the cytochrome c/cardiolipin complex peroxidase activity while protecting its ability to serve as an electron carrier, thus optimizing mitochondrial electron transport and ATP synthesis. This novel class of cardiolipin therapeutics has the potential to restore mitochondrial bioenergetics for treatment of numerous age-related diseases.

MiR-200 can repress breast cancer metastasis through ZEB1-independent but moesin-dependent pathways.

The microRNA-200 (miR-200) family has a critical role in regulating epithelial-mesenchymal transition and cancer cell invasion through inhibition of the E-cadherin transcriptional repressors ZEB1 and ZEB2. Recent studies have indicated that the miR-200 family may exert their effects at distinct stages in the metastatic process, with an overall effect of enhancing metastasis in a syngeneic mouse breast cancer model. We find in a xenograft orthotopic model of breast cancer metastasis that ectopic expression of members of the miR-200b/200c/429, but not the miR-141/200a, functional groups limits tumour cell invasion and metastasis. Despite modulation of the ZEB1-E-cadherin axis, restoration of ZEB1 in miR-200b-expressing cells was not able to alter metastatic potential suggesting that other targets contribute to this process. Instead, we found that miR-200b repressed several actin-associated genes, with the knockdown of the ezrin-radixin-moesin family member moesin alone phenocopying the repression of cell invasion by miR-200b. Moesin was verified to be directly targeted by miR-200b, and restoration of moesin in miR-200b-expressing cells was sufficient to alleviate metastatic repression. In breast cancer cell lines and patient samples, the expression of moesin significantly inversely correlated with miR-200 expression, and high levels of moesin were associated with poor relapse-free survival. These findings highlight the context-dependent effects of miR-200 in breast cancer metastasis and demonstrate the existence of a moesin-dependent pathway, distinct from the ZEB1-E-cadherin axis, through which miR-200 can regulate tumour cell plasticity and metastasis.

Mutant p53 drives invasion in breast tumors through up-regulation of miR-155.

Loss of p53 function is a critical event during tumorigenesis, with half of all cancers harboring mutations within the TP53 gene. Such events frequently result in the expression of a mutated p53 protein with gain-of-function properties that drive invasion and metastasis. Here, we show that the expression of miR-155 was up-regulated by mutant p53 to drive invasion. The miR-155 host gene was directly repressed by p63, providing the molecular basis for mutant p53 to drive miR-155 expression. Significant overlap was observed between miR-155 targets and the molecular profile of mutant p53-expressing breast tumors in vivo. A search for cancer-related target genes of miR-155 revealed ZNF652, a novel zinc-finger transcriptional repressor. ZNF652 directly repressed key drivers of invasion and metastasis, such as TGFB1, TGFB2, TGFBR2, EGFR, SMAD2 and VIM. Furthermore, silencing of ZNF652 in epithelial cancer cell lines promoted invasion into matrigel. Importantly, loss of ZNF652 expression in primary breast tumors was significantly correlated with increased local invasion and defined a population of breast cancer patients with metastatic tumors. Collectively, these findings suggest that miR-155 targeted therapies may provide an attractive approach to treat mutant p53-expressing tumors.

The role of microRNAs in metastasis and epithelial-mesenchymal transition.

For a tumour cell to metastasize it must successfully negotiate a number of events, requiring a series of coordinated changes in the expression of many genes. MicroRNAs are small non-coding RNA molecules that post-transcriptionally control gene expression. As microRNAs are now recognised as master regulators of gene networks and play important roles in tumourigenesis, it is no surprise that microRNAs have recently been demonstrated to have central roles during metastasis. Recent work has also demonstrated critical roles for microRNAs in epithelial-mesenchymal transition, a phenotypic change underlain by altered gene expression patterns that is believed to mirror events in metastatic progression. These findings offer new potential for improved prognostics through expression profiling and may represent novel molecular treatment targets for future therapy. In this review, we summarise the multistep processes of metastasis and epithelial-mesenchymal transition and describe the recent discoveries of microRNAs that participate in controlling these processes.

