Supracondylar osteotomy for the treatment of cubitus varus in children: a systematic review.

Cubitus varus is the most frequent complication following the treatment of supracondylar humeral fractures in children. We investigated data from publications reporting on the surgical management of cubitus varus found in electronic searches of Ovid/MEDLINE and Cochrane Library databases. In 894 children from 40 included studies, the mean age at initial injury was 5.7 years (3 to 8.6) and 9.8 years (4 to 15.7) at the time of secondary correction. The four osteotomy techniques were classified as lateral closing wedge, dome, complex (multiplanar) and distraction osteogenesis. A mean angular correction of 27.6º (18.5° to 37.0°) was achieved across all classes of osteotomy. The meta-analytical summary estimate for overall rate of good to excellent results was 87.8% (95% CI 84.4 to 91.2). No technique was shown to significantly affect the surgical outcome, and the risk of complications across all osteotomy classes was 14.5% (95% CI 10.6 to 18.5). Nerve palsies occurred in 2.53% of cases (95% CI 1.4 to 3.6), although 78.4% were transient. No one technique was found to be statistically safer or more effective than any other.

Not only in trauma patients: hospital-wide implementation of a massive transfusion protocol.

OBJECTIVES: To review outcomes of massive transfusion protocol (MTP) activation and determine the impact of MTP implementation on blood bank use. BACKGROUND: MTP has been established to rapidly provide plasma and packed red blood cells in ratios approaching 1 : 1. Due to availability, MTP has been utilised in non-traumatic haemorrhage despite evidence of benefit in this population. Our hospital-wide implementation of MTP was reviewed for propriety, outcomes and effect on blood bank resources. METHODS: Retrospective cohort study of patients receiving transfusion after MTP activation from October 2009 to 2011. Underlying medical conditions and baseline medication use were determined. In-hospital and 24-h mortality were compared with evaluation for confounding by Acute Physiology And Chronic Health Evaluation (APACHE) score and duration of MTP activation. Blood product use before and after MTP implementation was reviewed. RESULTS: MTP activation occurred in 62 trauma and 63 non-trauma patients. Non-trauma patients were older, had more underlying medical conditions and higher APACHE scores compared with trauma patients; 24-h mortality was higher in trauma compared with non-trauma patients (27·4 vs 11·1%, P = 0·02). There was no significant difference of in-hospital mortality. Transfusion ratio did not differ between trauma and non-trauma patients and was not associated with mortality even when MTP activation duration and APACHE score were considered. Hospital-wide blood product use did not change with MTP implementation. CONCLUSIONS: MTP may be successfully used in trauma and non-trauma settings without significantly impacting overall blood product utilisation. Inclusion of non-trauma patients into prospective studies of resuscitation with blood products is warranted to ensure benefit in these patients.

A blinded, randomized, controlled trial of three doses of high-dose insulin in poison-induced cardiogenic shock.

BACKGROUND: High dose insulin (HDI) has proven superior to glucagon and catecholamines in the treatment of poison-induced cardiogenic shock (PICS) in previous animal studies. Standard recommendations for dosing of insulin vary and the optimal dose of HDI in PICS has not been established. Our hypothesis was a dose of 10 U/kg/hr of HDI would be superior to 1 U/kg/hr with cardiac output (CO) as our primary outcome measure in pigs with propranolol-induced PICS. METHODS: This was a blinded, prospective, randomized trial with 4 arms consisting of 4 pigs in each arm. The arms were as follows: placebo (P), 1 U/kg/hr (HDI-1), 5 U/kg/hr (HDI-5), and 10 U/kg/hr (HDI-10). Cardiogenic shock was induced with a bolus of 0.5 mg/kg of propranolol followed by an infusion of 0.25 mg/kg/min until the point of toxicity, defined as 0.75 x (HR x MAP) was reached. At this point the propranolol infusion was decreased to 0.125 mg/kg/min and a 20 mL/kg bolus of normal saline (NS) was administered. The protocol was continued for 6 hours or until the animals died. RESULTS: 2 pigs died in the P arm, 1 pig died each in the HDI-1 and HDI-5 arms, and all pigs lived in the HDI-10 arm. There was a statistically significant difference in dose by time interaction on CO of 1.13 L/min over the 6 hr study period (p = < 0.001). There was also a statistically significant difference in dose by time interaction on MAP, HR, and systemic vascular resistance (SVR). No statistically significant difference was found between any of the arms regarding glucose utilization. CONCLUSION: HDI was statistically and clinically significantly superior to placebo in this propranolol model of PICS. Furthermore a dose response over time was found where CO increased corresponding to increases in doses of HDI.

