Changes in resting-state brain networks after cognitive-behavioral therapy for chronic pain.

BACKGROUND: Cognitive-behavioral therapy (CBT) is thought to be useful for chronic pain, with the pathology of the latter being closely associated with cognitive-emotional components. However, there are few resting-state functional magnetic resonance imaging (R-fMRI) studies. We used the independent component analysis method to examine neural changes after CBT and to assess whether brain regions predict treatment response. METHODS: We performed R-fMRI on a group of 29 chronic pain (somatoform pain disorder) patients and 30 age-matched healthy controls (T1). Patients were enrolled in a weekly 12-session group CBT (T2). We assessed selected regions of interest that exhibited differences in intrinsic connectivity network (ICN) connectivity strength between the patients and controls at T1, and compared T1 and T2. We also examined the correlations between treatment effects and rs-fMRI data. RESULTS: Abnormal ICN connectivity of the orbitofrontal cortex (OFC) and inferior parietal lobule within the dorsal attention network (DAN) and of the paracentral lobule within the sensorimotor network in patients with chronic pain normalized after CBT. Higher ICN connectivity strength in the OFC indicated greater improvements in pain intensity. Furthermore, ICN connectivity strength in the dorsal posterior cingulate cortex (PCC) within the DAN at T1 was negatively correlated with CBT-related clinical improvements. CONCLUSIONS: We conclude that the OFC is crucial for CBT-related improvement of pain intensity, and that the dorsal PCC activation at pretreatment also plays an important role in improvement of clinical symptoms via CBT.

Enthusiasm for homework and improvement of psychological distress in subthreshold depression during behavior therapy: secondary analysis of data from a randomized controlled trial.

BACKGROUND: Cognitive behavioral therapy (CBT) usually involves homework, the completion of which is a known predictor of a positive outcome. The aim of the present study was to examine the session-by-session relationships between enthusiasm to complete the homework and the improvement of psychological distress in depressed people through the course of therapy. METHODS: Working people with subthreshold depression were recruited to participate in the telephone CBT (tCBT) program with demonstrated effectiveness. Their enthusiasm for homework was enhanced with motivational interviewing techniques and was measured by asking two questions: "How strongly do you feel you want to do this homework?" and "How confident do you feel you can actually accomplish this homework?" at the end of each session. The outcome was the K6 score, which was administered at the start of each session. The K6 is an index of psychological distress including depression and anxiety. We used structural equation modeling (SEM) to elucidate the relationships between enthusiasm and the K6 scores from session to session. RESULTS: The best fitting model suggested that, throughout the course of behavior therapy (BT), enthusiasm to complete the homework was negatively correlated with the K6 scores for the subsequent session, while the K6 score measured at the beginning of the session did not influence the enthusiasm to complete the homeworks assigned for that session. CONCLUSIONS: Empirical data now support the practitioners of BT when they try to enhance their patient's enthusiasm for homework regardless of the participant's distress, which then would lead to a reduction in distress in the subsequent week. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT00885014 . April 20, 2009.

Depletion of homeodomain-interacting protein kinase 3 impairs insulin secretion and glucose tolerance in mice.

AIMS/HYPOTHESIS: Insufficient insulin secretion and reduced pancreatic beta cell mass are hallmarks of type 2 diabetes. Here, we focused on a family of serine-threonine kinases known as homeodomain-interacting protein kinases (HIPKs). HIPKs are implicated in the modulation of Wnt signalling, which plays a crucial role in transcriptional activity, and in pancreas development and maintenance. The aim of the present study was to characterise the role of HIPKs in glucose metabolism. METHODS: We used RNA interference to characterise the role of HIPKs in regulating insulin secretion and transcription activity. We conducted RT-PCR and western blot analyses to analyse the expression and abundance of HIPK genes and proteins in the islets of high-fat diet-fed mice. Glucose-induced insulin secretion and beta cell proliferation were measured in islets from Hipk3 ( -/- ) mice, which have impaired glucose tolerance owing to an insulin secretion deficiency. The abundance of pancreatic duodenal homeobox (PDX)-1 and glycogen synthase kinase (GSK)-3ß phosphorylation in Hipk3 ( -/- ) islets was determined by immunohistology and western blot analyses. RESULTS: We found that HIPKs regulate insulin secretion and transcription activity. Hipk3 expression was most significantly increased in the islets of high-fat diet-fed mice. Furthermore, glucose-induced insulin secretion and beta cell proliferation were decreased in the islets of Hipk3 ( -/- ) mice. Levels of PDX1 and GSK-3ß phosphorylation were significantly decreased in Hipk3 ( -/- ) islets. CONCLUSIONS/INTERPRETATION: Depletion of HIPK3 impairs insulin secretion and glucose tolerance. Decreased levels of HIPK3 may play a substantial role in the pathogenesis of type 2 diabetes.