Two days of pulsatile GnRH infusion beginning 4 days before weaning in sows initiates a wave of follicular growth that is not sustained after weaning.

Intervals to estrus and ovulation in weaned sows depend partially on the diameter of ovarian follicles at weaning. The objective was to determine if follicular diameter in sows could be increased by a 48h period of GnRH infusion before weaning and whether this pre-weaning growth would advance follicular development after weaning. The posterior vena cava was cannulated in eight sows at 10+/-1 day after farrowing. Sows were randomly assigned to receive intravenous treatment with either 2mL of GnRH (1microg/mL; n=4) or 2mL of saline (n=4) every 0.5h for 48h beginning 94h before weaning. The average follicular diameter and the number of follicles within diameter classes were determined daily by ultrasonography. Serum LH concentrations increased on the first infusion day but serum LH was equal to control on the last infusion day (P<0.077). The GnRH infusion increased the average diameter of ovarian follicles (P<0.001). Serum estradiol increased (P<0.001) and serum FSH decreased (P<0.016) coincident with GnRH-induced follicular development but these changes were reversed within 24h after the end of the infusion period. Follicles that grew in response to GnRH regressed and were replaced by a new population of follicles within 4 days after weaning. Within the experimental model for the present study, a GnRH infusion increased follicular growth in lactating sows but follicles could not be sustained beyond the end of GnRH infusion.

Regulation of ATR-dependent pathways by the FHA domain containing protein SNIP1.

The forkhead associated (FHA) domain-containing protein Smad nuclear interacting protein 1 (SNIP1) has multiple cellular functions, including the ability to interact with DNA-binding transcription factors and transcriptional coactivators. Moreover, we have demonstrated previously that SNIP1 regulates cyclin D1 expression and promoter activity. Here, we identify a new function for SNIP1 as a regulator of ATR checkpoint kinase-dependent pathways in human U-2 OS osteosarcoma cells: SNIP1 is required for p53 induction in response to ultraviolet light treatment and selectively regulates the phosphorylation of known ATR target proteins, including p53, Chk1 and the histone variant H2AX. These activities are independent of its ability to regulate cyclin D1 expression. Significantly, SNIP1 is also required for ATR-dependent functions of the human p14(ARF) tumour suppressor, including its ability to modulate the activity of the RelA(p65) NF-kappaB subunit. This, together with its other described functions, suggests that SNIP1 could have an important role during tumorigenesis and cancer therapy.

Decreased follicular size during late lactation caused by treatment with charcoal-treated follicular fluid delays onset of estrus and ovulation after weaning in sows.

The weaning to estrus and weaning to ovulation intervals in sows are controlled by ovarian follicular growth after weaning. Longer intervals could be caused by smaller diameter follicles at weaning that take more time to reach a preovulatory size. We addressed this hypothesis by decreasing the diameter of follicular populations before weaning and then measuring follicular development and interval to estrus and ovulation after weaning. The posterior vena cava, cranial to the entry of the ovarian vein, was cathetered for blood sampling and infusion in 20 sows at 12 +/- 1 d after farrowing. Sows were assigned randomly to receive either 30 mL of charcoal-treated follicular fluid (FF, n = 9; a treatment known to decrease serum FSH and follicular diameter) or 30 mL of saline (n = 11) by venous infusion thrice daily (0700, 1500, and 2300 h) for 96 h beginning at 14 +/- 1 d after farrowing. Sows were weaned 48 h after the last infusion. Blood samples were collected for FSH analysis thrice daily beginning on the day of catheterization and continuing until ovulation. Follicular diameter was determined once daily by transrectal ultrasonography. A treatment x time interaction was detected for serum FSH (P < 0.001) and follicular diameter (P < 0.001) because serum FSH and the diameter of follicular populations decreased in FF sows during the infusion period. After the infusion period, serum FSH rebounded in FF sows, and follicles resumed growth but grew at the same rate as those of saline-treated sows, thus failing to achieve equivalent diameters relative to saline-treated sows on a given day after weaning. As a result, sows treated with FF had longer (P < 0.05) weaning to estrus (6.1 +/- 0.4 d) and weaning to ovulation (8.6 +/- 0.5 d) intervals compared with saline-treated sows (4.7 +/- 0.4 d and 7.2 +/- 0.4 d, respectively). We conclude that the diameter of the follicular population at weaning is one factor that controls interval to estrus and ovulation in sows. Small follicles at weaning cannot undergo compensatory growth and require additional time to reach a preovulatory size.