Reprogramming and differentiation in mammals: motifs and mechanisms.

The natural reprogramming of the mammalian egg and sperm genomes is an efficient process that takes place in less than 24 hours and gives rise to a totipotent zygote. Transfer of somatic nuclei to mammalian oocytes also leads to their reprogramming and formation of totipotent embryos, albeit very inefficiently and requiring an activation step. Reprogramming of differentiated cells to induced pluripotent stem (iPS) cells takes place during a period of time substantially longer than reprogramming of the egg and sperm nuclei and is significantly less efficient. The stochastic expression of endogenous proteins during this process would imply that controlled expression of specific proteins is crucial for reprogramming to take place. The fact that OCT4, NANOG, and SOX2 form the core components of the pluripotency circuitry would imply that control at the transcriptional level is important for reprogramming to iPS cells. In contradistinction, the much more efficient reprogramming of the mammalian egg and sperm genomes implies that other levels of control are necessary, such as chromatin remodeling, translational regulation, and efficient degradation of no longer needed proteins and RNAs.

Synergistic cytotoxicity of buthionine sulfoximine (BSO) and intensive melphalan (L-PAM) for neuroblastoma cell lines established at relapse after myeloablative therapy.

Patients with high-risk neuroblastoma (NB) initially respond to aggressive, alkylator-based therapy only to die from recurrent disease that is refractory to chemotherapy, including alkylating agents. We examined the ability of buthionine sulfoximine (BSO)-mediated glutathione (GSH) depletion to modulate melphalan (L-PAM) resistance in five NB cell lines established after progressive disease following myeloablative therapy (high-dose melphalan, carboplatin, etoposide and total body irradiation) supported by autologous hematopoietic stem cell transplant (AHSCT), and in 15 NB cell lines established at diagnosis or after non-myeloablative therapy (pre-AHSCT). Four of five post-AHSCT NB cell lines and 10 of 15 pre-AHSCT NB cell lines were sensitive to single agent BSO (LC(90) <300 microM BSO), while two of five post-AHSCT lines and one of 15 pre-AHSCT lines showed high-level resistance to L-PAM (LC(90)>30 microM). Fixed ratio analysis demonstrated BSO/L-PAM synergy (combination index <1) for all five post-AHSCT and for all 15 pre-AHSCT cell lines tested. Multi-log cytotoxicity (often exceeding four logs of cell kill) was observed in post-AHSCT L-PAM-resistant cell lines (including p53 non-functional lines) only when clinically achievable concentrations of BSO were combined with myeloablative concentrations of L-PAM. We conclude that most neuroblastoma cell lines, including post-AHSCT NB cell lines that are highly resistant to myeloablative levels of L-PAM and lack p53 function, are sensitive to clinically achievable concentrations of L-PAM and BSO. However, some L-PAM-resistant NB cell lines (especially those lacking p53 function) require dose escalation of L-PAM to myeloablative concentrations in order to demonstrate significant synergistic cytotoxicity. Thus, optimal clinical application of BSO/L-PAM may require AHSCT.

Importance of dose intensity in neuro-oncology clinical trials: summary report of the Sixth Annual Meeting of the Blood-Brain Barrier Disruption Consortium.

