Histone H1 is a specific repressor of core histone acetylation in chromatin.

Although a link between histone acetylation and transcription has been established, it is not clear how acetylases function in the nucleus of the cell and how they access their targets in a chromatin fiber containing H1 and folded into a highly condensed structure. Here we show that the histone acetyltransferase (HAT) p300/CBP-associated factor (PCAF), either alone or in a nuclear complex, can readily acetylate oligonucleosomal substrates. The linker histones, H1 and H5, specifically inhibit the acetylation of mono- and oligonucleosomes and not that of free histones or histone-DNA mixtures. We demonstrate that the inhibition is due mainly to steric hindrance of H3 by the tails of linker histones and not to condensation of the chromatin fiber. Cellular PCAF, which is complexed with accessory proteins in a multiprotein complex, can overcome the linker histone repression. We suggest that linker histones hinder access of PCAF, and perhaps other HATs, to their target acetylation sites and that perturbation of the linker histone organization in chromatin is a prerequisite for efficient acetylation of the histone tails in nucleosomes.

Effect of lactation stage and concurrent pregnancy on milk composition in the bottlenose dolphin.

ALTHOUGH MANY TOOTHED WHALES (CETACEA: Odontoceti) lactate for 2-3 years or more, it is not known whether milk composition is affected by lactation stage in any odontocete species. We collected 64 pooled milk samples spanning 1-30 months postpartum from three captive bottlenose dolphins Tursiops truncatus. Milks were assayed for water, fat, crude protein (TN × 6.38) and sugar; gross energy was calculated. Ovulation and pregnancy were determined via monitoring of milk progesterone. Based on analysis of changes in milk composition for each individual dolphin, there were significant increases (P<0.05) in fat (in all three dolphins) and crude protein (in two of three), and a decrease (P<0.05) in water (in two of three) over the course of lactation, but the sugar content did not change. In all three animals, the energy content was positively correlated with month of lactation, but the percentage of energy provided by crude protein declined slightly but significantly (P<0.05). At mid-lactation (7-12 months postpartum, n=17), milk averaged 73.0±1.0% water, 12.8±1.0% fat, 8.9±0.5% crude protein, 1.0±0.1% sugar, 1.76±0.09 kcal g(-1) (=7.25 kJ g(-1)) and 30.3±1.3% protein:energy per cent. This protein:energy per cent was surprisingly high compared with other cetaceans and in relation to the growth rates of calves. Milk progesterone indicated that dolphins ovulated and conceived between 413 and 673 days postpartum, following an increase in milk energy density. The significance of these observed compositional changes to calf nutrition will depend on the amounts of milk produced at different stages of lactation, and how milk composition and yield are influenced by sampling procedure, maternal diet and maternal condition, none of which are known.

Parental communication deviance and affective style. Predictors of subsequent schizophrenia spectrum disorders in vulnerable adolescents.

In an attempt to assess the contributory role of family factors to the development of schizophrenia-like disorders, measures of parental communication deviance and affective styles of communication were obtained for a sample of families of disturbed but nonpsychotic adolescents. Outcome was assessed five years later. Absence of a pathologic affective style was associated with a benign outcome, but neither parental variable alone allowed precise identification of the schizophrenia-spectrum cases. However, an index using a combination of both variables was statistically predictive of subsequent psychiatric status at follow-up. Thus, adolescents whose parents had both a pathologic affective style of communication and a high level of communication deviance had schizophrenia-like disorders develop in young adulthood. Adolescents of parents who had both lower levels of communication deviance and a benign affective style had offspring with healthier outcomes.

Parental transactional style deviance as a possible indicator of risk for schizophrenia.

The presence of a pattern of parental transactional style deviance on the Thematic Apperception Test (TAT) (a significant attribute of parents of offspring with schizophrenia spectrum disorders) was used to identify a group of disturbed nonpsychotic adolescents hypothesized to be at high risk for subsequently developing schizophreniform psychopathology. High-risk male adolescents came from two symptom groups, withdrawn adolescents and adolescents in active family conflict, which are symptom patterns similar to the premorbid pictures of two schizophrenia subtypes. High-risk parents also tended to show transactional style deviance in direct interaction with their child and in a written statement describing their child's problem. The degree of risk was significantly related to the amount of therapy in which the family was subsequently engaged and, at a four-year follow-up, to the level of adjustment of the adolescents seen earlier in the project.

HMGN4, a newly discovered nucleosome-binding protein encoded by an intronless gene.

