HDAC inhibitors attenuate the development of hypersensitivity in models of neuropathic pain

Histone deacetylase inhibitors (HDACIs) interfere with the epigenetic process of histone acetylation and are known to have analgesic properties in models of chronic inflammatory pain. The aim of this study was to determine whether these compounds could also affect neuropathic pain. Different class I HDACIs were delivered intrathecally into rat spinal cord in models of traumatic nerve injury and antiretroviral drug-induced peripheral neuropathy (stavudine, d4T). Mechanical and thermal hypersensitivity was attenuated by 40% to 50% as a result of HDACI treatment, but only if started before any insult. The drugs globally increased histone acetylation in the spinal cord, but appeared to have no measurable effects in relevant dorsal root ganglia in this treatment paradigm, suggesting that any potential mechanism should be sought in the central nervous system. Microarray analysis of dorsal cord RNA revealed the signature of the specific compound used (MS-275) and suggested that its main effect was mediated through HDAC1. Taken together, these data support a role for histone acetylation in the emergence of neuropathic pain.

TGF-beta superfamily members and ovarian follicle development

In recent years, exciting progress has been made towards unravelling the complex intraovarian control mechanisms that, in concert with systemic signals, coordinate the recruitment, selection and growth of follicles from the primordial stage through to ovulation and corpus luteum formation. A plethora of growth factors, many belonging to the transforming growth factor-beta (TGF-beta) superfamily, are expressed by ovarian somatic cells and oocytes in a developmental, stage-related manner and function as intraovarian regulators of folliculogenesis. Two such factors, bone morphogenetic proteins, RMP-4 and BMP-7, are expressed by ovarian stromal cells and/or theca cells and have recently been implicated as positive regulators of the primordial-to-primary follicle transition. In contrast, evidence indicates a negative role for anti-Mullerian hormone (AMH, also known as Mullerian-inhibiting substance) of pre-granulosa/granulosa cell origin in this key event and subsequent progression to the antral stage. Two other TGF-beta superfamily members, growth and differentiation factor-9 (GDF-9) and BMP-15 (also known as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play key roles in promoting follicle growth beyond the primary stage; mice with null mutations in the gdf-9 gene or ewes with inactivating mutations in gdf-9 or bmp-15 genes are infertile with follicle development arrested at the primary stage. Studies on later stages of follicle development indicate positive roles for granulosa cell-derived activin, BMP-2, -5 and -6, theca cell-derived BMP-2, -4 and -7 and oocyte-derived BMP-6 in promoting granulosa cell proliferation, follicle survival and prevention of premature luteinization and/or atresia. Concomitantly, activin, TGF-beta and several BMPs may exert paracrine actions on theca cells to attenuate LH-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection in monovular species may depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Changes in intrafollicular activins, GDF-9, AMH and several BMPs may contribute to this selection process by modulating both FSH- and IGF-dependent signalling pathways in granulosa cells. Activin may also play a positive role in oocyte maturation and acquisition of developmental competence. in addition to its endocrine role to suppress FSH secretion, increased output of inhibin by the selected dominant follicle(s) may upregulate LH-induced androgen secretion that is required to sustain a high level of oestradiol secretion during the pre-ovulatory phase. Advances in our understanding of intraovarian regulatory mechanisms should facilitate the development of new approaches for monitoring and manipulating ovarian function and improving fertility in domesticated livestock, endangered species and man.

The anti-epileptic drug Valproic Acid (VPA) inhibits steroidogenesis in bovine theca and granulosa cells in vitro

Valproic acid (VPA) is used widely to treat epilepsy and bipolar disorder. Women undergoing VPA treatment reportedly have an increased incidence of polycystic ovarian syndrome (PCOS)-like symptoms including hyperandrogenism and oligo- or amenorrhoea. To investigate potential direct effects of VPA on ovarian steroidogenesis we used primary bovine theca (TC) and granulosa (GC) cells maintained under conditions that preserve their 'follicular' phenotype. Effects of VPA (7.8-500 µg/ml) on TC were tested with/without LH. Effects of VPA on GC were tested with/without FSH or IGF analogue. VPA reduced (P<0.0001) both basal (70% suppression; IC(50) 67±10 µg/ml) and LH-induced (93% suppression; IC(50) 58±10 µg/ml) androstenedione secretion by TC. VPA reduced CYP17A1 mRNA abundance (>99% decrease; P<0.0001) with lesser effects on LHR, STAR, CYP11A1 and HSD3B1 mRNA (<90% decrease; P<0.05). VPA only reduced TC progesterone secretion induced by the highest (luteinizing) LH dose tested; TC number was unaffected by VPA. At higher concentrations (125-500 µg/ml) VPA inhibited basal, FSH- and IGF-stimulated estradiol secretion (P<0.0001) by GC without affecting progesterone secretion or cell number. VPA reversed FSH-induced upregulation of CYP19A1 and HSD17B1 mRNA abundance (P<0.001). The potent histone deacetylase (HDAC) inhibitors trichostatin A and scriptaid also suppressed TC androstenedione secretion and granulosal cell oestrogen secretion suggesting that the action of VPA reflects its HDAC inhibitory properties. In conclusion, these findings refute the hypothesis that VPA has a direct stimulatory action on TC androgen output. On the contrary, VPA inhibits both LH-dependent androgen production and FSH/IGF-dependent estradiol production in this in vitro bovine model, likely by inhibition of HDAC.

