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In humans and other primates, blood platelets contain high concentrations of brain-derived neurotrophic factor due to the expression of the BDNF gene in megakaryocytes. By contrast, mice, typically used to investigate the impact of CNS lesions, have no demonstrable levels of brain-derived neurotrophic factor in platelets, and their megakaryocytes do not transcribe significant levels of the Bdnf gene. Here, we explore potential contributions of platelet brain-derived neurotrophic factor with two well-established CNS lesion models, using ‘humanized’ mice engineered to express the Bdnf gene under the control of a megakaryocyte-specific promoter. Retinal explants prepared from mice containing brain-derived neurotrophic factor in platelets were labelled using DiOlistics and the dendritic integrity of retinal ganglion cells assessed after 3 days by Sholl analysis. The results were compared with retinas of wild-type animals and with wild-type explants supplemented with saturating concentrations of brain-derived neurotrophic factor or the tropomyosin kinase B antibody agonist, ZEB85. An optic nerve crush was also performed, and the dendrites of retinal ganglion cells similarly assessed 7-day post-injury, comparing the results of mice containing brain-derived neurotrophic factor in platelets with wild-type animals. In mice engineered to contain brain-derived neurotrophic factor in platelets, the mean serum brain-derived neurotrophic factor levels were 25.74 ± 11.36 ng/mL for homozygous and 17.02 ± 6.44 ng/mL for heterozygous mice, close to those determined in primates. Retinal explants from these animals showed robust preservation of dendrite complexity, similar to that seen with wild-type explants incubated with medium supplemented with brain-derived neurotrophic factor or the tropomyosin receptor kinase B antibody agonist, ZEB85. The Sholl areas under curve were 1811 ± 258, 1776 ± 435 and 1763 ± 256 versus 1406 ± 315 in the wild-type control group (P ≤ 0.001). Retinal ganglion cell survival based on cell counts was similar in all four groups, showing ∼15% loss. A robust neuroprotective effect was also observed following optic nerve crush when assessing the dendrites of the retinal ganglion cells in the transgenic mouse, with Sholl area under the curve significantly higher compared to wild-type (2667 ± 690 and 1921 ± 392, P = 0.026), with no significant difference in the contralateral eye controls. Repeat experiments found no difference in cell survival, with both showing ∼50% loss. These results indicate that platelet brain-derived neurotrophic factor has a strong neuroprotective effect on the dendrite complexity of retinal ganglion cells in both an ex vivo and in vivo model, suggesting that platelet brain-derived neurotrophic factor is likely to be a significant neuroprotective factor in primates.

Aging and metabolic syndrome are associated with neurodegenerative pathologies including Alzheimer’s disease (AD) and there is growing interest in the prophylactic potential of probiotic bacteria in this area. In this study, we assessed the neuroprotective potential of the Lab4P probiotic consortium in both age and metabolically challenged 3xTg-AD mice and in human SH-SY5Y cell culture models of neurodegeneration. In mice, supplementation prevented disease-associated deteriorations in novel object recognition, hippocampal neurone spine density (particularly thin spines) and mRNA expression in hippocampal tissue implying an anti-inflammatory impact of the probiotic, more notably in the metabolically challenged setting. In differentiated human SH-SY5Y neurones challenged with β-Amyloid, probiotic metabolites elicited a neuroprotective capability. Taken together, the results highlight Lab4P as a potential neuroprotective agent and provide compelling support for additional studies in animal models of other neurodegenerative conditions and human studies.