Therapeutic options for the treatment of malignant brain tumors have been limited, in part, because of the presence of the blood-brain barrier. For this reason, the Sixth Annual Meeting of the Blood-Brain Barrier Disruption Consortium, the focus of which was the "Importance of Dose Intensity in Neuro-Oncology Clinical Trials," was convened in April 2000, at Government Camp, Mount Hood, Oregon. This meeting, which was supported by the National Cancer Institute, the National Institute of Neurological Disorders and Stroke, and the National Institute of Deafness and Other Communication Disorders, brought together clinicians and basic scientists from across the U.S. to discuss the role of dose intensity and enhanced chemotherapy delivery in the treatment of malignant brain tumors and to design multicenter clinical trials. Optimizing chemotherapy delivery to the CNS is crucial, particularly in view of recent progress identifying certain brain tumors as chemosensitive. The discovery that specific constellations of genetic alterations can predict which tumors are chemoresponsive, and can therefore more accurately predict prognosis, has important implications for delivery of intensive, effective chemotherapy regimens with acceptable toxicities. This report summarizes the discussions, future directions, and key questions regarding dose-intensive treatment of primary CNS lymphoma, CNS relapse of systemic non-Hodgkin's lymphoma, anaplastic oligodendroglioma, high-grade glioma, and metastatic cancer of the brain. The promising role of cytoenhancers and chemoprotectants as part of dose-intensive regimens for chemosensitive brain tumors and development of improved gene therapies for malignant gliomas are discussed.

Buthionine sulfoximine and myeloablative concentrations of melphalan overcome resistance in a melphalan-resistant neuroblastoma cell line.

BACKGROUND: Alkylator resistance contributes to treatment failure in high-risk neuroblastoma. Buthionine sulfoximine (BSO) can deplete glutathione and synergistically enhance in vitro sensitivity to the alkylating agent melphalan (L-PAM) for many neuroblastoma cell lines, but optimal use of this combination needs to be defined because clinical responses have been less frequent and not durable. PATIENTS AND METHODS: The authors established and characterized a neuroblastoma cell line (CHLA-171) from a patient who died of progressive disease after treatment with BSO and low-dose L-PAM. RESULTS: CHLA-171 lacks MYCN amplification, expresses PGP (P-glycoprotein) 9.5 RNA, and shows cell surface antigen expression (human leukocyte antigen class I weakly positive, but HSAN 1.2 (hybridoma, SAN 1.2) and anti-GD2 (anti-ganglioside GD2 antibody) strongly positive) characteristic of neuroblastoma cell lines. Twenty-four hours of BSO treatment (0-1,000 micromol/L) maximally depleted CHLA-171 glutathione to 36% of baseline. The cytotoxic response of CHLA-171 to BSO and L-PAM, alone and in combination, was measured by digital image microscopy (DIMSCAN) over a range of drug concentrations and compared with drug levels obtained in the patient during BSO/L-PAM therapy. As single agents, CHLA-171 was highly resistant to L-PAM (LD90 = 42 micromol/L; peak plasma concentration in the patient equals 3.9 micromol/L) and moderately resistant to BSO (LD90 = 509 micromol/L; steady-state concentration in the patient equals 397 micromol/L). Treatment with a 10:1 (BSO:L-PAM) fixed ratio combination synergistically overcame resistance (3-4 logs of cell kill, combination index <1) at clinically achievable levels of BSO (100-400 micromol/L) and levels of L-PAM (10-40 micromol/L) clinically achievable only with hematopoietic stem cell support. CONCLUSIONS: The in vitro results obtained for CHLA-171 suggest that BSO/L-PAM therapy may be optimally effective for drug-resistant neuroblastoma using myeloablative doses of L-PAM.

Synergism of buthionine sulfoximine and melphalan against neuroblastoma cell lines derived after disease progression.

BACKGROUND: Despite intensive-alkylator based regimens, >50% of patients with high-risk neuroblastoma (NB) die from recurrent disease that is probably due, in part, to acquired alkylator resistance. PROCEDURE: Using buthionine sulfoximine (BSO)-mediated, glutathione (GSH) depletion to modulate melphalan (L-PAM) resistance, we examined six NB cell lines established after progressive disease following either standard chemotherapy, BSO/L-PAM therapy, or myeloablative therapy and autologous hematopoietic stem cell transplant (AHSCT). RESULTS: Four of the six cell lines (three p53-nonfunctional and one p53-functional) showed high-level L-PAM resistance. CONCLUSIONS: Fixed ratio analysis demonstrated BSO/L-PAM synergy (combination index >1) for all cell lines tested. In L-PAM-resistant cell lines, the minimal cytotoxicity observed for BSO combined with nonmyeloablative concentrations of L-PAM was markedly enhanced (>4 logs total cell kill) when BSO was combined with myeloablative concentrations of L-PAM. In alkylator-resistant NB, the optimal use of BSO may require dose escalation of L-PAM to levels requiring AHSCT.

