Encenicline, an α7 Nicotinic Acetylcholine Receptor Partial Agonist, Reduces Immune Cell Infiltration in the Colon and Improves Experimental Colitis in Mice.

The α7 pentamer nicotinic acetylcholine receptors (nAChRs) are a target in transduction of anti-inflammatory signals from the central nervous system to the gastrointestinal (GI) tract. The aim of this study was to investigate the anti-inflammatory action of the novel α7 nAChR partial agonist encenicline and to determine the mechanism underlying its activity. Anti-inflammatory activity of encenicline was evaluated using trinitrobenzenesulfonic acid (TNBS)- and dextran sulfate sodium (DSS)-induced models of colitis. Macroscopic score, ulcer score, colon length and thickness, as well as myeloperoxidase (MPO) activity were recorded. Immunohistochemistry (IHC) was used to measure the infiltration of immune cells in the colon. Furthermore, we employed flow cytometry to determine the effect of encenicline on frequencies of FoxP3(+) and interleukin (IL)-17A(+) T cells in the mouse colon. Encenicline attenuated TNBS- and DSS-induced colitis in mice via α7 nAChRs, as indicated by significantly reduced macroscopic parameters and MPO activity. Treatment with encenicline significantly reduced the infiltration of macrophages, neutrophils, and B cells in the colon of TNBS-treated animals, as indicated by IHC. In the TNBS model encenicline reduced the frequency of FoxP3(+) IL-17A(+) T cells in the colon. In the DSS-model treatment encenicline increased the frequency of FoxP3(+) T cells and reduced IL-17A(+) T cells. Stimulation of α7 nAChR with partial agonist encenicline alleviates colitis via alteration of the number and/or activation status of the immune cells in the gut, emphasizing a potential role of α7 nAChRs as a target for anticolitic drugs.

FABP4 blocker attenuates colonic hypomotility and modulates white adipose tissue-derived hormone levels in mouse models mimicking constipation-predominant IBS.

BACKGROUND: The role of fatty acid binding protein 4 (FABP4) in lower gastrointestinal (GI) motility is unknown. We aimed to verify the effect of inhibition of FABP4 on GI transit in vivo, and to determine the expression of FABP4 in mouse and human tissues. METHODS: Fatty acid binding protein 4 inhibitor, BMS309403, was administered acutely or chronically for 6 and 13 consecutive days and its effect on GI transit was assessed in physiological conditions and in loperamide-induced constipation. Intracellular recordings were made to examine the effects of BMS309403 on colonic excitatory and inhibitory junction potentials. Abdominal pain was evaluated using behavioral pain response. Localization and expression of selected adipokines were determined in the mouse colon and serum using immunohistochemistry and Enzyme-Linked ImmunoSorbent Assay respectively. mRNA expression of FABP4 and selected adipokines in colonic and serum samples from irritable bowel syndrome (IBS) patients and control group were assessed. KEY RESULTS: Acute injection of BMS309403 significantly increased GI motility and reversed inhibitory effect of loperamide. BMS309403 did not change colonic membrane potentials. Chronic treatment with BMS309403 increased the number of pain-induced behaviors. In the mouse serum, level of resistin was significantly decreased after acute administration; no changes in adiponectin level were detected. In the human serum, level of adiponectin and resistin, but not of FABP4, were significantly elevated in patients with constipation-IBS (IBS-C). FABP4 mRNA expression was significantly downregulated in the human colon in IBS-C. CONCLUSIONS AND INFERENCES: Fatty acid binding protein 4 may be involved in IBS pathogenesis and become a novel target in the treatment of constipation-related diseases.

High activity of the endogenous opioid system and acute but not chronic stress influence experimental colitis development in mice.