Purpose: Serum lipids are modifiable, routinely collected blood test features associated with cardiovascular health. We examined the association of commonly collected serum lipid measures (total cholesterol [TC], high-density lipoprotein cholesterol [HDL-C], low-density lipoprotein cholesterol [LDL-C], and triglycerides) with intraocular pressure (IOP). Design: Cross-sectional study in the UK Biobank and European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk cohorts. Participants: We included 94 323 participants from the UK Biobank (mean age, 57 years) and 6230 participants from the EPIC-Norfolk (mean age, 68 years) cohorts with data on TC, HDL-C, LDL-C, and triglycerides collected between 2006 and 2009. Methods: Multivariate linear regression adjusting for demographic, lifestyle, anthropometric, medical, and ophthalmic covariables was used to examine the associations of serum lipids with corneal-compensated IOP (IOPcc). Main Outcome Measures: Corneal-compensated IOP. Results: Higher levels of TC, HDL-C, and LDL-C were associated independently with higher IOPcc in both cohorts after adjustment for key demographic, medical, and lifestyle factors. For each 1-standard deviation increase in TC, HDL-C, and LDL-C, IOPcc was higher by 0.09 mmHg (95% confidence interval [CI], 0.06–0.11 mmHg; P < 0.001), 0.11 mmHg (95% CI, 0.08–0.13 mmHg; P < 0.001), and 0.07 mmHg (95% CI, 0.05–0.09 mmHg; P < 0.001), respectively, in the UK Biobank cohort. In the EPIC-Norfolk cohort, each 1-standard deviation increase in TC, HDL-C, and LDL-C was associated with a higher IOPcc by 0.19 mmHg (95% CI, 0.07–0.31 mmHg; P = 0.001), 0.14 mmHg (95% CI, 0.03–0.25 mmHg; P = 0.016), and 0.17 mmHg (95% CI, 0.06–0.29 mmHg; P = 0.003). An inverse association between triglyceride levels and IOP in the UK Biobank (–0.05 mmHg; 95% CI, –0.08 to –0.03; P < 0.001) was not replicated in the EPIC-Norfolk cohort (P = 0.30). Conclusions: Our findings suggest that serum TC, HDL-C, and LDL-C are associated positively with IOP in 2 United Kingdom cohorts and that triglyceride levels may be associated negatively. Future research is required to assess whether these associations are causal in nature.

Purpose To describe and compare associations with macular retinal nerve fiber layer (mRNFL), ganglion cell complex (GCC), and ganglion cell–inner plexiform layer (GCIPL) thicknesses in a large cohort. Design Cross-sectional study. Participants We included 42 044 participants in the UK Biobank. The mean age was 56 years. Methods Spectral-domain OCT macular images were segmented and analyzed. Corneal-compensated intraocular pressure (IOPcc) was measured with the Ocular Response Analyzer (Reichert, Corp., Buffalo, NY). Multivariable linear regression was used to examine associations with mean mRNFL, GCC, and GCIPL thicknesses. Factors examined were age, sex, ethnicity, height, body mass index (BMI), smoking status, alcohol intake, Townsend deprivation index, education level, diabetes status, spherical equivalent, and IOPcc. Main Outcome Measures Thicknesses of mRNFL, GCC, and GCIPL. Results We identified several novel independent associations with thinner inner retinal thickness. Thinner inner retina was associated with alcohol intake (most significant for GCIPL: –0.46 μm for daily or almost daily intake compared with special occasion only or never [95% confidence interval (CI), 0.61–0.30]; P = 1.1×10–8), greater social deprivation (most significant for GCIPL: –0.28 μm for most deprived quartile compared with least deprived quartile [95% CI, –0.42 to –0.14]; P = 6.6×10–5), lower educational attainment (most significant for mRNFL: –0.36 μm for less than O level compared with degree level [95% CI, –0.45 to 0.26]; P = 2.3×10–14), and nonwhite ethnicity (most significant for mRNFL comparing blacks with whites: –1.65 μm [95% CI, –1.86 to –1.43]; P = 2.4×10–50). Corneal-compensated intraocular pressure was associated most significantly with GCIPL (–0.04 μm/mmHg [95% CI, –0.05 to –0.03]; P = 4.0×10–10) and was not associated significantly with mRNFL (0.00 μm/mmHg [95% CI, –0.01 to 0.01]; P = 0.77). The variables examined explained a greater proportion of the variance of GCIPL (11%) than GCC (6%) or mRNFL (7%). Conclusions The novel associations we identified may be important to consider when using inner retinal parameters as a diagnostic tool. Associations generally were strongest with GCIPL, particularly for IOP. This suggests that GCIPL may be the superior inner retinal biomarker for macular pathophysiologic processes and especially for glaucoma.

