Histone variant H2A.Z is required for early mammalian development.

Fundamental to the process of mammalian development is the timed and coordinated regulation of gene expression. This requires transcription of a precise subset of the total complement of genes. It is clear that chromatin architecture plays a fundamental role in this process by either facilitating or restricting transcription factor binding [1]. How such specialized chromatin structures are established to regulate gene expression is poorly understood. All eukaryotic organisms contain specialized histone variants with distinctly different amino acid sequences that are even more conserved than the major core histones [2]. On the basis of their highly conserved sequence, histone variants have been assumed critical for the function of mammalian chromatin; however, a requirement for a histone variant has not been shown in mammalian cells. Mice with a deletion of H1 degrees have been generated by gene targeting in ES cells, but these mice show no phenotypic consequences, perhaps due to redundancy of function [3]. Here we show for the first time that a mammalian histone variant, H2A.Z, plays a critical role in early development, and we conclude that this histone variant plays a pivotal role in establishing the chromatin structures required for the complex patterns of gene expression essential for normal mammalian development.

On the ordinality of numbers: A review of neural and behavioral studies.

The last several years have seen steady growth in research on the cognitive and neuronal mechanisms underlying how numbers are represented as part of ordered sequences. In the present review, we synthesize what is currently known about numerical ordinality from behavioral and neuroimaging research, point out major gaps in our current knowledge, and propose several hypotheses that may bear further investigation. Evidence suggests that how we process ordinality differs from how we process cardinality, but that this difference depends strongly on context-in particular, whether numbers are presented symbolically or nonsymbolically. Results also reveal many commonalities between numerical and nonnumerical ordinal processing; however, the degree to which numerical ordinality can be reduced to domain-general mechanisms remains unclear. One proposal is that numerical ordinality relies upon more general short-term memory mechanisms as well as more numerically specific long-term memory representations. It is also evident that numerical ordinality is highly multifaceted, with symbolic representations in particular allowing for a wide range of different types of ordinal relations, the complexity of which appears to increase over development. We examine the proposal that these relations may form the basis of a richer set of associations that may prove crucial to the emergence of more complex math abilities and concepts. In sum, ordinality appears to be an important and relatively understudied facet of numerical cognition that presents substantial opportunities for new and ground-breaking research.

Rate of psychiatric illness 1 year after traumatic brain injury.

OBJECTIVE: Neurobehavioral symptoms are not uncommon after a traumatic brain injury. However, psychiatric syndromes per se have rarely been studied in patients with such an injury. The purpose of this study was to evaluate the type and extent of psychiatric syndromes in patients with traumatic brain injury. METHOD: One hundred ninety-six hospitalized adults were studied 1 year after a traumatic brain injury with the use of a two-stage psychiatric diagnostic procedure. Psychiatric diagnoses were made according to ICD-10 criteria on the basis of data from the Schedules for Clinical Assessment in Neuropsychiatry interview. RESULTS: Of 164 patients interviewed, 30 (18.3%) had an ICD-10 diagnosis of a psychiatric illness. Among the 120 patients who were 18-64 years old, 21.7% had a psychiatric illness, compared with 16.4% in a study of the general population. A depressive illness was present in 13.9% of the traumatic brain injury patients, compared with 2.1% of the general population, and panic disorder was present in 9.0%, compared with 0.8% of the general population. CONCLUSIONS: In comparison with the general population, a higher proportion of adult patients had developed psychiatric illnesses 1 year after a traumatic brain injury; the rates of depressive episode and panic disorder were significantly higher in the study group. A history of psychiatric illness, an unfavorable global outcome according to the Glasgow Outcome Scale, a lower score on the Mini-Mental State examination, and fewer years of formal education seemed to be important risk factors in the development of a psychiatric illness. Compensation claims, however, were not associated with the rate of psychiatric illness.

Differential binding of thyroid hormone receptors to mouse glandular kallikrein gene promoters: evidence for multiple binding regions in the mGK-6 gene.