Nuclear accumulation of myocyte muscle LIM protein is regulated by heme oxygenase 1 and correlates with cardiac function in the transition to failure

Impaired mechanosensing leads to heart failure and we have previously shown that a decreased ratio of cytoplasmic to nuclear CSRP3/Muscle LIM protein (MLP ratio) is associated with a loss of mechanosensitivity. Here we tested whether passive or active stress/strain was important in modulating the MLP ratio and determined whether this correlated with heart function during the transition to failure. We exposed cultured neonatal rat myocytes to 10% cyclic mechanical stretch at 1 Hz, or electrically paced myocytes at 6.8 V (1 Hz) for 48 h. The MLP ratio decreased 50% (P < 0.05, n = 4) only in response to electrical pacing, suggesting impaired mechanosensitivity. Inhibition of contractility with 10 μM blebbistatin resulted in a ∼3 fold increase in the MLP ratio (n = 8, P < 0.05), indicating that myocyte contractility regulates nuclear MLP. Inhibition of histone deacetylase (HDAC) signaling with trichostatin A increased nuclear MLP following passive stretch, suggesting that HDACs block MLP nuclear accumulation. Inhibition of heme-oxygenase1 (HO-1) activity with PPZII blocked MLP nuclear accumulation. To examine how mechanosensitivity changes during the transition to heart failure, we studied a guinea pig model of angiotensin II infusion (400 ng/kg/min) over 12 weeks. Using subcellular fractionation we showed that the MLP ratio increased 88% (n = 4, P < 0.01) during compensated hypertrophy, but decreased significantly during heart failure (P < 0.001, n = 4). The MLP ratio correlated significantly with the E/A ratio (r = 0.71, P < 0.01 n = 12), a clinical measure of diastolic function. These data indicate for the first time that myocyte mechanosensitivity as indicated by the MLP ratio is regulated primarily by myocyte contractility via HO-1 and HDAC signaling.

Histone H4 gene expression and morphological changes on shoot apices of strawberry (Fragaria x ananassa Duch.) during floral induction

To investigate flower induction in June-bearing strawberry plants, morphological changes in shoot apices and Historic H4 expression in the central zone during flower initiation were observed. Strawberry plants were placed under flower inducible, short-day conditions (23 degrees C/17 degrees C, 10 h day length) for differing number of days (8, 16, 20, 24 or 32 days) and then these plants were transferred to non-inducible, long-day conditions (25 degrees C/20 degrees C, 14 h day length). The shoot apices of plants placed under short-day conditions for 8 days were flat, similar to shoot apices of plants in the vegetative phase of development, and Histone H4 was not expressed in the central zone during the experimental period. On the other hand, the shoot apices of plants placed under short-day conditions for 16 days remained flat, similar to shoot apices of plants placed under short-day conditions for 8 days, but Histone H4 was expressed in the central zone at the end of the short-day treatment. Morphological changes in the shoot apices of these plants were observed 8 days after the change in day-length. These plants developed differentiated flower organs after they were grown for another 30 days under long-day conditions. These results indicate that changes in the expression pattern of the Histone H4 gene occur before morphological changes during flower induction and that the expression of the gene in the central zone can be used as one of the indicators of the flowering process in strawberries. (c) 2006 Elsevier B.V. All rights reserved.