Optical coherence tomography (OCT) is an imaging technique based on interferometry of backscattered lights from materials and biological samples. For the quantitative evaluation of an OCT system, artificial optical samples or phantoms are commonly used. They mimic the structure of biological tissues and can provide a quality standard for comparison within and across devices. Phantoms contain medium matrix and scattering particles within the dimension range of target biological structures such as the retina. The aim was to determine if changes in speckle derived optical texture could be employed to classify the OCT phantoms based on their structural composition. Four groups of phantom types were prepared and imaged. These comprise different concentrations of a medium matrix (gelatin solution), different sized polystyrene beads (PBs), the volume of PBs and different refractive indices of scatterers (PBs and SiO2). Texture analysis was applied to detect subtle optical differences in OCT image intensity, surface coarseness and brightness of regions of interest. A semi-automated classifier based on principal component analysis (PCA) and support vector machine (SVM) was applied to discriminate the various texture models. The classifier detected correctly different phantom textures from 82% to 100%, demonstrating that analysis of the texture of OCT images can be potentially used to discriminate biological structure based on subtle changes in light scattering.

Nicotinamide adenine dinucleotide (NAD) is a REDOX cofactor and metabolite essential for neuronal survival. Glaucoma is a common neurodegenerative disease in which neuronal levels of NAD decline. Repleting NAD via dietary supplementation of nicotinamide (a precursor to NAD) is effective in preventing retinal ganglion cell neurodegeneration in mouse models. Supporting this, short-term oral nicotinamide treatment in human glaucoma patients provides a recovery of retinal ganglion cell function implying a protection of visual function. Despite this, the mechanism of neuroprotection and full effects of nicotinamide on retinal ganglion cells is unclear. Glaucoma is a complex neurodegenerative disease in which a mix of healthy, stressed, and degenerating retinal ganglion cells co-exist, and in which retinal ganglion cells display compartmentalized degeneration across their visual trajectory. Therefore, we assess the effects of nicotinamide on retinal ganglion cells in normal physiological conditions and across a range of glaucoma relevant insults. We confirm neuroprotection afforded by nicotinamide in rodent models which represent isolated ocular hypertensive, axon degenerative, and mitochondrial degenerative insults. We define a small molecular weight metabolome for the retina, optic nerve, and superior colliculus which demonstrates that ocular hypertension induces widespread metabolic disruption that can be prevented by nicotinamide. Nicotinamide provides these neuroprotective effects by increasing oxidative phosphorylation, buffering and preventing metabolic stress, and increasing mitochondrial size and motility whilst simultaneously dampening action potential firing frequency. These data support continued determination of the utility of long-term NAM treatment as a neuroprotective therapy for human glaucoma.One Sentence SummaryThe NAD precursor nicotinamide has a potent neuroprotective effect in the retina and optic nerve, targeting neuronal function, metabolism, and mitochondrial function.

We conducted a large multi-ethnic meta-analysis of genome-wide association studies for primary open-angle glaucoma (POAG) on a total of 34,179 cases vs 349,321 controls, and identified 127 independent risk loci, almost doubling the number of known loci for POAG. The majority of loci have broadly consistent effect across European, Asian and African ancestries. We identify a link, both genome-wide and at specific loci, between POAG and Alzheimer’s disease. Gene expression data and bioinformatic functional analyses provide further support for the functional relevance of the POAG risk genes. Several drug compounds target these risk genes and may be potential candidates for developing novel POAG treatments.