The hypoxia-inducible factors: key transcriptional regulators of hypoxic responses.

Oxygen depravation in mammals leads to the transcriptional induction of a host of target genes to metabolically adapt to this deficiency, including erythropoietin and vascular endothelial growth factor. This response is primarily mediated by the hypoxia-inducible factors (HIFs) which are members of the basic-helix-loop-helix/Per-ARNT-Sim (bHLH/PAS) transcription factor family. The HIFs are primarily regulated via a two-step mechanism of HIF post-translational modification, increasing both protein stability and transactivation capacity. This review aims to summarise our current understanding of these processes, and discuss the important role of the HIFs in the pathophysiology of many human diseases.

Effect of time of ovulation and sperm concentration on fertilization rate in gilts.

In normal production practices, sows and gilts are inseminated at least twice during estrus because the timing of ovulation is variable relative to the onset of estrus. The objective of this study was to determine if a normal fertilization rate could be achieved with a single insemination of low sperm number given at a precise interval relative to ovulation. Gilts (n=59) were randomly assigned to one of three treatment groups: low dose (LD; one insemination, 0.5 x 10(9) spermatozoa), high dose (HD; one insemination, 3 x 10(9) spermatozoa) or multiple dose (MD; two inseminations, 3 x 10(9) spermatozoa per insemination). Twice daily estrus detection (06:00 and 18:00 h) was performed using fenceline boar contact and backpressure testing. Transrectal ultrasonography was performed every 6 h beginning at the detection of the onset of standing estrus and continuing until ovulation. Gilts in the LD and HD groups were inseminated 22 h after detection of estrus; MD gilts received inseminations at 10 and 22 h after detection of estrus. Inseminations were administered by using an insemination catheter and semen was deposited into the cervix. The uterus was flushed on Day 5 after the onset of estrus and the number of corpora lutea, oocytes, and embryos were counted. Time of insemination relative to ovulation was designated as 40 to >24 h, 24 to >12 h, and 12 to 0 h before ovulation and >0 h after ovulation. The LD gilts had fewer embryos (P<0.04), more unfertilized oocytes (P<0.05) and a lower fertilization rate (P<0.07) compared to MD gilts. The effects of time of insemination relative to ovulation and the treatment by time interaction were not significant. We conclude that a cervical insemination with low spermatozoa concentration may not result in acceptable fertility even when precisely timed relative to ovulation.

Factors affecting follicular populations on Day 3 postweaning and interval to ovulation in a commercial sow herd.

Sows (n=146) in a commercial herd were studied to determine factors affecting follicular populations and interval to ovulation after weaning. Ovaries were examined daily by ultrasonography beginning on Day 3 postweaning and twice daily from Day 4.5 until ovulation. Ovarian images were recorded on videotape on Day 3 postweaning and follicles were counted. Subsequent ultrasounds were used to determine time of ovulation. Sows with short weaning to ovulation intervals (or=9 days) weaning to ovulation intervals (P<0.001). Follicular populations in sows with intermediate (7-8.5 days) intervals to ovulation were intermediate in diameter when compared to sows with short or long intervals to ovulation. Parity and body condition score (BCS) affected interval to ovulation; first parity and low body condition sows had longer intervals to ovulation (P<0.001 and 0.05, respectively). The longer intervals to ovulation in first parity and low body condition sows were associated with lesser follicular diameters on Day 3 after weaning. We conclude that follicular populations measured by ultrasonography on Day 3 after weaning were different for sows with different intervals to ovulation. Furthermore, production factors (i.e. parity and BCS) known to influence interval to ovulation were associated with differences in follicular growth within the first 3 days after weaning in sows.