Exposition to environmental factors is one of the major underlying causes in inflammatory bowel diseases (IBD), with several endogenous systems involved. Our aim was to characterize the impact of stress on the colitis development in relation to the endogenous opioid system (EOS) activity in mice. A unique mouse model of high and low activity of EOS (namely high (HA)/low (LA) stress-induced analgesia) was employed. Mice were bred using bidirectional selection and classified as HA or LA line based on the measurement of analgesia. Colitis was induced by instillation of trinitrobenzenesulfonic acid in 30% EtOH/0.9% NaCl. After 4 days, the macroscopic score was assessed and samples for molecular and histological studies were collected. To evaluate the influence of stress on colitis development, chronic mild stress (exposure to stress stimuli for 2 and 5 weeks) and acute stress (short restraint over 3 days) were applied before colitis induction. We observed a difference in the colitis development between non-stressed HA and LA mice, as indicated by macroscopic and ulcer scores. Acute stress improved colitis in HA mice but did not change the inflammation score in LA line as compared to respective non-stressed mice. Chronic mild stress had no influence on colitis in either of mouse lines. Our study supports the hypothesis that the activity of EOS may be crucial in IBD development. We also evidence that acute, but not chronic stress influenced IBD exacerbation, depending on EOS function.

Systemic Administration of Sialorphin Attenuates Experimental Colitis in Mice via Interaction With Mu and Kappa Opioid Receptors.

BACKGROUND AND AIMS: Pharmacological treatment and/or maintenance of remission in inflammatory bowel disease [IBD] is currently one of the biggest challenges in the field of gastroenterology. Here we aimed to assess the anti-inflammatory effect and the mechanism of action of sialorphin, the natural blocker of the endogenous opioid peptide-degrading enzymes neprilysin [NEP] and aminopeptidase N [APN], in mouse models of IBD and the changes in the expression of these enzymes in IBD patients. METHODS: We used two models of experimental colitis in mice [2,4,6-trinitrobenzene sulphonic acid [TNBS]- and dextran sulphate sodium [DSS]-induced]. Macroscopic score, ulcer score, colonic wall thickness, and myeloperoxidase [MPO] activity were recorded. Additionally, we measured the expression of NEP and APN in the colon of IBD patients and healthy controls. RESULTS: We showed that sialorphin attenuated acute, semichronic, and relapsing TNBS-induced colitis in mice after systemic administration, and its anti-inflammatory action is associated with mu and kappa opioid receptors. CONCLUSIONS: We show that indirect stimulation of opioid receptors by the blockade of NEP and APN is a promising pharmacological strategy for the treatment of IBD, and may become of greater importance than the use of classical opioid agonists.

Colorectal cancer-associated nuclear antigen.

By using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting assays in the presence of polyclonal antiserum raised against electrophoretically specific polypeptides of colorectal cancer nuclear polypeptides with M(r) of 35-40 kDa, we have identified p36 protein whose expression accompanies tumorigenesis of large intestine. Immunological analysis of 35 nuclear protein preparations has indicated expression of p36 antigen in nine of 11 right-sided (81.8%) and 21 of 24 (87.5%) left-sided colorectal tumor cases, but not in any control tissue samples. In this study, we have identified p36 antigen in two colon tumor cell lines, i.e., SW620 and HT29 as well. Fractionation experiments based on selective extraction of nuclei isolated from cancerous specimens, which enables their separation into chromatin, nuclear matrix and its subfraction, i.e., internal and peripheral matrix have revealed the concentration of this particular antigen in the internal matrix.

Transient receptor potential vanilloid 4 inhibits mouse colonic motility by activating NO-dependent enteric neurotransmission.

UNLABELLED: Recent studies implicate TRPV4 receptors in visceral pain signaling and intestinal inflammation. Our aim was to evaluate the role of TRPV4 in the control of gastrointestinal (GI) motility and to establish the underlying mechanisms. We used immunohistochemistry and PCR to study TRPV4 expression in the GI tract. The effect of TRPV4 activation on GI motility was characterized using in vitro and in vivo motility assays. Calcium and nitric oxide (NO) imaging were performed to study the intracellular signaling pathways. Finally, TRPV4 expression was examined in the colon of healthy human subjects. We demonstrated that TRPV4 can be found on myenteric neurons of the colon and is co-localized with NO synthase (NOS-1). In vitro, the TRPV4 agonist GSK1016790A reduced colonic contractility and increased inhibitory neurotransmission. In vivo, TRPV4 activation slowed GI motility and reduced stool production in mouse models mimicking pathophysiological conditions. We also showed that TRPV4 activation inhibited GI motility by reducing NO-dependent Ca(2+) release from enteric neurons. In conclusion, TRPV4 is involved in the regulation of GI motility in health and disease. KEY MESSAGES: • Recent studies implicate TRPV4 in pain signaling and intestinal inflammation. • Our aim was to characterize the role of TRPV4 in the control of GI motility. • We found that TRPV4 activation reduced colonic contractility. • Our studies also showed altered TRPV4 mRNA expression in IBS-C patients. • TRPV4 may be a novel pharmacological target in functional GI diseases.