Purpose: "She wouldn't remember. Even when I go through, and she's decided to go to bed, I'll say I'll come and do her drops. If I didn't say that, they wouldn't be done." Dementia is widely considered as a key factor in whether patients take their medication as prescribed. However, few studies have examined the effect of dementia on medication management strategies for glaucoma including how patient and carer needs impact adherence and long-term prognosis. We report findings from a qualitative grounded theory study incorporating the views of patients, carers, and healthcare professionals.Methods: Eighty-three semistructured interviews were conducted with 35 patients, 22 lay carers, and nine healthcare professionals across sites in Wales and Scotland. These explored understanding of eye drop regimens, barriers, and facilitators to drop administration, as well as attitudes toward glaucoma, dementia, and other comorbidities.Results: Using Pound's synthesis of adherence behavior, we identified categories of active and passive acceptance of medicines, alongside modification or rejection of eye drop regimens. In relation to dementia, participants highlighted transitions between such categories, with a shift from active to passive acceptance commonly reported. This loss of self-medicating capability was referred to as the precipice of care, where entwinement of multiple conditions (eg, heart disease, glaucoma, and dementia) and sociocultural influences (eg, living alone) contributed to accelerated health declines. That said, numerous factors mitigated this, with a key role being the lay carer. Spouses and family members often acted as the monitor of eye drops for patients, seeking intervention when any behavioral changes influenced their administration.Conclusion: Though dementia was associated with progression toward the precipice of care, factors such as communication with healthcare professionals appeared to affect patient adherence. Recommendations for healthcare practice include better recording of dementia diagnoses and integrating eye drops into preexisting routines.

We used cultured adult mouse retinae as a model system to follow and quantify the retraction of dendrites using diolistic labelling of retinal ganglion cells (RGCs) following explantation. Cell death was monitored in parallel by nuclear staining as ‘labelling’ with RGC and apoptotic markers was inconsistent and exceedingly difficult to quantify reliably. Nuclear staining allowed us to delineate a lengthy time window during which dendrite retraction can be monitored in the absence of RGC death. The addition of brain‐derived neurotrophic factor (BDNF) produced a marked reduction in dendritic degeneration, even when application was delayed for 3 days after retinal explantation. These results suggest that the delayed addition of trophic factors may be functionally beneficial before the loss of cell bodies in the course of conditions such as glaucoma.

Restraint has been used as a procedure to study the effects of stress on gestation outcome in rodents. The effects of restraint could potentially be used as a model for the impact of general stress produced by high doses of toxicants and other interventions. In mice, restraint in the peri-implantation period leads to implantation failure, and restraint at appropriate times in organogenesis produces cleft palate, supernumerary ribs, and resorption. In rats, there is some evidence for an association with restraint for implantation failure, but not for the morphological anomalies. Restraint in late gestation alters adult sexual behavior of male rat offspring, but consequences for their fertility are not known. Intrauterine growth retardation is not commonly associated with gestational restraint. In the few studies where they have been directly compared, different restraint procedures produced graded, qualitatively different, or no effects. Adrenocortical hormones have been implicated as mediating the effect of restraint on cleft palate, but not on supernumerary ribs, implantation failure, or sexual differentiation. Given the variety of restraint procedures and the varying species-dependent consequences, it is not possible to infer a generalizable pattern of developmental effects due to gestational stress from the restraint literature. As an alternative approach, contemporary methods in gene expression and developmental biology could profitably be applied to understanding different patterns of stress-mediated effects of toxicant exposures on intrauterine development.

Retinal ganglion cell degeneration has been reported in a range of experimental models of glaucoma. Manifest as pruning of retinal ganglion cell dendrites, it is likely to influence both the function and viability of affected cells. Electrophysiological studies in primate glaucoma have shown that affected cells retain some function and could therefore form a neural substrate for the recovery of visual function in glaucoma. Clinical studies in which the intraocular pressure is reduced have suggested that some improvement in retinal function may be possible in hypotensive eyes. These experimental studies highlight the importance of establishing the extent to which retinal ganglion cell degeneration occurs in human glaucoma. If substantial numbers of degenerating retinal ganglion cells are present in glaucoma, they could present an ideal target for the recovery of vision.