Effects of age on gene expression during estrogen-induced synaptic sprouting in the female rat.

Age and estrogen treatment influenced fiber outgrowth and compensatory neuronal sprouting after unilateral entorhinal cortex lesions (ECL) which model Alzheimer disease-like deafferentation in the dentate gyrus of the hippocampus. In young F344 rats (3 months old), ovariectomy (OVX) decreased reactive fiber outgrowth by 60%. Sprouting in middle-aged rats (18 months old) was reduced in intact females; no further reduction was caused by OVX. Several astrocyte mRNAs were measured in the dentate gyrus of young and middle-aged female rats in three different estrogen states (sham OVX, OVX, or OVX + estradiol) 1 week after ECL. Glial fibrillary acidic protein (GFAP) mRNA was twofold greater in middle-aged rats than young, although both ages showed threefold increases in response to ECL. In prior studies GFAP was found to be decreased by estradiol treatment 3-4 days after ECL; in this study GFAP mRNA had returned to sham OVX levels in young rats by 7 days post-ECL. Surprisingly, estradiol treatment increased GFAP mRNA levels by 25% above OVX in middle-aged rats. Apolipoprotein E (apoE) mRNA was decreased 20% by age in the dentate, although both age groups showed a 25% increase in apoE mRNA in response to ECL. Apolipoprotein J (apoJ) mRNA was increased 20% in the dentate gyrus of middle-aged rats, and both age groups responded to ECL with a 65% increase in apoJ mRNA. The estrogen state did not alter levels of either apolipoprotein mRNA in the deafferented dentate. The data suggest that the estrogen-induced decrease of GFAP in response to lesions does not persist at 7 days post-ECL during sprouting. Overall effects of age on the dentate gyrus include elevated GFAP mRNA and decreased apoE mRNA. The cortical wound site showed consistent enhancement of GFAP mRNA in both age groups by estradiol above sham OVX and greater responses in middle-aged rats.

Variations of synaptotagmin I, synaptotagmin IV, and synaptophysin mRNA levels in rat hippocampus during the estrous cycle.

Periodic changes in ovarian steroid levels during fertility cycles affect learning both in humans and in rats in parallel with electrophysiological and morphological fluctuations in selective neuronal populations. In particular, during the estrous cycle of the female rat, hippocampal CA1 region undergoes cyclic modifications in synaptic density. To investigate the molecular mechanisms involved in synaptic remodeling during the estrous cycle, we analyzed the expression of three presynaptic markers, synaptotagmin I, synaptotagmin IV, and synaptophysin, in the female adult rat brain by in situ hybridization. Relative abundance in mRNA for these three markers was quantified at four phases of the estrous cycle: diestrus, proestrus (AM and PM), and estrus. mRNA levels for syt1 exhibited cyclic variations in pyramidal neurons of the CA3 region of hippocampus during the estrous cycle, while mRNA levels for syt4 and SYN were relatively invariant in this or other regions of the hippocampus. Because CA3 pyramidal neurons make synaptic contacts in CA1, modulation of syt1 expression in CA3 may participate in the changes in synaptic density observed in CA1 during the estrous cycle. Furthermore, both syt1 and SYN mRNA varied cyclically in layer II, but not in layer III of entorhinal cortex, while syt4 remained unchanged throughout the cycle. These data suggest that regular variations in steroid hormone levels during fertility cycles may alter the properties of several networks involved in information processing and learning and memory through altered levels of presynaptic proteins.

Glial fibrillary acidic protein transcription responses to transforming growth factor-beta1 and interleukin-1beta are mediated by a nuclear factor-1-like site in the near-upstream promoter.