A method for efficient isotopic labeling of recombinant proteins.

A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for 13C labeling of the C-terminal domain of angiopoietin-2, 15N labeling of ubiquitin and for 2H/13C/15N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.

NMR spin relaxation methods for characterization of disorder and folding in proteins.

The flexibility and dynamics of proteins directly influence the processes of protein folding, recognition, and function. NMR spin relaxation methods are used to assess the dynamics and mobility of proteins, for fast ps and ns motions as well as slower microsecond and ms events. The degree of protein flexibility and disorder as well as the changes in protein flexibility can be assessed by NMR spin relaxation methods at individual residues within the protein. In addition to probing protein dynamics, the changes in the NMR-derived order parameters can be used to estimate the entropic contributions of order-disorder transitions. Furthermore, kinetic processes in the ms time regime may be directly investigated to extract the rates of conformational interconversion, ligand binding, and protein folding processes. We show how a variety of dynamical information can be obtained from NMR relaxation measurements. We present examples that illustrate the use of NMR spin relaxation analysis for investigation of folding and disorder in proteins.

The DNA-binding domain in the Bacillus subtilis transition-state regulator AbrB employs significant motion for promiscuous DNA recognition.

AbrB is a Bacillus subtilis protein responsible for regulating a diverse array of unrelated genes during periods of sub-optimal growth conditions. DNA binding by AbrB is unique in that sequence recognition is specific, yet no obvious consensus sequence of bound promoter regions is apparent. The N-terminal domain is a recently characterized representative of a novel class of DNA-binding proteins that possess a looped-hinge helix DNA-binding topology. Although the structural characterization of this DNA-binding topology contributed to an understanding of the architectural basis for recognition of DNA target sequences, specific mechanisms responsible for promiscuity in DNA sequence recognition still were not apparent. Analysis of (15)N backbone relaxation parameters shows that dynamic motion of regions directly linked to DNA binding show concerted motion on the microsecond-millisecond timescale. Furthermore, dynamic motion of the hinge region suggests that the DNA-binding region is capable of conformational orientations that allow it to accommodate DNA sequence variability in the cognate binding sites.

Ovarian follicular growth in sows.

The resumption of ovarian follicular development during lactation and after weaning in sows is a complex process that ultimately determines rebreeding efficiency of sows. Ovarian follicular development before weaning is heterogeneous because multiple patterns of development are observed when individual sows are compared. Sows can have relatively inactive ovaries before weaning with follicles of < 2 mm in diameter. Other sows have non-ovulatory follicular waves in which follicles grow to approximately 5 mm and subsequently regress before weaning. Sows may also have preovulatory follicular development and ovulation, or may develop cystic ovaries before weaning. Weaning is a random event relative to follicular development on the ovary. Therefore, variation in the weaning to oestrus interval in sows is caused by weaning at random stages of follicular development. Most sows experience a rapid period of follicular growth after weaning and return to oestrus within 3-7 days. Delayed intervals to oestrus after weaning are associated with inactive ovaries before weaning (follicles < 2 mm in diameter) or weaning during the regression phase of a follicular wave. An integrated model for follicular growth and oestrus in weaned sows should include endocrine mechanisms (that is, individual differences in insulin, insulin-like growth factor I (IGF-I), LH and FSH), behavioural mechanisms (relationship between follicular growth and the initiation of oestrus) and morphological mechanisms (that is, timing of weaning relative to ovarian follicular development).

Buried polar interactions and conformational stability in the simian immunodeficiency virus (SIV) gp41 core.