Diversity of nuclear protein fractions of hamster liver and hepatoma produced by DNaseI.

The structural and functional diversity between active and inactive chromatin is thought to depend, in part, upon differences in DNA-bound protein composition, including changes in the number of sulfhydryl groups. The aim of the present study was to compare protein composition in untreated nuclei, DNaseI-released and resistant nuclear fractions of hamster liver and Kirkman-Robbins hepatoma cells. Electrophoretic analysis of nuclear proteins showed some evident quantitative and qualitative differences between normal and neoplastic cells. The most significant diversities were noticed in DNaseI solubilized fraction of both types of cells. Nuclease attack released a characteristic set of non-histones with mol. wt 37,000, 50,000, 74,000 and 130,000-160,000 from transformed cells, and polypeptides of mol. wt 45,000 and 76,000 from normal cells. Cell-specific distribution within examined nuclear polypeptides was revealed using selective staining of their protein-bound sulfhydryls. Immunoblot analysis demonstrated that a non-histone protein with mol. wt 48,000, overexpressed in rodent tumour cells, was exclusively concentrated in liver DNaseI-sensitive fraction, which amounted only to 8.3% +/- 2.0% of total nuclear DNA. In hepatoma cells, however, this particular polypeptide is distributed between nuclease-sensitive and nuclease-resistant nuclear fractions. Non-histone protein of mol. wt 48,000 appeared to contain free sulfhydryl groups. In summary, these results show molecular specificity of nuclear proteins from normal and tumour cells and differences in their distribution among nuclease-released and nuclease-resistant nuclear fractions. The diversity in molecular characteristics and sulfhydryl group patterns observed among the examined proteins of normal and neoplastic cells may suggest their involvement in some changes in the rearrangement of nuclei during neoplastic transformation.

p53 protein detection by the western blotting technique in normal and neoplastic specimens of human endometrium.

The aim of the study was to investigate p53 protein expression by the Western blotting technique (estimated by integrated optical density - IOD) in normal (n = 13) and neoplastic (n = 40) human endometrial tissues as well as in a case of uterine carcinosarcoma and in a specimen of the botryoid sarcoma of the uterine cervix. p53 protein levels were correlated with patients' age as well as with conventionally used clinicopathological features of the endometrial neoplasm. A statistically significant difference was noted in p53 levels in the nuclear, but not in the cytoplasmatic, fraction between the normal endometria and endometrial cancer tissues (P < 0.0001). In the neoplastic endometria, nuclear p53 protein expression was higher than in cytoplasmatic fraction, and the difference was significant (P < 0.05). Higher nuclear p53 protein levels correlated with advanced histological grading of endometrioid endometrial carcinomas, but no relationship was noted between p53 protein expression and patients' age, clinical stage, histological type or depth of myometrial invasion. A case of uterine carcinosarcoma and a specimen of a botryoid sarcoma of the uterine cervix expressed nuclear p53 oncoprotein (57 IOD and 89 IOD, respectively). In conclusion, we found a statistically higher nuclear p53 levels in malignant as compared to normal human endometrial specimens by the Western blotting technique. Although there were no significant differences between p53 expression and clinicopathological features of the neoplasm (except poor histological grading), further studies are necessary to evaluate the influence of p53 nuclear/cytoplasmatic levels on the clinical outcome of Polish patients suffering from endometrial cancer.

High mobility group non-histone chromosomal proteins from chicken thrombocytes and erythrocytes.

The high mobility group non-histone chromosomal proteins (HMG) from chicken thrombocytes were compared with those from chicken erythrocytes. In terms of their extractability, electrophoretic behaviour in polyacrylamide gels containing 2.5 M-urea, and amino acid composition, the HMG proteins of these cells were indistinguishable. However, microcomplement fixation assay performed in the presence of antiserum against HMG-1 and HMG-2 revealed that these high molecular weight HMG proteins of chicken thrombocytes and erythrocytes are not cross-reactive. Electrophoretic analysis of HMG-1 and HMG-2 from these two types of cells in polyacrylamide gels containing 6.25 M-urea suggested differences in the HMG-2 patterns.