Elevated expression of glial fibrillary acidic protein (GFAP) is associated with astrocyte activation during responses to injury in the CNS. Because transforming growth factor-beta1 (TGF-beta1) and interleukin-1beta (IL-1beta) are released during neural responses to injury and because these cytokines also modulate GFAP mRNA levels, it is of interest to define their role in GFAP transcription. The increases of GFAP mRNA in response to TGF-beta1 and decreases in response to IL-1beta were shown to be transcriptionally mediated in rat astrocytes transfected with a luciferase-reporter construct containing 1.9 kb of 5'-upstream rat genomic DNA. Constructs containing sequential deletions of the rat GFAP 5'-upstream promoter identified a short region proximal to the transcription start (-106 to -53 bp) that provides full responses to TGF-beta1 and IL-1beta. This region contains an unusual sequence motif with overlapping nuclear factor-1 (NF-1)- and nuclear factor-kappaB (NF-kappaB)-like binding sites and homology to known TGF-beta response elements. Mutagenesis (3-bp exchanges) in -70 to -68 bp blocked the induction of GFAP by TGF-beta1 and the repression by IL-1beta. Gel shift experiments showed that the DNA segment -85 to -63 bp was bound by a factor(s) in nuclear extracts from astrocytes. The concentrations of these DNA binding factors were increased by treatment of astrocytes with TGF-beta1 and decreased by IL-1beta. Binding of these nuclear factors was blocked by mutation of -70 to -68 bp. Despite homology to NF-1 or NF-kappaB binding sites in the GFAP promoter at segment -79 to -67 bp, anti-NF-kappaB or anti-NF-1 antibodies did not further retard the gel shift of the nuclear factors/DNA complex. Moreover, astrocytic nuclear proteins do not compete for the specific binding to NF-1 consensus sequence. Thus, nuclear factors from astrocytes that bind to the -85- to -63-bp promoter segment might be only distantly related to NF-1 or NF-kappaB. These findings are pertinent to the use of GFAP promoter constructs in transgenic animals, because cisacting elements in the GFAP promoter are sensitive to cytokines that may be elaborated in response to expression of transgene products.

Depletion of glutathione by buthionine sulfoxine is cytotoxic for human neuroblastoma cell lines via apoptosis.

Buthionine sulfoximine (BSO) selectively inhibits glutathione (GSH) synthesis and has been used to sensitize tumor cells to alkylating agents, but has minimal single-agent cytotoxicity for most cell types. We determined the cytotoxicity of BSO for 18 (12 MYCN amplified; 6 MYCN nonamplified) human neuroblastoma cell lines using DIMSCAN, a digital image microscopy cytotoxicity assay. D-L(R:S) BSO was highly cytotoxic (>3 logs of cell kill) for most neuroblastoma cell lines, with 17/18 cell lines having IC90 values (range 2. 1->1000 microM) below equivalent steady state plasma levels of L(R:S) BSO reported in adult human trials. Cell lines with genomic amplification of MYCN were more sensitive to BSO than MYCN nonamplified cell lines (P = 0.04). D-L(R:S) BSO (500 microM for 72 h) induced apoptosis as detected by DNA laddering, nuclear morphology, and TUNEL staining of DNA fragments using flow cytometry. Maximal cell killing occurred within 48 h and was antagonized byic value in neuroblastoma.

Bidirectional transcription regulation of glial fibrillary acidic protein by estradiol in vivo and in vitro.

Glial fibrillary acidic protein (GFAP) expression shows cyclic variation in the rat hypothalamus and hippocampus during the normal estrous cycle. To elucidate the role of transcription in the regulation of GFAP, we examined levels of GFAP intron 1 by in situ hybridization in the hypothalamus and hippocampus of normal, cycling rats. On the afternoon of proestrus, when plasma estradiol levels are highest, GFAP transcription and messenger RNA were both increased in the arcuate nucleus of the hypothalamus and decreased in the outer molecular layer of the dentate gyrus. In the hilus of the hippocampus, neither GFAP transcription nor messenger RNA changed during the estrous cycle. In vitro, astrocytes showed bidirectional responses, such that estradiol treatment increased GFAP transcription in monotypic astrocytic cultures but decreased GFAP transcription in astrocytes cocultured with neurons. The functionality of an estrogen response element in the 5'-upstream region of the GFAP promoter was established by site-directed mutagenesis and binding of human recombinant estrogen receptor in gel shift assays. We conclude that estrogen may act directly upon astrocytes by estrogen receptor binding, and that the direction of the transcriptional response is influenced by astrocyte-neuron interactions.