For human (HIV) and simian (SIV) immunodeficiency viruses, the gp41 envelope protein undergoes a receptor-activated conformational change from a labile native structure to an energetically more stable fusogenic conformation, which then mediates viral-cell membrane fusion. The core structure of fusion-active gp41 is a six-helix bundle in which three antiparallel carboxyl-terminal helices are packed against an amino-terminal trimeric coiled coil. Here we show that a recombinant model of the SIV gp41 core, designated N36(L6)C34, forms an alpha-helical trimer that exhibits a cooperative two-state folding-unfolding transition. We investigate the importance of buried polar interactions in determining the overall fold of the gp41 core. We have replaced each of four polar amino acids at the heptad a and d positions of the coiled coil in N36(L6)C34 with a representative hydrophobic amino acid, isoleucine. The Q565I, T582I, and T586I variants form six-helix bundle structures that are significantly more stable than that of the wild-type peptide, whereas the Q575I variant misfolds into an insoluble aggregate under physiological conditions. Thus, the buried polar residues within the amino-terminal heptad repeat are important determinants of the structural specificity and stability of the gp41 core. We suggest that these conserved buried polar interactions play a role in governing the conformational state of the gp41 molecule.

Characterization of millisecond time-scale dynamics in the molten globule state of alpha-lactalbumin by NMR.

The motional dynamics of the molten globule (MG) state of alpha-lactalbumin have been characterized using (15)N transverse relaxation rates (R2). A modified version of the Carr-Purcell-Meiboom-Gill (CPMG) R2 pulse sequence is proposed in order to overcome the loss of sensitivity that arises from extreme line broadening due to complex dynamics on the millisecond time-scale. Using this pulse sequence, chemical exchange rates were extracted by examining the (15)N transverse relaxation rates as a function of CPMG delay values. The results clearly illustrate that pervasive conformational exchange of 0.2-0.5 ms in the (15)N backbone resonances of the molten globule state of alpha-lactalbumin. The temperature dependence of the conformational exchange rates display standard Arrhenius kinetic behavior between 10 and 30 degrees C. Estimates of the activation energies range from 0.8 to 4. 4 kcal/mol, indicating a low energetic barrier to conformational fluctuations relative to native state proteins. The fluctuations and low energetic barriers may be critical for directing the search for contacts that will result in the transition from the MG state to the native state.

Use of heme-protein complexes by the Yersinia enterocolitica HemR receptor: histidine residues are essential for receptor function.

The abilities of two bacterial active heme transporters, HmbR of Neisseria meningitidis and HemR of Yersinia enterocolitica, to use different heme sources were compared. While HmbR-expressing cells used only hemoglobin (Hb) and heme, HemR-expressing bacteria were able to grow on Hb, heme, myoglobin, hemopexin, catalase, human and bovine serum albumin-heme, and haptoglobin-hemoglobin complexes as sources of iron. Expression of functional HemR allowed Escherichia coli cells to respond to heme-containing peptides, microperoxidases MP-8, MP-9, and MP-11, suggesting the ability of HemR to transport heme covalently linked to other molecules. Comparison of HemR with other heme receptors identified several highly conserved histidine residues as well as two conserved amino acid motifs, the FRAP and NPNL boxes. A site-directed mutagenesis approach was used to investigate the roles of His128, His192, His352, and His461 residues in HemR function. The HemR receptor with histidine changed to lysine at position 128 (HemR(H128K)), HemR(H461L), HemR(H461A), and HemR(H128A,H461A) mutant receptors were unable to use Hb, human serum albumin-heme, and myoglobin as sources of porphyrin and iron. Utilization of free heme was also severely affected, with some residual heme uptake in cells expressing HemR(H128K), HemR(H461A), and HemR(H461L). Conversely, the HemR(H192T), HemR(H352A), HemR(H352K), and HemR(H192T,H352K) mutant receptors were fully functional. All mutant HemR proteins were expressed in the outer membrane at levels similar to that of the wild-type HemR receptor. Nonfunctional HemRs were able to bind heme- and Hb-agarose. A hypothetical model of the HemR function in which two conserved histidine residues, His128 and His461, participate in the transport of heme through the receptor pore is postulated.

Reduced growth hormone receptor (GHR) messenger ribonucleic acid in liver of periparturient cattle is caused by a specific down-regulation of GHR 1A that is associated with decreased insulin-like growth factor I.