We have used a DNA-cellulose competition assay to investigate the binding of thyroid hormone receptors to fragments of the mouse glandular kallikrein genes and the human and rat GH genes. Nuclear extracts from human lymphoblastoid IM-9 cells were incubated with [125I]tri-iodothyronine [( 125I]T3) and DNA-cellulose. The ability of cloned gene fragments to compete for radiolabelled receptors bound to DNA-cellulose was compared with that of DNA from pBR322. As previously observed, a 900 bp fragment from the human GH gene showed preferential binding to the thyroid hormone receptor. High-affinity binding was observed with a synthetic fragment of the rat GH gene encompassing positions -163 to -192 but not with a similar fragment from positions -224 to -192. Preferential binding was also observed with fragments of the mouse glandular kallikrein gene, mGK-6. Binding to the entire gene and fragments containing 2300 and 776 bp of the promoter region was identical. Detectable but reduced binding was seen with a shorter fragment. These results suggest that the T3 receptor binds to multiple sites within the first 776 bp of the mGK-6 gene promoter. Potential thyroid hormone response elements can be identified within this region of the gene. In contrast, the kallikrein gene mGK-3, which shows a different response to thyroid hormone from that of mGK-6, showed no significant binding in the comparable promoter region.

The alpha-1,3-galactosyltransferase knockout mouse. Implications for xenotransplantation.

Organ xenografts in discordant combinations such as pig-to-man undergo hyperacute rejection due to the presence of naturally occurring human anti-pig xenoantibodies. The galactose alpha(1,3)-galactose epitope on glycolipids and glycoproteins is the major porcine xenoantigen recognized by these xenoantibodies. This epitope is formed by alpha(1,3)-galactosyltransferase, which is present in all mammals except man, apes, and Old World monkeys. We have generated mice lacking this major xenoantigen by inactivating the alpha(1,3)-galactosyltransferase gene. These mice are viable and have normal organs but develop cataracts. Substantially less xenoantibody from human serum binds to cells and tissues of these mice compared with normal mice. Similarly, there is less activation of human complement on cells from mice lacking the galactose alpha(1,3)-galactose epitope. These mice confirm the importance of the galactose alpha(1,3)-galactose epitope in human xenoreactivity and the logic of continuing efforts to generate pigs that lack this epitope as a source of donor organs.

Site-specific transgene insertion: an approach.

Methods to improve the production of transgenic animals are being developed. Conventional transgenesis, involving microinjection of DNA into fertilized eggs, has a number of limitations. These result from the inability to control both the site of transgene insertion and the number of gene copies inserted. The approach described seeks to overcome these problems and to allow single copy insertion of transgenes into a defined site in animal genomes. The method involves the use of embryonic stem cells, gene targeting and the FLP recombinase system.

The effect of extracts from human dental plaque, oral streptococci and actinomyces bacteria on HELA cell growth, [3H]-thymidine uptake and 51Cr release.

Dialysed extracts from dental plaque and a number of species of oral streptococci and actinomycetes have been investigated for their effects on cell growth, [3H]-thymidine uptake and 51Cr release from HeLa cell cultures. Eight of the 15 extracts inhibited cell growth and all but one inhibited [3H]-thymidine incorporation. None of the extracts stimulated 51Cr release from prelabelled cells. These results suggest that many of the species of streptococci and actinomyces found in the mouth and in dental plaque are capable of producing high mol. wt substances that interfere with HeLa cell cultures by inhibiting [3H]-thymidine uptake and DNA synthesis but do not affect the degree of intactness of the external cell membrane.

Neuropsychiatric sequelae one year after a minor head injury.

OBJECTIVE: To assess neuropsychiatric sequelae 1 year after minor head injury in a cross sectional study using home interviews with patients and their relatives at 1 year after head injury. METHODS: The study cohort included 148 adults who were admitted to hospital after a minor head injury between 1 July 1994 and 30 June 1995 and showed clinical or radiological evidence of brain injury. Main outcome measures used in the study were the Glasgow outcome scale, Edinburgh rehabilitation status scale, Barthel index, clinical interview schedule-revised, mini mental state examination, and assessment of symptoms of postconcussional syndrome. RESULTS: At one year follow up, four (2.9%) patients had a severe disability, 35 (25.5%) had a moderate disability, and 95 (69.3%) had no disability according to the Glasgow outcome scale. A slightly higher proportion (33.3%, n=45) showed disability according to the Edinburgh rehabilitation status scale. Thirty one patients (23.1%) scored <24 in the mini mental state examination. These were mostly patients over the age of 65. Twenty three patients (17.2%) were diagnosed as psychiatric cases according to the clinical interview schedule-revised scale. Seventy four (55.2%) patients showed one of the symptoms of postconcussional syndrome. The most commonly shown neurobehavioural problems were irritability (30%), sleep disturbance (29%), and impatience (27%). CONCLUSION: One year after a minor head injury, a substantial proportion of patients showed neuropsychiatric sequelae.