Increased synaptic sprouting in response to estrogen via an apolipoprotein E-dependent mechanism: implications for Alzheimer's disease.

Estrogen replacement therapy appears to delay the onset of Alzheimer's disease (AD), but the mechanisms for this action are incompletely known. We show how the enhancement of synaptic sprouting by estradiol (E2) in response to an entorhinal cortex (EC) lesion model of AD may operate via an apolipoprotein E (apoE)-dependent mechanism. In wild-type (WT) mice, ovariectomy decreased commissural/associational sprouting to the inner molecular layer of the dentate gyrus, with synaptophysin (SYN) as a marker. E2 replacement returned SYN in the inner layer to levels of EC-lesioned, ovary-bearing controls and increased the area of compensatory synaptogenesis in the outer molecular layer. In EC-lesioned apoE-knock-out (KO) mice, however, E2 did not enhance sprouting. We also examined apoJ (clusterin) mRNA, which is implicated in AD by its presence in senile plaques, its transport of Abeta across the blood-brain barrier, and its induction by neurodegenerative lesioning. ApoJ mRNA levels were increased by E2 replacement in EC-lesioned WT mice but not in apoE-KO mice. These data suggest a mechanism for the protective effects of estrogens on AD and provide a link between two important risk factors in the etiology of AD, the apoE epsilon4 genotype and an estrogen-deficient state. This is also the first evidence that SYN, a presynaptic protein involved in neurotransmitter release, is regulated by E2 in the adult brain, and that apoE is necessary for the induction of apoJ mRNA by E2 in brain injury.

Astrocytes and microglia respond to estrogen with increased apoE mRNA in vivo and in vitro.

This study examined the regulation of apolipoprotein E (apoE) by 17beta-estradiol (E2) in brain glia, using rats with regular ovulatory cycles as an in vivo model and cultured astrocytes and mixed glia as in vitro models. Two brain regions were examined which had demonstrated transient synaptic remodeling during the estrous cycle. In the hippocampal CA1 region and the hypothalamic arcuate nucleus, apoE mRNA was elevated at proestrus when plasma E2 was high and synaptic density was increasing. Both astrocytes and microglia contributed to this increase in apoE mRNA. In vitro, E2 treatment had no effect on apoE mRNA levels in monotypic cultures of either astrocytes or microglia. In contrast, mixed glial cultures responded to E2 with increased apoE mRNA and protein, suggesting that heterotypic cellular interactions are important in the brain response to estrogens. In situ hybridization in combination with cell-specific markers showed that E2 increased apoE mRNA levels in both astrocytes and microglia. These results, which are the first evidence of apoE mRNA localization to microglia in vivo and the control of apoE expression in brain cells by estrogens, are discussed in terms of the possible protective role of E2 in Alzheimer's disease and prior findings that emphasize the expression of apoE mRNA in astrocytes within the brain.

Buthionine sulphoximine alone and in combination with melphalan (L-PAM) is highly cytotoxic for human neuroblastoma cell lines.

Buthionine sulphoximine (BSO) selectively inhibits glutathione (GSH) synthesis and may enhance the antineuroblastoma activity of melphalan (L-PAM). We determined the cytotoxicity of BSO (dose range 0-1000 microM) alone and in combination with L-PAM (dose range 0-0 microM) in a panel of 18 human neuroblastoma cell lines. BSO alone was highly cytotoxic with 16/18 neuroblastoma cell lines having IC90 values (range 2.1- > 1000 microM) below the clinically achievable steady-state plasma level of 500 microM BSO. Maximal cell killing correlated with GSH levels decreased to less than 10% baseline, and was partially reversed by the addition of exogenous anti-oxidants (GSH, vitamin E and ascorbate). Fluorocytometric analysis of DNA fragments by the Tunnel method detected 92% of a BSO sensitive cell line in apoptosis after a 48 h exposure to 500 microM BSO. The combination of L-PAM and BSO synergistically enhanced the cell killing of L-PAM alone by > 1-3 logs (combination index < 1). We conclude that BSO has significant single-agent cytotoxicity against neuroblastoma and enhances cell killing when combined with L-PAM.

Methylation of the rat glial fibrillary acidic protein gene shows tissue-specific domains.