Activation of natural killer T cells by α-carba-GalCer (RCAI-56), a novel synthetic glycolipid ligand, suppresses murine collagen-induced arthritis.

Alpha-carba-GalCer (RCAI-56), a novel synthetic analogue of α-galactosylceramide (α-GalCer), stimulates invariant natural killer T (NK T) cells to produce interferon (IFN)-γ. IFN-γ exhibits immunoregulatory properties in autoimmune diseases by suppressing T helper (Th)-17 cell differentiation and inducing regulatory T cells and apoptosis of autoreactive T cells. Here, we investigated the protective effects of α-carba-GalCer on collagen-induced arthritis (CIA) in mice. First, we confirmed that α-carba-GalCer selectively induced IFN-γ in CIA-susceptible DBA/1 mice in vivo. Then, DBA/1 mice were immunized with bovine type II collagen (CII) and α-carba-GalCer. The incidence and clinical score of CIA were significantly lower in α-carba-GalCer-treated mice. Anti-IFN-γ antibodies abolished the beneficial effects of α-carba-GalCer, suggesting that α-carba-GalCer ameliorated CIA in an IFN-γ-dependent manner. Treatment with α-carba-GalCer reduced anti-CII antibody production [immunoglobulin (Ig)G and IgG2a] and CII-reactive interleukin (IL)-17 production by draining lymph node (DLN) cells, did not induce apoptosis or regulatory T cells, and significantly increased the ratio of the percentage of IFN-γ-producing T cells to IL-17-producing T cells (Th1/Th17 ratio). Moreover, the gene expression levels of IL-6 and IL-23p19, Th17-related cytokines, were reduced significantly in mice treated with α-carba-GalCer. In addition, we observed higher IFN-γ production by NK T cells in α-carba-GalCer-treated mice in the initial phase of CIA. These findings indicate that α-carba-GalCer polarizes the T cell response toward Th1 and suppresses Th17 differentiation or activation, suggesting that α-carba-GalCer, a novel NK T cell ligand, can potentially provide protection against Th17-mediated autoimmune arthritis by enhancing the Th1 response.

In vivo deep-flexion kinematics in patients with posterior-cruciate retaining and anterior-cruciate substituting total knee arthroplasty.

BACKGROUND: Posterior-cruciate ligament retaining total knee arthroplasty designs have long been used with excellent clinical success, but often have shown kinematics and flexion performance that are significantly different from the natural knee. The purpose of this study was to compare deep-flexion knee kinematics in patients with two types of posterior-cruciate ligament retaining total knee arthroplasty. METHODS: One group received a traditional curved symmetric articular configuration, and one group received a design incorporating a lateral compartment which constrains the lateral condyle to the antero-posterior center of the tibial plateau in extension, but allows translation in flexion--roughly approximating the role of the anterior cruciate ligament. In vivo kinematics were analyzed using three-dimensional model registration and plain radiographs of kneeling and squatting activities in 20 knees in 18 patients. FINDINGS: Knees with the anterior cruciate ligament substituting design exhibited greater flexion, femoral antero-posterior translation and tibial internal rotation. INTERPRETATION: Geometric features intended to improve knee flexion, including greater antero-posterior stability, a more posterior tibial sulcus, and reshaped femoral condyles, do provide measurable and significant differences in deep-flexion knee kinematics.

Replication study for the association of new meta-analysis-derived risk loci with susceptibility to type 2 diabetes in 6,244 Japanese individuals.

