In avian erythroid cells the erythrocyte-specific histone H5 is involved, like H1, in the packing of nucleosomes in the 25-nm chromatin fibers. In this study the distribution of histone H5 along the polynucleosomal chains was visualized by immunoelectron microscopy.Trinucleosomes from chicken erythrocytes and liver were used in order to test the specificity of the reaction with purified rabbit anti-H5 antibodies at various ionic strengths (5 -80 mM). Long-chain chromatin was then reacted with anti-H5 antibodies and with sorted monomeric ferritin conjugate under chosen conditions.The antigenic determinants of histone H5 in the 25-nm fiber of long-chain chromatin (at 80 mM NaCl) are as accessible to the specific antibodies as in trinucleosomes.When the immunocomplexes were examined by electron microscopy in a low-ionic-strength buffer, permitting maximum extension of the chromatin structure on the grid, clusters of compacted nucleosomes were seen, separated by short regions of relaxed nucleosomes. Single nucleosomes enlarged by the antibodies are sometimes visible in the extended domains. We conclude that histone H5 is located primarily on series of adjacent nucleosomes but it can also be found on single nucleosomes located in the H1-enriched extended domains.Chicken erythrocytes contain a unique histone H5 [1,2], which also exists in the nucleated erythrocytes of a variety of non-mammalian vertebrates and even invertebrates in place of histone HI. The histone H5 may be considered as an H1 variant emerging in the earliest forms of erythroid cells in connection with cell specialization in embryonic [3] and adult life [4]. As erythropoiesis proceeds, the early dividing cells differentiate into non-dividing reticulocytes and then into no longer transcribing mature erythrocytes.All the histones in normal dividing cells are synthesized at the time when DNA replication occurs, while histone H5 is the only one synthesized in the non-dividing reticulocytes [5,6]. During the terminal stages of erythrocyte differentiation the histone H5 accumulated up to a ratio of 3: l for H5/Hl in mature erythrocytes, accompanied by a transition to supercompaction of the chromatin and reduced transcriptional activity. An unanswered question is how do the H5 molecules, synthesized last, replace the HI molecules and how are these histones distributed along the polynucleosomal chains of mature erythrocyte chromatin. We approached this question by immunoelectron microscopy, whereas Pospelov et al. [7] have recently investigated the lateral proximity of the two histone types by cross-linking experiments. Our results led to similar conclusions. MATERIALS AND METHODS Preparative ProceduresHistone H5 was extracted from purified nuclei of chicken erythrocytes as described previously [8,9]. The purity of the protein was assessed by polyacrylamide gel electrophoresis according to Weintraub [lo]. Antibodies against H5 were elicited by injection of rabbits with histone-H5 -RNA complexes as recommended by Stollar and Ward [ll]. Pure antibodies against...

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Studies in rodents and humans have suggested that the pineal gland and its secretory product melatonin play an important role in the modulation of the immune system. In this study, we tested the hypothesis of a difference in immune parameters between ewes with naturally high vs. low circulating melatonin. Thus, two comparable groups of 10 Ile-de-France sheep were selected from a large flock, for their naturally high and low plasma concentrations of melatonin. The mean plasma melatonin concentrations during daytime (09:00 hr) and nighttime (24:00 hr) were, respectively, 9 and 664 pg/mL (high group) and 5 and 169 pg/mL (low group; P<0.01). Animals from both groups were subjected to various in vitro and in vivo measurements of the characteristics of their immune system. The total number of white blood cells (lymphocytes, polymorphonuclears, and monocytes) and the assessment of the sub-populations of blood lymphocytes (T4, T8, T19, B, and monocytes) did not show any significant differences between the two groups, sampled during day or night. The level of blood leukocytes proliferation after in vitro culture with ConA, LPS, or CWF stimulation, before or after experimental immunization, did not reveal any differences. No significant differences were registered in the production of antibodies between the two groups of animals. The results of the present experiment suggest that in natural conditions a high level of circulating melatonin does not modify the activity of the immune system in sheep.

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