Neurobehavioural symptoms one year after a head injury.

BACKGROUND: Neurobehavioural symptoms are common immediately after a minor head injury but have not been studied one year after the injury. AIMS: To estimate the rate and pattern of neurobehavioural symptoms one year after a head injury of varying severity. METHOD: Adults who had been hospitalised after a head injury (n = 196, 164 of whom had a face-to-face interview) and showed indirect evidence of brain assault were assessed for the presence of neurobehavioural symptoms with the help of a behaviour rating scale. RESULTS: About 40% had three or more symptoms. Individual symptoms varied among 3% (social disinhibition), 15% (lack of initiative) and 35% (irritability) of the cohort. Premorbid factors such as lower social class and lower educational achievement, head-injury-related factors such a low Glasgow coma score, and outcome-related factors such as the presence of a disability according to the Edinburgh Rehabilitation Status Scale and psychiatric caseness according to the Clinical Interview Schedule--Revised, significantly influenced the rate and the pattern of behavioural symptoms. The pattern of symptoms varied between age groups and according to the severity of the head injury. CONCLUSIONS: A significant proportion of patients with varying degrees of severity of head injury showed behavioural symptoms after one year of head injury.

Tissue-specific and hormonal regulation of angiotensinogen minigenes in transgenic mice.

Angiotensinogen is the precursor of the potent vasoactive peptide angiotensin II, and is therefore an important determinant of blood pressure and electrolyte homeostasis. In order to map the tissue-specific and inducible enhancer elements governing angiotensinogen gene expression in transgenic mice, we constructed minigenes containing either 0.75 kb or 4 kb or 5' flanking DNA from the BALB/c angiotensinogen gene. Sequences necessary and sufficient to mediate induction by glucocorticoids, oestrogen and bacterial endotoxin were contained on the minigene bearing 0.75 kb of DNA upstream of the capsite. This construct was also able to confer tissue specificity in the majority of organs producing angiotensinogen. In the testis and salivary gland, differences between the donor (BALB/c) and recipient (Swiss) strains were responsible for the apparently aberrant expression of the minigene constructs. The genetic lesion responsible for these expression polymorphisms has been characterized using recombinant inbred mice. An EcoRI restriction fragment length polymorphism which co-segregates with the angiotensinogen expression phenotypes into many inbred mouse strains is also described.

A comparison of bovine lymphocyte antigens.

In Canberra, 31 antigens have been described on the surface of bovine lymphocytes. Seven antigens are subgroups of other antigens. Eleven antigens are similar to the eleven antigens which have been described in Melbourne. Fourteen antigens are similar to twelve international-workshop antigens and two European-workshop antigens.

Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants.

We have isolated two murine homeobox genes, Nkx-2.5 and Nkx-2.6, that are new members of a sp sub-family of homeobox genes related to Drosophila NK2, NK3 and NK4/msh-2. In this paper, we focus on the Nkx-2.5 gene and its expression pattern during post-implantation development. Nkx-2.5 transcripts are first detected at early headfold stages in myocardiogenic progenitor cells. Expression preceeds the onset of myogenic differentiation, and continues in cardiomyocytes of embryonic, foetal and adult hearts. Transcripts are also detected in future pharyngeal endoderm, the tissue believed to produce the heart inducer. Expression in endoderm is only found laterally, where it is in direct apposition to promyocardium, suggesting an interaction between the two tissues. After foregut closure, Nkx-2.5 expression in endoderm is limited to the pharyngeal floor, dorsal to the developing heart tube. The thyroid primordium, a derivative of the pharyngeal floor, continues to express Nkx-2.5 after transcript levels diminish in the rest of the pharynx. Nkx-2.5 transcripts are also detected in lingual muscle, spleen and stomach. The expression data implicate Nkx-2.5 in commitment to and/or differentiation of the myocardial lineage. The data further demonstrate that cardiogenic progenitors can be distinguished at a molecular level by late gastrulation. Nkx-2.5 expression will therefore be a valuable marker in the analysis of mesoderm development and an early entry point for dissection of the molecular basis of myogenesis in the heart.

Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene.