Gene conversion and evolution of Xq28 duplicons involved in recurring inversions causing severe hemophilia A

Inversions breaking the 1041 bp int1h-1 or the 9.5-kb int22h-1 sequence of the F8 gene cause hemophilia A in 1/30,000 males. These inversions are due to homologous recombination between the above sequences and their inverted copies on the same DNA molecule, respectively, int1h-2 and int22h-2 or int22h-3. We find that (1) int1h and int22h duplicated more than 25 million years ago; (2) the identity of the copies (>99%) of these sequences in humans and other primates is due to gene conversion; (3) gene conversion is most frequent in the internal regions of int22h; (4) breakpoints of int22h-related inversions also tend to involve the internal regions of int22h; (5) sequence variations in a sample of human X chromosomes defined eight haplotypes of int22h-1 and 27 of int22h-2 plus int22h-3; (6) the latter two sequences, which lie, respectively, 500 and 600 kb telomeric to int22h-1 are five-fold more identical when in cis than when in trans, thus suggesting that gene conversion may be predominantly intrachromosomal; (7) int1h, int22h, and flanking sequences evolved at a rate of about 0.1% substitutions per million years during the divergence between humans and other primates, except for int1h during the human-chimpanzee divergence, when its rate of evolution was significantly lower. This is reminiscent of the slower evolution of palindrome arms in the male specific regions of the Y chromosome and we propose, as an explanation, that intrachromosomal gene conversion and cosegregation of the duplicated regions favors retention of the ancestral sequence and thus reduces the evolution rate.

Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

Background: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose ( to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods: We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results: On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion: Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.

A test of the master gene hypothesis for interspersed repetitive DNA sequences

Many families of interspersed repetitive DNA elements, including human Alu and LINE (Long Interspersed Element) elements, have been proposed to have accumulated through repeated copying from a single source locus: the "master gene." The extent to which a master gene model is applicable has implications for the origin, evolution, and function of such sequences. One repetitive element family for which a convincing case for a master gene has been made is the rodent ID (identifier) elements. Here we devise a new test of the master gene model and use it to show that mouse ID element sequences are not compatible with a strict master gene model. We suggest that a single master gene is rarely, if ever, likely to be responsible for the accumulation of any repeat family.

A genetic linkage map of microsatellite, gene-specific and morphological markers in diploid Fragaria

Diploid Fragaria provide a potential model for genomic studies in the Rosaceae. To develop a genetic linkage map of diploid Fragaria, we scored 78 markers (68 microsatellites, one sequence-characterised amplified region, six gene-specific markers and three morphological traits) in an interspecific F2 population of 94 plants generated from a cross of F.vesca f. semperflorens × F. nubicola. Co-segregation analysis arranged 76 markers into seven discrete linkage groups covering 448 cM, with linkage group sizes ranging from 100.3 cM to 22.9 cM. Marker coverage was generally good; however some clustering of markers was observed on six of the seven linkage groups. Segregation distortion was observed at a high proportion of loci (54%), which could reflect the interspecific nature of the progeny and, in some cases, the self-incompatibility of F. nubicola. Such distortion may also account for some of the marker clustering observed in the map. One of the morphological markers, pale-green leaf (pg) has not previously been mapped in Fragaria and was located to the mid-point of linkage group VI. The transferable nature of the markers used in this study means that the map will be ideal for use as a framework for additional marker incorporation aimed at enhancing and resolving map coverage of the diploid Fragaria genome. The map also provides a sound basis for linkage map transfer to the cultivated octoploid strawberry.

Dispersal of NK homeobox gene clusters in amphioxus and humans

The Drosophila melanogaster genome has six physically clustered NK-related homeobox genes in just 180 kb. Here we show that the NK homeobox gene cluster was an ancient feature of bilaterian animal genomes, but has been secondarily split in chordate ancestry. The NK homeobox gene clusters of amphioxus and vertebrates are each split and dispersed at two equivalent intergenic positions. From the ancestral NK gene cluster, only the Tlx-Lbx and NK3-NK4 linkages have been retained in chordates. This evolutionary pattern is in marked contrast to the Hox and ParaHox gene clusters, which are compact in amphioxus and vertebrates, but have been disrupted in Drosophila.

Dietary vitamin E modulates differential gene expression in the rat hippocampus: potential implications for its neuroprotective properties

A wide range of cell culture, animal and human epidemiological studies are suggestive of a role of vitamin E (VE) in brain function and in the prevention of neurodegeneration. However, the underlying molecular mechanisms remain largely unknown. In the current investigation Affymetrix gene chip technology was utilised to establish the impact of chronic VE deficiency on hippocampal genes expression. Male albino rats were fed either a VE deficient or standard diet (60 mg/kg feed) for a period of 9 months. Rats were sacrificed, the hippocampus removed and genes expression established in individual animals. VE deficiency showed to have a strong impact on genes expression in the hippocampus. An important number of genes found to be regulated by VE was associated with hormones and hormone metabolism, nerve growth factor, apoptosis, dopaminergic neurotransmission, and clearance of amyloid-beta and advanced glycated endproducts. In particular, VE strongly affected the expression of an array of genes encoding for proteins directly or indirectly involved in the clearance of amyloid beta, changes which are consistent with a protective effect of VE on Alzheimer's disease progression.