In this paper we present a novel method for performing image registration of different modalities. Mutual Information (MI) is an established method for performing such registration. However, it is recognised that standard MI is not without some problems, in particular it does not utilise spatial information within the images. Various modifications have been proposed to resolve this, however these only offer slight improvement to the accuracy of registration. We present Feature Neighbourhood Mutual Information (FNMI) that combines both image structure and spatial neighbourhood information which is efficiently incorporated into Mutual Information by approximating the joint distribution with a covariance matrix (c.f. Russakoff's Regional Mutual Information). Results show that our approach offers a very high level of accuracy that improves greatly on previous methods. In comparison to Regional MI, our method also improves runtime for more demanding registration problems where a higher neighbourhood radius is required. We demonstrate our method using retinal fundus photographs and scanning laser ophthalmoscopy images, two modalities that have received little attention in registration literature. Registration of these images would improve accuracy when performing demarcation of the optic nerve head for detecting such diseases as glaucoma.

In developmental and reproductive toxicity studies, drinking water is a common means of delivering the test agent. Reduced consumption of toxicant-containing water raises questions about indirect effects of reduced maternal fluid consumption resulting from unpalatability, versus direct effects of the test compound. Issues to consider include: objective assessment of dehydration and thirst, the relative contributions of innate and learned behaviors to drinking behavior and flavor preference, and the objective assessment of physiologic stress. Not only do lab animals under ad lib conditions consume more water than the minimum required to maintain fluid balance, animals faced with water restriction have substantial physiologic capacity for protection of metabolic processes. Measures of blood biochemistry can provide quantifiable, objective indications of fluid balance, but changes in these parameters could result from other causes such as effects of a test toxicant. Consummatory behaviors in response to perceived need are highly influenced by learning. Hence, the drinking behavior, water intake, and flavor acceptance/preference of animals used in toxicology experiments could be subject to learning experiences with the test compound. Physiological symptoms of stress produced by water deprivation may be distinguishable from the symptoms associated with other generalized stressors, such as food deprivation, but doing so may be beyond the scope of most developmental or reproductive toxicity studies. Use of concurrent controls, paired to test groups for water consumption, could help distinguish between the direct effects of a test toxicant as opposed to effects of reduced water consumption alone.

Two new photoaffinity derivatives of polyamines have been synthesized by the reaction of spermine or spermidine with methyl 4-azidobenzimidate. The new compounds were purified chromatographically and characterized by several methods including proton magnetic resonance spectroscopy. The spermine derivative is N1-ABA-spermine [(azidobenzamidino)spermine], and the spermidine derivative is a mixture of N1- and N8-ABA-spermidine. ABA-spermine stabilizes nucleosome core particles in thermal denaturation experiments, with similar but not identical effects when compared with the parent polyamine, spermine. In circular dichroism experiments, ABA-spermine was capable of producing a B----Z transition in poly(dG-m5dC) at a concentration of 30 microM, compared with 5 microM required to produce the same effect with spermine. On the other hand, ANB-spermine [(azidonitrobenzoyl)spermine; Morgan, J. E., Calkins, C. C., & Matthews, H. R. (1989) Biochemistry 28, 5095-5106] stabilized the B form of poly(dG-br5dC). ABA-spermine is a potent inhibitor of ornithine decarboxylase from Escherichia coli, giving 50% inhibition at 0.12 mM, while ANB-spermine is a modest inhibitor, comparable to spermine or spermidine. Under conditions of nitrogen-limited growth, yeast take up ABA-spermine and ABA-spermidine at approximately one-third to half the rate of spermidine or spermine. In contrast, ANB-spermine was not significantly taken up. The photoaffinity polyamines were used to photoaffinity label the DNA in nucleosome core particles, and the sites of labeling were determined by exonuclease protection. All photoaffinity reagents showed both nonspecific labeling and specific sites of higher occupancy. However, the positions of the sites varied: the ANB-spermine sites confirmed those previously reported (Morgan et al., 1989); the ABA-spermine and ABA-spermidine sites were spaced at 9.8 bp intervals from the 3' end of each DNA strand. This observation, together with the effect of spermine on the circular dichroism of DNA in nucleosome core particles, implies that polyamines alter the helical twist of DNA in nucleosome core particles. The ABA-polyamines are offered as general-purpose photoaffinity polyamine reagents.