AIMS/HYPOTHESIS: Additional susceptibility loci for type 2 diabetes have been identified by a meta-analysis of genome-wide association studies (GWASs) in European populations. To examine further the roles of these new loci, we performed a replication study for the association of these single-nucleotide polymorphism (SNP) loci with the disease in three independent Japanese populations. METHODS: We genotyped seven of the 11 SNPs that emerged in stage 2 of the meta-analysis for European GWASs (rs864745 in JAZF1, rs12779790 near CDC123/CAMK1D, rs7961581 near TSPAN8/LGR5, rs4607103 near ADAMTS9, rs10923931 in NOTCH2, rs1153188 near DCD and rs9472138 near VEGFA) for three independent Japanese populations (first set, 1,630 type 2 diabetes patients vs 1,064 controls; second set, 1,272 type 2 diabetes patients vs 856 controls; third set, 486 type 2 diabetes patients vs 936 controls) using a TaqMan assay. The association of the SNP loci in each population was analysed using a logistic regression analysis, adjusting for age, sex and BMI, and the data were evaluated by a meta-analysis. RESULTS: A meta-analysis for the three case-control studies identified a nominal association of rs864745 in JAZF1 with type 2 diabetes (OR 1.148, 95% CI 1.034-1.275, p = 0.0098, corrected p = 0.069). The association of other loci did not reach statistically significant levels (nominal p > 0.05). CONCLUSIONS/INTERPRETATION: From these results the contribution of these seven loci in conferring susceptibility to type 2 diabetes is considered minor in the Japanese population, if they are present.

Histone chaperones: 30 years from isolation to elucidation of the mechanisms of nucleosome assembly and disassembly.

Some three decades have passed since the discovery of nucleosomes in 1974 and the first isolation of a histone chaperone in 1978. While various types of histone chaperones have been isolated and functionally analyzed, the elementary processes of nucleosome assembly and disassembly have been less well characterized. Recently, the tertiary structure of a hetero-trimeric complex composed of the histone chaperone CIA/ASF1 and the histone H3-H4 dimer was determined, and this complex was proposed to be an intermediate in nucleosome assembly and disassembly reactions. In addition, CIA alone was biochemically shown to dissociate the histone (H3-H4)2 tetramer into two histone H3-H4 dimers. This activity suggested that CIA regulates the semi-conservative replication of nucleosomes. Here, we provide an overview of prominent histone chaperones with the goal of elucidating the mechanisms that preserve and modify epigenetic information. We also discuss the reactions involved in nucleosome assembly and disassembly.

Variations in the HHEX gene are associated with increased risk of type 2 diabetes in the Japanese population.

AIMS/HYPOTHESIS: Recently, several groups have carried out whole-genome association studies in European and European-origin populations and found novel type 2 diabetes-susceptibility genes, fat mass and obesity associated (FTO), solute carrier family 30 (zinc transporter), member 8 (SLC30A8), haematopoietically expressed homeobox (HHEX), exostoses (multiple) 2 (EXT2), CDK5 regulatory subunit associated protein 1-like 1 (CDKAL1), cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) (CDKN2B) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), which had not been in the list of functional candidates. The aim of this study was to determine the association between single nucleotide polymorphisms (SNPs) in these genes and type 2 diabetes in participants from the Japanese population. METHODS: Sixteen previously reported SNPs were genotyped in 864 Japanese type 2 diabetes individuals (535 men and 329 women; age 63.1 +/- 9.5 years (mean+/-SD), BMI 24.3 +/- 3.9 kg/m(2)) and 864 Japanese control individuals (386 men and 478 women; age 69.5 +/- 6.8 years, BMI 23.8 +/- 3.7 kg/m(2)). RESULTS: The SNPs rs5015480 [odds ratio (OR) = 1.46 (95% CI 1.20-1.77), p = 2.0 x 10(-4)], rs7923837 [OR = 1.40 (95% CI 1.17-1.68), p = 2.0 x 10(-4)] and rs1111875 [OR = 1.30 (95% CI 1.11-1.52), p = 0.0013] in HHEX were significantly associated with type 2 diabetes with the same direction as previously reported. SNP rs8050136 in FTO was nominally associated with type 2 diabetes [OR = 1.22 (95% CI 1.03-1.46), p = 0.025]. SNPs in other genes such as rs7756992 in CDKAL1, rs10811661 in CDKN2B and rs13266634 in SLC30A8 showed nominal association with type 2 diabetes. rs7756992 in CDKAL1 and rs10811661 in CDKN2B were correlated with impaired pancreatic beta cell function as estimated by the homeostasis model assessment beta index (p = 0.023, p = 0.0083, respectively). CONCLUSIONS/INTERPRETATION: HHEX is a common type 2 diabetes-susceptibility gene across different ethnic groups.