Immunological analysis of histone H2A from the slime mold Physarum polycephalum.

Specific antibodies against the histone H2A from calf thymus were generated by injecting rabbits with complexes: histone H2A-RNA with a protein to RNA ratio of 3:1. In the microcomplement fixation assay the antibodies against the histone H2A from calf thymus immuno-reacted with the histone H2A from calf thymus but not with H2A from Physarum polycephalum. The histone H2A from calf thymus therefore appears to have an immunological determinant(s) which does not exist in H2A from Physarum polycephalum.

Molecular characterization of non-histone chromatin proteins from experimental tumours.

Using two-dimensional (2-D) electrophoresis, two non-histone chromatin protein fractions (NHCP1 and NHCP2) from three animal tumours (Kirkman-Robbins hepatoma, Morris hepatoma 7777 and Ehrlich ascites cells) and normal hamster liver were analyzed. Apart from many common components several tissue specific polypeptides of the NHCP1 and NHCP2 fractions were detected. It was found that some spots present in electropherograms of non-histone proteins of tumour cells (M X 10(-3)/pI): 17-24/4.9-6.5 (NHCP1 and NHCP2); 34-41/4.9-6.0 (HCP1 and NHCP2); 44-46/5.3-7.5 (HCP2); 46-49/5.0-7.5 (NHCP1); 49/5.9-7.5 (NHCP2) and 102-134/5.6-7.0 (NHCP1) were absent from normal liver.

Molecular characterization of cellular proteins from colorectal tumors.

AIMS AND BACKGROUND: Recent evidence has suggested that progressive stages of colorectal tumorigenesis can be defined by a sequence of genetic events characterized by altered expression of certain genes and the appearance of cancer-specific proteins. Although the significance of these events is still not clear, expression of cancer-specific protein components may be directly involved in the neoplastic transformation. The purpose of the present study was to compare molecular characteristics of cellular proteins from human colorectal tumors and normal colonic mucosa. METHODS: Normal mucosa and colorectal tumors from 18 patients were fractionated by a differential centrifugation scheme into four cellular fractions, i.e., nuclear, mitochondrial (10P), microsomal (100P) and cytosolic (100S). The proteins of these fractions from normal and tumorigenic mucosa were analyzed by one-dimensional polyacrylamide gel electrophoresis followed by Coomassie brilliant blue R-250 and silver nitrate staining. Nuclear proteins from normal and neoplastic tissues which had revealed the most significant diversities were further characterized by two-dimensional gel electrophoresis. Electrophoretically cancer-specific nuclear proteins in the molecular mass zone 35-40 kDa were used as immunogen to produce rabbit polyclonal antibodies. RESULTS: Electrophoretic analysis by one-dimensional gel electrophoresis showed clear differences in molecular characteristics of cellular proteins between normal and tumorigenic mucosa, especially among nuclear fractions. The latter were also confirmed by their two-dimensional electrophoresis results. Rabbit antibodies raised against electrophoretically specific nuclear proteins characterized by molecular mass of 35-40 kDa cross-reacted with 36 kDa polypeptide in 15 of 18 (83.3%) studied nuclear fractions of colorectal tumors but not with any normal mucosa. In some cases, nuclear cancer-associated components of 38 and 40 kDa were also recognized by these antibodies. CONCLUSIONS: During colorectal carcinogenesis, specific expression of several nuclear proteins takes place. One of them, the polypeptide of 36 kDa not found in normal colonic epithelium, was shared by over 83% of the studied carcinomas despite variations in detailed cancer properties. This particular nuclear protein may be considered as a potential marker for the colon malignancy.

Zinc and cadmium analysis in human prostate neoplasms.

The objective of this study was to test the hypothesis that prostatic cancer is associated with the changes of zinc (Zn) and cadmium (Cd) concentration. Normal prostate, benign prostatic hyperplasia (BPH), and prostatic carcinoma (PCA) were analyzed for Zn and Cd by atomic absorption spectrometry. Cd level was measured using a graphite furnace and Zn level was measured by flame mode. Metal content was assessed in whole tissues and in nuclear, plasma membrane, and cytosolic fractions. An increase of Zn content in BPH, but a decrease in PCA as compared to normal tissue, was observed. Cd concentration appeared to be higher in BPH and PCA than in normal tissue. No correlation between Zn and Cd level was found in BPH specimens obtained from the same patients. Probability values of p < or = 0.05 were considered to indicate significant differences. Obtained results seem to support the hypothesis of Cd carcinogenicity and preventing function of Zn in prostatic cancer. Plasma membrane fraction corresponding to lysosomal, mitochondrial, and microsomal subcellular compartments are probably critical in Zn and Cd participation in human prostate neoplasms.