The scl gene encodes a basic-helix-loop-helix transcription factor which was identified through its involvement in chromosomal translocations in T-cell leukemia. To elucidate its physiological role, scl was targeted in embryonic stem cells. Mice heterozygous for the scl null mutation were intercrossed and their offspring were genotyped. Homozygous mutant (scl-/-) pups were not detected in newborn litters, and analysis at earlier time points demonstrated that scl-/- embryos were dying around embryonic day 9.5. The scl-/- embryos were pale, edematous, and markedly growth retarded after embryonic day 8.75. Histological studies showed complete absence of recognizable hematopoiesis in the yolk sac of these embryos. Early organogenesis appeared to be otherwise normal. Culture of yolk sac cells of wild-type, heterozygous, and homozygous littermates confirmed the absence of hematopoietic cells in scl-/- yolk sacs. Reverse transcription PCR was used to examine the transcripts of several genes implicated in early hematopoiesis. Transcripts of GATA-1 and PU.1 transcription factors were absent from RNA from scl-/- yolk sacs and embryos. These results implicate scl as a crucial regulator of early hematopoiesis.

Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5.

The murine homeo box gene Nkx2-5 is expressed in precardiac mesoderm and in the myocardium of embryonic and fetal hearts. Targeted interruption of Nkx2-5 resulted in abnormal heart morphogenesis, growth retardation and embryonic lethality at approximately 9-10 days postcoitum (p.c.). Heart tube formation occurred normally in mutant embryos, but looping morphogenesis, a critical determinant of heart form, was not initiated at the linear heart tube stage (8.25-8.5 days p.c.). Commitment to the cardiac muscle lineage, expression of most myofilament genes and myofibrillogenesis were not compromised. However, the myosin light-chain 2V gene (MLC2V) was not expressed in mutant hearts nor in mutant ES cell-derived cardiocytes. MLC2V expression normally occurs only in ventricular cells and is the earliest known molecular marker of ventricular differentiation. The regional expression in mutant hearts of two other ventricular markers, myosin heavy-chain beta and cyclin D2, indicated that not all ventricle-specific gene expression is dependent on Nkx2-5. The data demonstrate that Nkx2-5 is essential for normal heart morphogenesis, myogenesis, and function. Furthermore, this gene is a component of a genetic pathway required for myogenic specialization of the ventricles.

Hlx homeo box gene is essential for an inductive tissue interaction that drives expansion of embryonic liver and gut.

The divergent murine homeo box gene Hlx is expressed in restricted hematopoietic cell types and, during embryogenesis, prominently in visceral mesenchyme of the developing liver, gall bladder, and gut. Targeted disruption of the gene has now established that it plays a key role in visceral organogenesis. Embryos homozygous for the mutation died around embryonic day 15 with anemia and severe hypoplasia of the liver and gut. Liver ontogeny commenced normally with formation of the liver diverticulum and differentiation of hepatocytes, but the organ failed to expand and reached only 3% of normal size. The apparent liver hypoplasia was not associated with a notable increase in apoptotic cells. Gut development also began normally, but the intestines failed to undergo extensive elongation and looping and reached only a quarter of normal length. The anemia resulted from a deficiency in the fetal form of hematopoiesis, which occurs in the liver, but no intrinsic defect in Hlx-/- hematopoietic cells was observed in vitro, and liver-derived Hlx-/- hematopoietic stem cells that were transplanted to irradiated normal mice could fully reconstitute hematopoiesis. The impaired fetal hematopoiesis therefore reflects insufficient support function provided by the minute liver. Hlx is normally expressed in visceral mesenchyme lying adjacent to the developing liver and gut epithelia affected by the mutation, but not in the epithelia themselves. Hence, Hlx regulates a mesenchymal-epithelial interaction that drives a vital growth phase in visceral organogenesis. Moreover, because mutation of Hlx blocked liver growth but not its specification, early morphogenesis, or differentiation, development of this organ appears to occur by step-wise inductive interactions under separate genetic control.

In vitro development of porcine nuclear transfer embryos constructed using fetal fibroblasts.