We describe a newly discovered nuclear protein, HMGN4, that is closely related to the canonical HMGN2 nucleosome-binding protein. The protein is encoded by an intronless gene, which, in humans, is located in the hereditary hemochromatosis [correction of hemachromatosis] region at position 6p21.3. A single approximately 2-kb HMGN4 mRNA was found to be expressed, in variable amounts, in all human tissues tested; however, the HMGN4 transcript was significantly less abundant than that of HMGN2. The HMGN4 protein could be detected in HeLa cells by Western analysis with an antibody elicited against a unique region of the protein. Transfection of HeLa cells with a plasmid expressing HMGN4-GFP indicated that the protein localizes to the nucleus. Our results expand the multiplicity of the HMGN protein family and increase the known cellular repertoire of nucleosome-binding proteins.

The impact of education about schizophrenia on relatives varying in expressed emotion.

The demand for information by relatives and the success of family intervention programs with an initial didactic component has resulted in a proliferation of educational interventions in schizophrenia. The present study assesses the impact of a single educational session on relatives of recent-onset schizophrenic patients. Results suggest that relatives who participated in family education experience an increased sense of support from the treatment team and a nearly significant tendency toward a decrease in self-blame regarding the schizophrenic illness. Despite findings in previous studies suggesting information acquisition immediately after education and retention after 6 months, the present study found no information retention after a 2-month period. After family education, relatives rated as high in expressed emotion (EE) reported a significantly increased sense of understanding of the illness and expressed increased feelings of support from the treatment team, whereas low EE relatives did not change significantly in these attitudes as a function of the educational session. Low EE relatives demonstrated more actual information about the illness and were less likely to perceive the symptoms as being done intentionally to bother them.

HMGN proteins play roles in DNA repair and gene expression in mammalian cells.

HMGN (high-mobility-group N) family members are vertebrate proteins that unfold chromatin and promote transcription and replication of chromatin templates in vitro. However, their precise roles in vivo have been elusive until recently. This paper summarizes recent advances from studies of Hmgn1 knockout mice and genetically engineered cell lines that are beginning to reveal the diverse roles that HMGN proteins play in DNA repair and transcription within mammalian cells.

Human DNA topoisomerase IIbeta binds and cleaves four-way junction DNA in vitro.

We have used gel retardation analysis to show that human DNA topoisomerase IIbeta can bind a 40 bp linear duplex containing a single DNA topoisomerase IIbeta cleavage site. Furthermore, we demonstrate for the first time that human DNA topoisomerase IIbeta binds to four-way junction DNA. This supports previous suggestions that topoisomerase II may be targeted to supercoiled DNA through the recognition of DNA cruciforms, helix-helix crossovers and hairpins. DNA topoisomerase IIbeta had a 4-fold higher affinity for the four-way junction than for the linear duplex, as demonstrated by protein titration and competition analysis. Furthermore, the DNA topoisomerase IIbeta:four-way junction complex was significantly more salt stable than the complex with linear DNA. The four-way junction contained potential topoisomerase IIbeta cleavage sites straddling the points of strand exchange, and indeed, topoisomerase IIbeta was able to cleave three of these four predicted sites. This indicates that topoiso-merase IIbeta can bind to the centre of the junction. Topoisomerase II has to bind both the transported and the gated DNA helices prior to strand passage, and it is possible that both helices are provided by the four-way junction in this case. The stable complex of DNA topoisomerase IIbeta with four-way junction DNA may provide an ideal substrate for further studies into the mechanism of substrate recognition and binding by DNA topoisomerase II.

The subacute hospital treatment of the borderline patient. II: An integrated focus on splitting.

A psychiatric inpatient unit with expertise in family treatment has developed an integrated effort to provide a one to three month treatment program for hospitalized borderline patients. Treatment goals are more ambitious than those obtainable by simple crisis intervention. Appreciation of staff splitting is enhanced by an intergenerational family systems approach. Early and sustained focus on splitting is aided by the use of a 15 minute educational tape and a weekly therapy group required for all nonpsychotic patients and recommended for their families. Cases illustrating the family perspective and a group therapy session are presented in detail. The therapy group, run by an interdisciplinary team (the authors) requires active staff initiative and confrontation to avoid responsibility-evading patient coalitions. Diagnostic considerations and impact of the program on patients and on staff are discussed.

HMGN3a and HMGN3b, two protein isoforms with a tissue-specific expression pattern, expand the cellular repertoire of nucleosome-binding proteins.