A genetic variation of the transcription factor 7-like 2 gene is associated with risk of type 2 diabetes in the Japanese population.

AIMS/HYPOTHESIS: It has been suggested that transcription factor 7-like 2 protein (TCF7L2) plays an important role in glucose metabolism by regulating the production level of glucagon-like peptide-1, a hormone which modifies glucose-dependent insulin secretion. Recently, variants of TCF7L2 gene were reported to confer an increased risk of type 2 diabetes in three different samples from European and European-origin populations. We studied whether the single nucleotide polymorphisms (SNPs) in TCF7L2 were associated with type 2 diabetes in samples from a Japanese population. METHODS: Five SNPs were genotyped in three different sample sets. Association with type 2 diabetes was investigated in each, as well as in combined sample sets. RESULTS: The SNP rs7903146 was nominally associated with type 2 diabetes in the initial (p = 0.08) and two replication sample sets (p = 0.05 and 0.06). For the combined sample set, in which we successfully genotyped 1,174 type 2 diabetes patients and 823 control subjects, rs7903146 showed a significant association with type 2 diabetes (odds ratio = 1.69 [95% CI 1.21-2.36], p = 0.002) with the same direction as the previous reports in samples from European and European-origin populations. SNPs rs7903146 and rs7901695 were in complete linkage disequilibrium. The rest of the five SNPs (rs7895340, rs11196205 and rs12255372) did not show any significant associations with type 2 diabetes. CONCLUSIONS/INTERPRETATION: The consistent association between rs7903146 in TCF7L2 and type 2 diabetes in different ethnic groups, including the Japanese population, suggests that TCF7L2 is a common susceptibility gene for type 2 diabetes.

Absence of an association between the polymorphisms in the genes encoding adiponectin receptors and type 2 diabetes.

AIMS/HYPOTHESIS: Secreted by adipocytes, adiponectin is a hormone that acts as an antidiabetic and anti-atherogenic adipokine. We recently cloned the genes encoding two adiponectin receptors (ADIPOR1 and ADIPOR2). The aim of this study was to examine whether ADIPOR1 and/or ADIPOR2 play a major role in genetic susceptibility to insulin resistance or type 2 diabetes in the Japanese population. METHODS: By direct sequencing and a search of public databases, we identified single nucleotide polymorphisms (SNPs) in ADIPOR1 and ADIPOR2, and investigated whether these SNPs are associated with insulin resistance and type 2 diabetes in the Japanese population. RESULTS: The linkage disequilibrium (LD) in the chromosomal region of ADIPOR1 was almost completely preserved, whereas the LD in ADIPOR2 was less well preserved. None of the SNPs in ADIPOR1 or ADIPOR2 were significantly associated with insulin resistance or type 2 diabetes. No differences in ADIPOR1 or ADIPOR2 haplotype frequencies were observed between type 2 diabetic and non-diabetic subjects. CONCLUSIONS/INTERPRETATION: Genetic variations in ADIPOR1 or ADIPOR2 are unlikely to lead to a common genetic predisposition to insulin resistance or type 2 diabetes in the Japanese population.

Cell death with predominant apoptotic features in Saccharomyces cerevisiae mediated by deletion of the histone chaperone ASF1/CIA1.