Evidence for associations between common polymorphisms of estrogen receptor beta gene with homocysteine and nitric oxide

Background Homocysteine and asymmetric dimethylarginine (ADMA) affect nitric oxide (NO) concentration, thereby contributing to cardiovascular disease (CVD). Both amino acids can be reduced in vivo by estrogen. Variation in the estrogen receptor (ER) may influence homocysteine and ADMA, yet no information is available on associations with single nucleotide polymorphisms in the estrogen receptor genes ER alpha (PvuII and XbaI) and ER beta (1730G -> A and cx+56 G -> A). Objective To find relationships between common polymorphisms associated with cardiovascular disease and cardiovascular risk factors homocysteine and ADMA. Methods In a cross-sectional study with healthy postmenopausal women (n = 89), homocysteine, ADMA, nitric oxide metabolites (NOx), plasma folate and ER alpha and beta polymorphisms ER alpha PvuII, ER alpha XbaI; ER beta 1730G -> A (AluI), ER beta cx+56 G -> A (Tsp5091) were analyzed. Results Women who are homozygotic for ER beta cx+56 G -> A A/A exhibited higher homocysteine (p = 0.012) and NOx (p = 0.056) levels than wildtype or heterozygotes. NOx concentration was also significantly affected by ER beta 1730 G -> A polymorphism (p = 0.025). The ER beta (p < 0.001) and ER alpha (p < 0.001) polymorphisms were in linkage disequilibrium. Conclusions Women who are homozygotic for ER beta cx+S6 G -> A A/A may be at increased risk for cardiovascular disease due to higher homocysteine levels.

Identification of hepatic molecular mechanisms of action of alpha-tocopherol using global gene expression profile analysis in rats

The recent discovery that vitamin E (VE) regulates gene activity at the transcriptional level indicates that VE may exert part of its biological effects by mechanisms which may be independent of its well-recognised antioxidant function. The objective of this study was the identification of hepatic vitamin E-sensitive genes and examination of the effects of VE on their corresponding biological endpoints. Two groups of male rats were randomly assigned to either a VE-sufficient diet or to a control diet deficient in VE for 290 days. High-density oligonucleotide microarrays comprising over 7000 genes were used to assess the transcriptional response of the liver. Differential gene expression was monitored over a period of 9 months, at four different time-points, and rats were individually profiled. This experimental strategy identified several VE-sensitive genes, which were chronically altered by dietary VE. VE supplementation down-regulated scavenger receptor CD36, coagulation factor IX and 5-alpha-steroid reductase type 1 mRNA levels while hepatic gamma glutamyl-cysteinyl synthetase was significantly up-regulated. Measurement of the corresponding biological endpoints such as activated partial thromboplastin time, plasma dihydrotestosterone and hepatic glutathione substantiated the gene chip data which indicated that dietary VE plays an important role in a range of metabolic processes within the liver. (C) 2004 Elsevier B.V. All rights reserved.

Expression of the efflux pump genes cmeB, cmeF and the porin gene porA in multiple-antibiotic-resistant Campylobacter jejuni

Aims: In Escherichia coli, increased expression of efflux pumps and/or decreased expression of porins can confer multiple antibiotic resistance (MAR), causing resistance to at least three unrelated classes of antibiotics, detergents and dyes. It was hypothesized that in Campylobacter jejuni, the efflux systems CmeABC, CmeDEF and the major outer membrane porin protein, MOMP (encoded by porA) could confer MAR. Methods: The expression of cmeB, cmeF and porA in 32 MAR C. jejuni isolated from humans or poultry was determined by comparative (C)-reverse transcriptase (RT)-PCR and denaturing DHPLC. A further 13 ethidium bromide-resistant isolates and three control strains were also investigated. Accumulation of ciprofloxacin carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) was also determined for all strains. Results: Although resistance to ethidium bromide has been associated with MAR, expression of all three genes was similar in the ethidium bromide-resistant isolates. These data indicate that CmeB, CmeF and MOMP play no role in resistance to this agent in C. jejuni. Six MAR isolates over-expressed cmeB, 3/32 over-expressed cmeB and cmeF. No isolates over-expressed cmeF alone. Expression of porA was similar in all isolates. All nine isolates that over-expressed cmeB contained a mutation in cmeR, substituting glycine 86 with alanine. All cmeB over-expressing isolates also accumulated low concentrations of ciprofloxacin, which were restored to wild-type levels in the presence of CCCP. Conclusions: These data indicate that over-expression of cmeB is associated with MAR in isolates of C. jejuni. However, as cmeB was over-expressed by only one-third (9/32) of MAR isolates, these data also indicate other mechanisms of MAR in C. jejuni.