GH receptor (GHR) messenger RNA (mRNA) is transcribed from at least three different promoters within the liver of cattle. The first promoter (P1) is liver specific and alternatively splices exon 1A onto the GHR mRNA (GHR 1A mRNA). The second and third promoters (P2 and P3) have constitutive activity in many tissues and alternatively splice exons 1B and 1C onto the GHR mRNA (GHR 1B and GHR 1C mRNA). The total amount of GHR in the liver partially determines liver insulin-like growth factor I (IGF-I) synthesis in response to GH. Two studies were conducted to characterize the changes in GHR 1A mRNA, alternatively spliced GHR mRNA, and IGF-I mRNA during late pregnancy and early lactation in dairy cattle. Liver RNA was isolated from pregnant Holstein cattle (Bos taurus) on days -14, 0, and 21 relative to parturition (study 1) or days -14, 0, 15, 30, 60, and 90 relative to parturition (study 2). Ribonuclease protection assays were used to quantify total GHR (all GHR variants) as well as liver-specific GHR 1A and alternatively spliced GHR mRNA. Likewise, total IGF-I as well as alternatively spliced IGF-I mRNA (class 1 and class 2 transcripts) were measured. A decrease in total GHR mRNA at parturition (P < 0.01) was associated with a specific decrease in GHR 1A mRNA (P < 0.001). The amount of alternatively spliced GHR mRNA (including GHR 1B and GHR 1C mRNA) did not change at parturition (P > 0.10). Total liver IGF-I mRNA and blood IGF-I concentrations were also decreased at parturition (P < 0.05 and P < 0.01, respectively). However, a decrease in IGF-I mRNA was observed for both class 1 and class 2 IGF-I transcripts (P < 0.01 and P < 0.05, respectively). We conclude that the reduced amount of GHR mRNA during early lactation is caused by a specific down-regulation of GHR 1A mRNA that was associated with decreased liver IGF-I mRNA and decreased blood IGF-I concentrations. These data provide evidence for independent regulation of GHR mRNA by mechanisms that discriminate between GHR P1 (transcribes GHR 1A) and alternative promoters that transcribe constitutive GHR mRNA.

Temperature dependence of intramolecular dynamics of the basic leucine zipper of GCN4: implications for the entropy of association with DNA.

The basic leucine zipper domain of the yeast transcription factor GCN4 consists of a C-terminal leucine zipper and an N-terminal basic DNA-binding region that achieves a stable structure only after association with DNA. Backbone dynamics of a peptide encompassing the basic and leucine zipper bZip domain (residues 226-281) are described using NMR spectroscopy. The 15N longitudinal relaxation rates, 15N transverse relaxation rates, and {1H}-15N nuclear Overhauser effects were measured for the backbone amide nitrogen atoms at 290 K, 300 K, and 310 K. The relaxation data were interpreted using reduced spectral density mapping to determine values of the spectral density function, J(omega), at the frequencies 0, omegaN, and 0.87omegaH to characterize overall and intramolecular motions on picosecond-nanosecond timescales. To account for the temperature dependence of overall rotational diffusion, the J(0) values were normalized using Stoke's Law. At 310 K, the 13Calpha and 13CO chemical shifts in conjunction with the spectral density values indicate that the leucine zipper sequence forms a highly ordered alpha-helix, while the basic region populates an ensemble of highly dynamic transient structures with substantial helical character. The normalized values of J(0) and the values of J(0.87omegaH) for residues in the leucine zipper dimerization domain are independent of temperature. In contrast, residues in the basic region exhibit pronounced increases in the normalized J(0) and decreases in J(0.87omegaH) as temperature is decreased. A strong correlation exists between the temperature dependence of 13CO chemical shifts and of J(0.87omegaH). These results suggest that, for the basic region, lowering the temperature increases the population of transient helical conformations, and concomitantly reduces the amplitude or timescale of conformational fluctuations on picosecond-nanosecond timescales. Changes in the conformational dynamics of the peptide backbone of the basic region that accompany DNA binding contribute to the overall thermodynamics of complex formation. The change in backbone conformational entropy derived from NMR spin-relaxation data agrees well with the result calculated from calorimetric measurements. Restriction of the conformational space accessible to the basic region may significantly reduce the entropic cost associated with formation of the basic region helices consequent to DNA binding.