The in vitro development of porcine nuclear transfer embryos constructed using primary cultures from day 25 fetal fibroblasts which were either rapidly dividing (cycling) or had their cell-cycle synchronized in G0/G1 using serum starvation (serum-starved) was examined. Oocyte-karyoplast complexes were fused and activated simultaneously and then cultured in vitro for seven days to assess development. Fusion rates were not different for either cell population. The proportion of reconstructed embryos that cleaved was higher in the cycling group compared to the serum-starved group (79 vs. 56% respectively; P < 0.05). Development to the 4-cell stage was not different using either population. Both treatments supported similar rates of development to the morula (1.5 vs. 7%, cycling vs. serum-starved) and blastocyst stage (1.5 vs. 3%, cycling vs. serum-starved). The blastocyst produced using cycling cells had a total cell number of 10. Total cell numbers for the three blastocysts produced serum-starved cells were 22, 24, and 33. These blastocysts had inner cell mass numbers of 0, 15, and 4, respectively. Six hundred and thirty-five nuclear transfer embryos reconstructed using serum-starved cells were transferred to 15 temporarily mated recipients for 3-4 days. Of these, 486 were recovered (77% recovery rate) of which 106 (22%) had developed to the 4-cell stage or later. These were transferred to a total of 15 recipients which were either unmated or mated. Seven recipients farrowed a total of 51 piglets. Microsatellite analysis revealed that none of these were derived from the nuclear transfer embryos transferred.

Transgenic mice expressing a hemopoietic growth factor gene (GM-CSF) develop accumulations of macrophages, blindness, and a fatal syndrome of tissue damage.

Transgenic mice carrying the murine granulocyte-macrophage colony stimulating factor (GM-CSF) gene expressed from a retroviral promoter exhibit elevated levels of GM-CSF in the serum, urine, peritoneal cavity, and eye. The eyes of transgenic mice are opaque, contain accumulations of macrophages, and develop retinal damage. Similarly, lesions containing macrophages develop in striated muscle. The mice also display an accumulation of large, often multinucleate, activated macrophages in the peritoneal and pleural cavities. The transgene is transcribed in peritoneal cells, as well as in eyes and infiltrated striated muscle. A high proportion of transgenic mice die with muscle wasting when aged 2-4 months, possibly because of macrophage activation resulting from the high levels of GM-CSF.

ATP2A2 mutations in Darier's disease and their relationship to neuropsychiatric phenotypes.

Darier's disease (DD) is a rare, dominantly inherited disorder that affects the skin producing a variety of types of lesion. Close examination of lesional DD skin shows the presence of abnormal keratinization (epidermal differentiation) and acantholysis (loss of cohesion) of keratinocytes. A number of clinical studies have described the co-occurrence of various neurological and psychiatric symptoms with DD, including mood disorders, epilepsy, mental retardation and a slowly progressive encephalopathy. A single locus for DD has been mapped to chromosome 12q23-q24.1, and a variety of missense, nonsense, frameshift and splicing mutations in the ATP2A2 gene have been described recently in families with DD. This gene encodes the sarcoplasmic/endoplasmic reticulum calcium-pumping ATPase SERCA2, which has a central role in intra-cellular calcium signalling. In this study, we performed mutation analysis on ATP2A2 in 19 unrelated DD patients, of whom 10 had neuropsychiatric phenotypes. We identified and verified 17 novel mutations predicting conservative and non-conservative amino acid changes, potential premature translation terminations and potential altered splicing. Our findings confirm that mutations in ATP2A2 are associated with DD. In neuropsychiatric cases, there was a non-random clustering of mutations in the 3' end of the gene ( P = 0.01), and a predominance of the missense type (70% versus 38% in DD patients). This supports the hypothesis that the DD gene has pleiotropic effects in brain and that mutations in SERCA2 are implicated in the pathogenesis of neuropsychiatric disorders.

MnDPDP enhanced magnetic resonance imaging of focal liver lesions.

Mangafodipir trisodium (MnDPDP) is a contrast agent for use in magnetic resonance imaging (MRI) of the liver. The agent is taken up by normal hepatocytes resulting in increased signal on T1-weighted imaging, and is excreted in the biliary system. Hepatocyte-containing liver neoplasms such as hepatomas or focal nodular hyperplasia (FNH), take up MnDPDP and demonstrate varying degrees of enhancement. Metastatic liver deposits and primary liver tumours of non-hepatocyte origin do not typically enhance with MnDPDP thus increasing their conspicuity compared with pre-contrast T1-weighted images. Metastases may demonstrate rim enhancement particularly on delayed imaging at 24 h, which can increase their conspicuity, thus allowing better visualization of small lesions. Functional biliary obstruction due to liver metastases can also result in wedge shaped areas of parenchymal enhancement. The MRI features of various focal liver after continuance with lesions following MnDPDP are discussed and illustrated including primary lesions such as hepatoma and secondary metastases.