BACKGROUND: Although no potential homologues of multicellular apoptotic genes (e.g. Bax, Bak, Bcl-2, caspases and p53) have been identified in a unicellular eukaryote, previous reports contain several implications of the apoptotic behaviour of yeasts (i.e. Saccharomyces cerevisiae and Schizosaccharomyces pombe). Therefore, whether or not yeast undergoes apoptosis has been a topic of some debate. hCCG1, which is the largest subunit of TFIID and a histone acetyltransferase, appears to be involved in the regulation of apoptosis. The factor hCIA interacts with hCCG1 and functions as a histone chaperone in mammalian cells; its homologue in yeast is Asf1p/Cia1p. Therefore, we anticipated that a yeast mutant in Asf1p/Cia1p would be a valuable tool for studying apoptosis in yeast. RESULTS: We established a strain of S. cerevisiae lacking the histone chaperone ASF1/CIA1. This disruptant, asf1/cia1, arrested preferentially at the G2/M-phase and died. We systematically analysed the phenotype associated with the death of this mutant yeast and identified many changes, such as fragmentation of the nuclei, condensation and fragmentation of chromatin, reduction of the mitochondrial membrane-potential, dysfunction of the mitochondrial proton pump, and a discernible release of cytochrome c to cytoplasm that resembles those in apoptotic multicellular organisms. Other changes potentially associated with the death in our mutant included a reduction in the vacuolar membrane potential, dysfunction of the vacuolar proton pump, reduction of endocytosis, and the presence of many autophagic bodies. However, these mutant yeast cells also showed cellular enlargement, which is characteristic of necrosis. CONCLUSIONS: Cell death in S. cerevisiae occurs with a phenotype that largely resembles apoptosis in multicellular organisms, but that has some features of necrosis. Therefore, we indicate that yeast undergoes a 'prototypal active cell death' that retains some characteristics of passive cell death (necrosis). In addition, we think that active cell death is ubiquitously the essential attribute of life. Although such an active cell death system in yeast remains open to confirmation, we speculate that deletion of the histone chaperone Asf1p/Cia1p inhibits the normal assembly/disassembly of nucleosomes in yeast and thereby initiates the active cell death system.

Serine acetyltransferase involved in cysteine biosynthesis from spinach: molecular cloning, characterization and expression analysis of cDNA encoding a plastidic isoform.

A cDNA clone that encodes a chloroplast-localizing isoform of serine acetyltransferase (SATase) (EC 2.3.1.30) was isolated from spinach (Spinacia oleracea L.). The cDNA encodes a polypeptide of 347 amino acids containing a putative transit peptide of ca. 60-70 amino acids at the N-terminal. Deduced amino acid sequence of SATase from spinach exhibited homology with other SATases from plants. DNA blot hybridization analysis showed the presence of 2-3 copies of Sat gene in the genome of spinach. RNA blot hybridization analysis indicated the constitutive expression of Sat gene in green and etiolated seedlings of spinach. Bacterial expression of the cDNA could directly rescue the cysteine auxotrophy of Escherchia coli caused by a lack of SATase locus (cysE). Catalytically active SATase protein was produced in E. coli cells. L-Cysteine, an end product of the cysteine biosynthetic pathway, inhibited the activity of recombinant spinach SATase, indicating the regulatory function of SATase in this metabolic pathway. A chloroplastic localization of this spinach SATase was revealed by the analyses of transgenic plant expressing transit peptide of SATase-beta-glucuronidase (GUS) fusion protein, and transient expression using the transit peptide-green fluorescent protein (GFP) fusion protein. The result from in vitro translation analysis suggests that this cDNA may encode both plastidic and cytosolic SATases.

Identification of mouse Jun dimerization protein 2 as a novel repressor of ATF-2.

A mouse cDNA that encodes a DNA-binding protein was identified by yeast two-hybrid screening, using activating transcription factor-2 (ATF-2) as the bait. The protein contained a bZIP (basic amino acid-leucine zipper region) domain and its amino acid sequence was almost identical to that of rat Jun dimerization protein 2 (JDP2). Mouse JDP2 interacted with ATF-2 both in vitro and in vivo via its bZIP domain. It was encoded by a single gene and various transcripts were expressed in all tested tissues of adult mice, as well as in embryos, albeit at different levels in various tissues. Furthermore, mouse JDP2 bound to the cAMP-response element (CRE) as a homodimer or as a heterodimer with ATF-2, and repressed CRE-dependent transcription that was mediated by ATF-2. JDP2 was identified as a novel repressor protein that affects ATF-2-mediated transcription.

Cerebrospinal fluid immunocytochemical analysis and neuroimaging in the diagnosis of primary leptomeningeal melanoma. Case report.