The gene for glial fibrillary acidic protein (GFAP) was compared for CpG sites that are potential locations of methylated cytosine (mC). GFAP sequences in the 5'-upstream promoter and in exon 1 of rat, mouse, and human showed extensive similarity in the locations of CpG sites in the promoter and in exon 1, implying conservation. The methylation of mC at 9 CpG sites in the promoter and 10 sites in exon 1 was analyzed in F344 male rats by a quantitative application of ligation-mediated polymerase chain reaction (LMPCR). CpG sites with varying mC in different tissues were found in the GFAP promoter and in a CpG island in exon 1. In the brain, the promoter had about 40% less mC than in testis and liver. The degree of methylation varied strikingly between adjacent sites within and between tissues. Testis GFAP exon 1 had a gradient of mC from 5' to 3' across the exon that was absent in liver, brain, and cultured neurons and astrocytes. Among brain regions, the hippocampus had 10-40% less mC at 12 CpG sites than in hypothalamus; the other sites (7/19) showed smaller differences between these brain regions. In DNA from primary cultures, astrocytes had slightly less mC than neurons at all sites. Because neuron-rich hippocampal subregions and primary neurons cultures had less methylation than nonneural tissues, we hypothesize that neuroectodermal derivatives tend to be less methylated, whether or not GFAP is expressed. Four domains of methylated CpG sites are proposed on the basis of tissue and cell-type distribution: I) a constitutively methylated domain in the mid-upstream promoter; II) a testis-specific gradient of methylation in exon 1; III) a hypomethylated domain found in neuroectodermal derivatives; and IV) subsets of sites in the promoter and in exon 1 that have the least methylation in astrocytes, and therefore may be astrocyte-specific domains.

Age-related increases in glial fibrillary acidic protein do not show proportionate changes in transcription rates or DNA methylation in the cerebral cortex and hippocampus of male rats.

Age-related increases in the expression of glial fibrillary acidic protein (GFAP) in many brain regions are observed in short- and long-lived mammals. Possible genomic mechanisms for the increase of GFAP mRNA and protein were studied in the hippocampus and cortex of male F344 rats and a longer-lived hybrid F1 (F344 x Brown Norway). No age-related changes were found in the extent of cytosine methylation at 19 CpG sites in the 5'-upstream GFAP promoter and in exon 1. With the nuclear runon assay, no change was found in the transcription rate of GFAP in the cerebral cortex or hippocampus. Thus, age-related increases in GFAP are not associated with proportionate changes in transcription rates or DNA methylation. However, the transcription of glutamine synthetase was increased by about 60%. These findings contrast with age-related loss of bulk tissue DNA methylation and decreased transcription rates of other genes reported in non-neural tissues.

Regulation of an Arabidopsis oleosin gene promoter in transgenic Brassica napus.

Progressive deletions of the 5'-flanking sequences of an Arabidopsis oleosin gene were fused to beta-glucuronidase (GUS) and introduced into Brassica napus plants using Agrobacterium-mediated transformation. The effect of these deletions on the quantitative level of gene expression, organ specificity and developmental regulation was assessed. In addition, the influence of abscisic acid (ABA), jasmonic acid (JA), sorbitol and a combined ABA/sorbitol treatment on gene expression was investigated. Sequences that positively regulate quantitative levels of gene expression are present between -1100 to -600 and -400 to -200 of the promoter. In addition, sequences present between -600 and -400 down-regulate quantitative levels of expression. In transgenic B. napus plants, the oleosin promoter directs seed-specific expression of GUS which is present at early stages of seed development and increases throughout seed maturation. Sequences present between -2500 and -1100 of the promoter are involved in modulating the levels of expression at early stages of embryo development. Histochemical staining of embryos demonstrated that expression is uniform throughout the tissues of the embryo. Sequences involved in the response to ABA and sorbitol are present between -400 and -200. The induction of GUS activity by a combined ABA/sorbitol treatment is additive suggesting that ABA is not the sole mediator of osmotically induced oleosin gene expression. A response to JA was only observed when the oleosin promoter was truncated to -600 suggesting that the reported effect of JA on oleosin gene expression may be at a post-transcriptional level.