Intraocular pressure (IOP), an important risk factor for glaucoma, is a continuous trait determined by a complex set of genetic and environmental factors that are largely unknown. Genetic studies in laboratory animals may facilitate the identification of genes that affect IOP. We examined the use of the rebound tonometer for measuring IOP in non-anaesthetised birds, along with the device’s robustness to alignment errors. Calibration curves were obtained by measuring the IOP of cannulated chicken eyes with the rebound tonometer over a range of pressures. To simulate different types of alignment errors that might be expected with measurement of IOP in alert chickens, for some calibrations the tonometer was positioned (1) at various distances from the cornea, (2) laterally displaced from the visual axis, or (3) angled away from the visual axis. In vivo measurements were taken on three-week-old alert chickens from a layer line, a broiler line, and a layer-broiler ‘‘advanced intercross line’’ (AIL) designed to facilitate QTL mapping. The rebound tonometer showed excellent linearity (R2 ¼ 0.95e0.99) during calibration, as well as robustness to variation in the probe-to-cornea distance over the range 3e5 mm and to lateral displacement over the range 0e2 mm. However, the tonometer appeared less robust to off-axis misalignment over the range 0e20? (P < 0.05). Also, the slope of calibration curves sometimes differed between eyes (P < 0.001), presumably reflecting differences in ocular structure. The IOP measured in non-anaesthetised three-week-old AIL chickens was 17.51 ? 0.13 mmHg (mean ? S.E.; N ¼ 105 birds). IOP was significantly associated with corneal thickness (P < 0.05) and body weight (P < 0.001) in a regression model. Replicate measurements were necessary in order to gauge IOP accurately in individual birds; a series of seven tonometry sessions over a 12-h period during the light phase of the light/dark cycle permitted IOP to be measured with a 95% CI of ?0.7 mmHg. IOP did not differ significantly between the broiler and layer chicken lines which served as the progenitor lines for the AIL. In conclusion, the rebound tonometer permits rapid estimation of IOP in chickens and is well tolerated. The small alignment errors that are expected when taking measurements in non-anaesthetised animals are unlikely to affect accuracy. Since high IOP is a major risk factor for glaucoma, identifying QTL controlling IOP may offer future health benefits. However, our preliminary findings highlight several obstacles to mapping such QTL using the chicken advanced intercross line evaluated here.

To determine if a computer-based stereoscopic teaching program could improve optic cup/disc ratio (CDR) agreement between student observers and an expert.Experimental study.Six student observers (A-F) assessed at least 30 digital stereoscopic optic disc images using a digital stereoscopic analysis program. All observers made 36 CDR measurements (every 10 degrees ) for each disc image that they assessed.Disc images were divided into 3 sets of 10 images (image sets 1, 2, and 3). Observers A, B, and C determined CDRs for all 3 image sets. Set 1 was examined without access to a teaching program, set 2 using a teaching program that allowed the observer to view an expert assessment of the optic disc, and set 3 after using the teaching program. Observers D, E, and F evaluated image sets 1 and 3 only and did not have access to the teaching program. Ten months later, these same observers (D-F) viewed image set 2 using the teaching program and then reassessed image set 3.Standard deviation (SD) of CDR differences between observers and an expert, the percentage of CDR observations differingor =0.20 from those of the expert, and intraclass correlation coefficients (ICCs) between observers.Before teaching, the average SD of differences between all observers and the expert was 0.10. After teaching, the average SD of differences between all observers and the expert was 0.057. For observers D, E, and F, the average SD of differences for image set 3 without teaching was 0.074. The percentage of all observer measurements that differedor =0.20 from those of the expert for image set 1 was 16.7%; for set 3 after teaching, 1.7%; and for set 3 for observers D, E, and F without teaching, 10%. Interobserver ICC values for all observers were 0.37 before teaching and 0.76 after. For observers D, E, and F, the ICC value for set 3 without access to teaching was 0.69.This study suggests that an interactive digital stereoscopic teaching program improves agreement between observers and an expert when assessing CDRs.