Experimental colitis in mice is attenuated by changes in the levels of endocannabinoid metabolites induced by selective inhibition of fatty acid amide hydrolase (FAAH).

BACKGROUND AND AIMS: Pharmacological treatment and/or maintenance of remission in inflammatory bowel diseases (IBD) is currently one of the biggest challenge in the field of gastroenterology. Available therapies are mostly limited to overcoming the symptoms, but not the cause of the disease. Recently, the endocannabinoid system has been proposed as a novel target in the treatment of IBD. Here we aimed to assess the anti-inflammatory action of the novel fatty acid amide hydrolase (FAAH) inhibitor PF-3845 and its effect on the endocannabinoid and related lipid metabolism during the course of experimental colitis. METHODS: We used two models of experimental colitis in mice (TNBS- and DSS-induced) and additionally, we employed LC/MS/MS spectrometry to determine the changes in biolipid levels in the mouse colon during inflammation. RESULTS: We showed that the FAAH inhibitor PF-3845 reduced experimental TNBS-induced colitis in mice and its anti-inflammatory action is associated with altering the levels of selected biolipids (arachidonic and oleic acid derivatives, prostaglandins and biolipids containing glycine in the mouse colon). CONCLUSIONS: We show that FAAH is a promising pharmacological target and the FAAH-dependent biolipids play a major role in colitis. Our results highlight and promote therapeutic strategy based on targeting FAAH-dependent metabolic pathways in order to alleviate intestinal inflammation.

Activation of the endogenous nociceptin system by selective nociceptin receptor agonist SCH 221510 produces antitransit and antinociceptive effect: a novel strategy for treatment of diarrhea-predominant IBS.

BACKGROUND: Diarrhea-predominant irritable bowel syndrome (IBS-D) is a functional gastrointestinal (GI) disorder, defined by the presence of loose stools and abdominal pain. In search for a novel anti-IBS-D therapy, here we investigated the nociceptin receptor (NOP)-dependent effects in the GI tract. METHODS: A novel potent and selective NOP agonist SCH 221510 was used in the study. The effect of NOP activation on mouse intestinal motility was characterized in vitro and in vivo, in physiological conditions and in animal models of hypermotility and diarrhea. Well-established mouse models of visceral pain were used to characterize the antinociceptive effect of the NOP activation. To provide additional evidence that the endogenous nociceptin system is a relevant target for IBS, NOP expression and nociceptin levels were quantified in serum and colonic biopsies from IBS-D patients. KEY RESULTS: SCH 221510 produced a potent NOP-mediated inhibitory effect on mouse intestinal motility in vitro and in vivo in physiological conditions. The NOP agonist displayed an antidiarrheal and analgesic action after oral administration in animal models mimicking the symptoms of IBS-D. Studies on human samples revealed a strong decrease in endogenous nociceptin system expression in IBS-D patients compared with healthy controls. CONCLUSIONS & INFERENCES: Collectively, mouse and human data suggest that the endogenous nociceptin system is involved in IBS-D and may become a target for anti-IBS-D treatments using potent and selective synthetic NOP agonists.

Polyphenol extract from evening primrose pomace alleviates experimental colitis after intracolonic and oral administration in mice.