A 20-year-old man presented with slowly progressing symptoms indicative of increased intracranial pressure. Two weeks later he underwent surgery for placement of a ventriculoperitoneal shunt. Cytological examination of the patient's cerebrospinal fluid (CSF) revealed atypical cells that contained no detectable melanin deposits, but proved to be immunocytochemically positive for monoclonal antibodies to melanocytic cells (HMB-45) and S-100 protein. Dermatological and ophthalmological examinations failed to demonstrate any abnormalities. On the basis of these findings, a diagnosis of primary leptomeningeal melanoma was made. Gadolinium-enhanced magnetic resonance (MR) images of the brain and spinal regions obtained 2 months after admission demonstrated typical widespread leptomeningeal enhancement. Results of technetium-99m-hexakis (2-methoxyisobutyl isonitrile) single-photon emission computerized tomography (99mTc-MIBI SPECT) scanning revealed intense uptake of the isotope in the leptomeningeal regions and some cisterns. The patient's condition progressively worsened and he died 5 months after admission. The diagnosis was confirmed at autopsy. Immunocytochemical analysis of CSF performed using HMB-45 and S-100 protein antibodies is important for the diagnosis of leptomeningeal melanoma because of the test's simplicity, high specificity, and sensitivity. Gadolinium-enhanced MR imaging is used to demonstrate the extent of the leptomeningeal melanoma. An additional and supplemental neuroimaging modality, 99mTc-MIBI SPECT scanning has good potential for the detection and diagnosis of leptomeningeal melanoma.

Purification, crystallization and preliminary X-ray crystallographic analysis of human CCG1-interacting factor B.

A novel human factor CIB (CCG1-interacting factor B) has been isolated using the yeast two-hybrid system. The 22 kDa CIB protein has been expressed in Escherichia coli, purified to homogeneity and crystallized in a form suitable for crystallographic studies. The protein was crystallized in the orthogonal space group P2(1)2(1)2(1), with unit-cell parameters a = 43.60 (2), b = 44.45 (1), c = 110.70 (5) A and one molecule in the asymmetric unit. The crystal diffracted beyond 2.2 A resolution using synchrotron radiation.

Involvement of the TIP60 histone acetylase complex in DNA repair and apoptosis.

It is well known that histone acetylases are important chromatin modifiers and that they play a central role in chromatin transcription. Here, we present evidence for novel roles of histone acetylases. The TIP60 histone acetylase purifies as a multimeric protein complex. Besides histone acetylase activity on chromatin, the TIP60 complex possesses ATPase, DNA helicase, and structural DNA binding activities. Ectopic expression of mutated TIP60 lacking histone acetylase activity results in cells with defective double-strand DNA break repair. Importantly, the resulting cells lose their apoptotic competence, suggesting a defect in the cells' ability to signal the existence of DNA damage to the apoptotic machinery. These results indicate that the histone acetylase TIP60-containing complex plays a role in DNA repair and apoptosis.

Functional interaction of general transcription initiation factor TFIIE with general chromatin factor SPT16/CDC68.

BACKGROUND: Transcriptional initiation of class II genes is one of the major targets for the regulation of gene expression and is carried out by RNA polymerase II and many auxiliary factors, which include general transcription initiation factors (GTFs). TFIIE, one of the GTFs, functions at the later stage of transcription initiation. As recent studies indicated the possibility that TFIIE may have a role in chromatin transcriptional regulation, we isolated TFIIE-interacting factors which have chromatin-related functions. RESULTS: Using the yeast two-hybrid screening system, we isolated the C-terminal part of the human homologue of Saccharomyces cerevisiae (y) Spt16p/Cdc68p, a general chromatin factor. The C-terminal part of human SPT16/CDC68 directly interacts with TFIIE, and ySpt16p/Cdc68p also interacts with yTFIIE (Tfa1p/Tfa2p), thus indicating the existence of an evolutionarily conserved interaction between TFIIE and SPT16/CDC68. Functional interaction of yTFIIE and ySpt16p/Cdc68p was examined using a conditional yTFIIE-alpha mutant strain. Over-expression of ySpt16p/Cdc68p suppressed the phenotype of cold sensitivity of the yTFIIE-alpha-cs mutant strain, and in vitro binding assays revealed that yTFIIE-alpha-cs mutant protein showed diminished binding affinity to ySpt16p/Cdc68p. CONCLUSIONS: These observations indicate that general transcription initiation factor TFIIE functionally interacts with general chromatin factor SPT16/CDC68, a finding which provides new insight into the involvement of TFIIE in chromatin transcription. This may well lead to a breakthrough in relationships between the transcription initiation process and structural changes in chromatin.