In mouse hepatoma cells, both the regulatory and the transcribed regions of the cyp1a1 gene assume a nucleosomal configuration when the gene is silent; two nucleosomes occupy specific sites at the transcriptional promoter. Activation of transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin is accompanied by changes in chromatin structure, which depend upon a functional aromatic hydrocarbon (Ah) receptor. In the transcribed region of the gene, nucleosome disruption occurs as a consequence of RNA elongation. In contrast, at the promoter, loss of positioned nucleosome sis independent of transcription and represents an event in the mechanism by which the liganded Ah receptor enhances transcriptional initiation.

Putrescine (a diamine) raises the thermal denaturation temperature of mononucleosomes but produces only minor changes in the overall shape of the thermal denaturation curve. This is similar to the effect of sodium ions and is consistent with nonspecific binding to the DNA of the nucleosome. At very low levels of spermidine or spermine the same simple rise in thermal denaturation temperature is seen but at higher levels (above 1 microM for total spermidine concentration) the thermal denaturation curve becomes substantially sharper and the premelt region of the curve diminishes in area. The acetylspermidines display intermediate effects. The change in shape of the thermal denaturation curve was resolved into components (R1 and R2) due to mononucleosomes in their original conformation plus a component (T) induced by the presence of spermidine or spermine. The proportion of component T was substantially reduced with acetylspermidine, compared to equivalent concentrations of spermidine. Hence, we suggest that spermidine acetylation in vivo has the potential to partially destabilise the nucleosome structure, possibly in coordination with histone acetylation.

The interactions of spermine (4+ charge at physiological pH), N1-acetylspermine(3+), spermidine(3+), N1- and N8-acetylsperimidine(2+), putrescine(2+), hexaamminecobalt(3+), and magnesium(2+) with nucleosome core particles have been examined by using thermal denaturation and circular dichroism. Tetra- and triamines were 2-3 times more effective than diamines at stabilizing core particles against thermal denaturation. Secondary effects were also observed, with acetylpolyamines slightly less effective than unmodified polyamines of equivalent charge. Hexaamminecobalt(3+) was less effective than the triamines, while magnesium had essentially no effect. This is surprising since magnesium is more effective than diamines at stabilizing naked DNA. All the cations tested altered the circular dichroism spectra of the core particles in the DNA region (284 nm). The peak at 284 nm was suppressed by tetra- and trivalent compounds to approximately twice the extent of divalent compounds. Magnesium appears to suppress the peak by a lesser extent than the diamines. This indicates that the DNA twist and/or folding is changed by these cations. A plateau of both thermal denaturation and circular dichroism effects was observed at cation concentrations where 30-40% of the total DNA negative charges could be neutralized by the added cations. We suggest that polyamine and histone acetylation function in concert to lower the stability and change the conformation of the nucleosome core, thus facilitating replication and transcription in vivo.

Histone H4 is a good substrate in vitro for the protein histidine kinase activity found both in Physarum polycephalum nuclear extracts and in Saccharomyces cerevisiae cell extracts. However, histone H4 in nucleosome core particles is not a substrate for these kinases. Isolated chromatin was also not a substrate for the protein histidine kinase. The results significantly limit possible interpretations of histidine phosphorylation on histone H4 in vivo and provide a new, sharper focus for future work. In addition, a polynucleotide kinase activity was identified in the Physarum extracts.