HMGN1 (HMG-14) and HMGN2 (HMG-17) are nuclear proteins that bind specifically to nucleosomes, reduce the compactness of the chromatin fiber, and enhance transcription from chromatin templates. Here we report that many vertebrates contain an additional type of HMGN protein named HMGN3 (Trip 7). The human HMGN3 gene is located on chromosome 6 and spans 32 kilobase pairs, which is nearly 10-fold longer than the closely related HMGN2 gene. However, the intron/exon boundaries of the HMGN3 gene are identical to those of HMGN1 and HMGN2. Unique within the HMGN family, the HMGN3 transcript undergoes alternative splicing and generates two different variants, HMGN3a and HMGN3b. The shorter variant, HMGN3b, arises from an additional splice site that truncates exon V and causes a frameshift. The resulting HMGN3b protein lacks the majority of the C-terminal chromatin-unfolding domain. Both splice variants are found in many vertebrates from frogs to man and are expressed in many tissues. The pattern of tissue-specific expression differs considerably from those of HMGN1 and HMGN2 at both the mRNA and the protein level. Our results expand the multiplicity of the HMGN protein family and raise the possibility that these nucleosome-binding proteins function as co-activators in tissue-specific gene expression.

Concentrations of progesterone in milk from bottlenose dolphins during different reproductive states.

There are few published reports of an alternative, less invasive method than blood sampling to obtain reproductive hormone concentrations from captive dolphins. The aims of this study were to: (1) validate milk as an effective alternative to blood plasma for determining progesterone concentrations; and (2) utilize milk samples collected frequently to obtain progesterone concentration profiles and determine reproductive status. During the course of this study 16 plasma/milk sample pairs were collected from four adult bottlenose dolphins to correlate plasma and milk concentrations of progesterone. Milk samples were also collected approximately weekly for 4-5 months during three independent lactational periods. Additionally, milk samples were collected daily for approximately 1 year during three other independent lactational periods. A highly significant correlation was found between progesterone concentrations in plasma and milk (r(2) = 0.91, P < 0. 01). Progesterone contained in milk whey, fat, and solids were 3.95 +/- 1.3, 8.5 +/- 1.1, and 52.0 +/- 0.6%, respectively. Progesterone profiles from milk samples collected from two dolphins during 1995 indicated pregnancies (with progesterone concentrations between 8 and 46.5 ng/ml) which resulted in parturition. High progesterone concentrations in a third dolphin that did not give birth indicated a possible pseudopregnancy or fetal resorption. A possible ovulation not resulting in pregnancy was evident in one female in 1998, follicular activity in another female in 1998, and a year-long anestrous period in the third animal studied in 1998. It is confirmed that dolphins can become pregnant while lactating and that the approximate time of conception is identifiable in milk profiles, illustrating the potential application of this method in pregnancy detection and reproductive monitoring.

Mutagenesis of E477 or K505 in the B' domain of human topoisomerase II beta increases the requirement for magnesium ions during strand passage.

A type II topoisomerase is essential for decatenating DNA replication products, and it accomplishes this task by passing one DNA duplex through a transient break in a second duplex. The B' domain of topoisomerase II contains three highly conserved motifs, EGDSA, PL(R/K)GK(I/L/M)LNVR, and IMTD(Q/A)DXD. We have investigated these motifs in topoisomerase II beta by mutagenesis, and report that they play a critical role in establishing the DNA cleavage-religation equilibrium. In addition, the mutations E477Q (EGDSA) and K505E (PLRGKILNVR) increase the optimal magnesium ion concentration for strand passage, without affecting the Mg(2+) dependence of ATP hydrolysis. It is likely that the binding affinity of the magnesium ion(s) specifically required for DNA cleavage has been reduced by these mutations. The crystal structure of yeast topo II indicates that residues E477 and K505 may help to position the three aspartate residues of the IMTD(Q/A)DXD motif for magnesium ion coordination, and we propose two possible locations for the magnesium ion binding site(s). These observations are consistent with a previous model in which the B' domain is positioned such that these acidic residues lie next to the active site tyrosine residue. A magnesium ion bound by these aspartate residues could therefore mediate the DNA cleavage-religation reaction.

Ethylene oxide resistance of nondesiccated and desiccated spores of Bacillus subtilis var. niger hermetically sealed in various polymeric films.

The resistance to destruction of spores of Bacillus subtilis var. niger hermetically sealed in various polymeric films and exposed to ethylene oxide with and without relative humidity was determined. The effect of desiccation was also determined. The order of increased resistance to sterilization with regard to type of polymeric film was found to be: polyethylene equal to polyvinyl chloride, less than nylon, less than cellophane/polyethylene laminate, less than phenoxy, less than mylar/polyethylene laminate. Desiccated spores sealed in various polymeric films were much more resistant to ethylene oxide sterilization than nondesiccated spores. Relative humidity was an important factor in ethylene oxide sterilization with spores not sealed in polymeric films. However, with spores hermetically sealed in polyethylene, added relative humidity was an insignificant factor in the sterilization process.