Oenothera paradoxa (EP) preparations are commonly used in folk medicine to treat skin diseases, neuralgia, and gastrointestinal (GI) disorders. Several reports suggested that EP preparations exhibit potent anti-inflammatory and antioxidant activities both in vitro and in vivo. Here, we aimed to characterize the action of EP pomace polyphenol extract in mouse model of colitis. We analyzed the composition of EP pomace polyphenol extract using reversed phase HPLC system and ultra-performance liquid chromatography (UPLC) system coupled with a quadrupole-time of flight (Q-TOF) MS instrument. Then, we used a well-established animal model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis to determine the anti-inflammatory action of EP pomace polyphenol extract. We also investigated the effect of the EP pomace polyphenol extract on pro-inflammatory (IL-1ß and TNF-α) cytokine mRNA levels and hydrogen peroxide concentration in the inflamed colon. Administration of EP pomace polyphenol extract significantly improved macroscopic and microscopic damage scores, as well as myeloperoxidase (MPO) activity in TNBS-treated mice. The anti-inflammatory effect of the extract was observed after intracolonic and oral administration and was dose-dependent. Significant reduction of tissue hydrogen peroxide level after treatment with EP pomace polyphenol extract suggests that its therapeutic effect is a result of free radical scavenging. This novel finding indicates that the application of the EP pomace polyphenol extract in patients with inflammatory bowel diseases (IBDs) may become an attractive supplementary treatment for conventional anti-inflammatory therapy.

Transient receptor potential vanilloid 4 blockade protects against experimental colitis in mice: a new strategy for inflammatory bowel diseases treatment?

Recent reports suggested that the activation of Transient Receptor Potential Vanilloid 4 (TRPV4) receptors in the gastrointestinal tract has pro-inflammatory effects. In this study, we demonstrated for the first time that TRPV4 mRNA expression is up-regulated in patients with inflammatory bowel diseases (IBD). Furthermore, selective blockade of TRPV4 in the 2,4,6-trinitrobenzenesulfonic acid animal model alleviates colitis and pain associated with the intestinal inflammation. Our study indicates that TRPV4 may play a role in mechanisms of defense in intestinal inflammation and that TRPV4 may be an attractive target for future systemic or topic anti-inflammatory treatment in patients with IBD.

The expression of TLR pathway molecules in peripheral blood mononuclear cells and their relationship with tumor invasion and cytokine secretion in laryngeal carcinoma.

PURPOSE: The aim of this study was to analyze of TLRs mRNA expression in peripheral blood mononuclear cells as potential biomarkers of neoplastic lesions progress and to evaluate their role in the possible mechanisms responsible for the secretion of cytokines in laryngeal cancer. MATERIAL/METHODS: The analysis of TLR2, TLR4, TRAF6, IRAK1 expression in isolated PBMCs by the reverse transcription PCR (RT-PCR) analysis as well as IL-6, IL-8 and TNF-α levels in supernatants of peripheral blood mononuclear cells from 55 patients with carcinoma of the larynx was performed by ELISA. The invasiveness of carcinoma was evaluated according to tumor front grading, TFG. RESULTS: We noted that tumors with a well-defined borderline were characterized by significantly higher values of the average expression of TRAF6. Our research also confirmed that more aggressive carcinomas according to TFG, with a more dispersed type of invasion were characterized by significantly lower values of the average expression of IRAK1. Moreover, we observed that tumors with the invasion of cartilage were characterized by significantly lower values of the average expression of TLR4. In addition, the relationships of TLR2 with IL-6 and TNF-α level were highlighted. Significant interconnections were also found between the TLR4 and IL-8, TNF-α, IL-6 secretion after stimulation. The relationships of TRAF6 with IL-8 production after stimulation were noted. CONCLUSIONS: Our findings confirmed the implication of the TLRs pathway molecules in proinflammatory cytokine secretions and their importance as encouraging potential indicators for assessment of the degree of aggressive tumor phenotype.

Regulation of transcription in eukaryotes by DNA-binding proteins.

1. The recognition of DNA by gene regulatory proteins is often mediated by structural motifs that comprise a protein DNA-binding domain. 2. Although binding of these proteins to DNA is not itself sufficient to affect transcription it is a necessary prerequisite. 3. This review summarizes recent studies that define structural motifs for DNA binding function of eukaryotic transcription factors.

Immunospecificity of nonhistone chromatin proteins tightly bound to DNA from chicken thrombocytes and erythrocytes.

Erythroid cell-specific antisera capable of detecting tightly bound nonhistone chromatin protein--DNA complexes were obtained by injecting rabbits with dehistonized chicken reticulocyte chromatin. The antisera showed no crossreactivity with chromatin of thrombocytes which are regarded as cells genealogically closely related with erythrocytes. The lack of thrombocyte chromatin immunoactivity was not caused by conformational constrains. Tightly bound nonhistone protein--DNA complexes isolated from thrombocyte chromatin showed no immunological similarity with these of erythrocyte chromatin.