Differential Gene Expression in the Liver of the African Lungfish,Protopterus annectens,after 6 Months of Aestivation in Air or 1 Day of Arousal from 6 Months of Aestivation

The African lungfish, Protopterus annectens, can undergo aestivation during drought. Aestivation has three phases: induction, maintenance and arousal. The objective of this study was to examine the differential gene expression in the liver of P. annectens after 6 months (the maintenance phase) of aestivation as compared with the freshwater control, or after 1 day of arousal from 6 months aestivation as compared with 6 months of aestivation using suppression subtractive hybridization. During the maintenance phase of aestivation, the mRNA expression of argininosuccinate synthetase 1 and carbamoyl phosphate synthetase III were up-regulated, indicating an increase in the ornithine-urea cycle capacity to detoxify ammonia to urea. There was also an increase in the expression of betaine homocysteine-S-transferase 1 which could reduce and prevent the accumulation of hepatic homocysteine. On the other hand, the down-regulation of superoxide dismutase 1 expression could signify a decrease in ROS production during the maintenance phase of aestivation. In addition, the maintenance phase was marked by decreases in expressions of genes related to blood coagulation, complement fixation and iron and copper metabolism, which could be strategies used to prevent thrombosis and to conserve energy. Unlike the maintenance phase of aestivation, there were increases in expressions of genes related to nitrogen, carbohydrate and lipid metabolism and fatty acid transport after 1 day of arousal from 6 months aestivation. There were also up-regulation in expressions of genes that were involved in the electron transport system and ATP synthesis, indicating a greater demand for metabolic energy during arousal. Overall, our results signify the importance of sustaining a low rate of waste production and conservation of energy store during the maintenance phase, and the dependence on internal energy store for repair and structural modification during the arousal phase, of aestivation in the liver of P. annectens.     

Molecular characterization of argininosuccinate synthase and argininosuccinate lyase from the liver of the African lungfish Protopterus annectens,and their mRNA expression levels in the liver,kidney, brain and skeletal muscle during aestivation

Argininosuccinate synthase (Ass) and argininosuccinate lyase (Asl) are involved in arginine synthesis for various purposes. The complete cDNA coding sequences of ass and asl from the liver of Protopterus annectens consisted of 1,296 and 1,398 bp, respectively. Phylogenetic analyses revealed that the deduced Ass and Asl of P. annectens had close relationship with that of the cartilaginous fish Callorhinchus milii. Besides being strongly expressed in the liver, ass and asl expression were detectable in many tissues/organs. In the liver, mRNA expression levels of ass and asl increased significantly during the induction phase of aestivation, probably to increase arginine production to support increased urea synthesis. The increases in ass and asl mRNA expression levels during the prolonged maintenance phase and early arousal phase of aestivation could reflect increased demand on arginine for nitric oxide (NO) production in the liver. In the kidney, there was a significant decrease in ass mRNA expression level after 6 months of aestivation, indicating possible decreases in the synthesis and supply of arginine to other tissues/organs. In the brain, changes in ass and asl mRNA expression levels during the three phases of aestivation could be related to the supply of arginine for NO synthesis in response to conditions that resemble ischaemia and ischaemia–reperfusion during the maintenance and arousal phase of aestivation, respectively. The decrease in ass mRNA expression level, accompanied with decreases in the concentrations of arginine and NO, in the skeletal muscle of aestivating P. annectens might ameliorate the potential of disuse muscle atrophy.     

Molecular characterization and mRNA expression of carbamoyl phosphate synthetase III in the liver of the African lungfish, <Emphasis Type="Italic">Protopterus annectens</Emphasis>, during aestivation or exposure to ammonia

This study aimed to obtain the full sequence of carbamoyl phosphate synthetase III (cps III) from, and to determine the mRNA expression of cps III in, the liver of P. annectens during aestivation in air, hypoxia or mud, or exposure to environmental ammonia (100 mmol l⁻¹ NH₄Cl). The complete coding cDNA sequence of cps III from the liver of P. annectens consisted of 4530 bp, which coded for 1,510 amino acids with an estimated molecular mass of 166.1 kDa. The Cps III of P. annectens consisted of a mitochondrial targeting sequence of 44 amino acid residues, a GAT domain spanning from tyrosine 45 to isoleucine 414, and a methylglyoxal synthase-like domain spanning from valine 433 to arginine 1513. Two cysteine residues (cysteine 1337 and cysteine 1347) that are characteristic of N-acetylglutamate dependency were also present. The critical Cys-His-Glu catalytic triad (cysteine 301, histidine 385 and glutamate 387) together with methionine 302 and glutamine 305 affirmed that P. annectens expressed Cps III and not Cps I. A comparison of the translated amino acid sequence of Cps III from P. annectens with CPS sequences from other animals revealed that it shared the highest similarity with elasmobranch Cps III. A phylogenetic analysis indicates that P. annectens CPS III could have evolved from Cps III of elasmobranchs. Indeed, Cps III from P. annectens used mainly glutamine as the substrate, and its activity decreased significantly when glutamine and ammonia were included together in the assay system. There were significant increases (9- to 12-fold) in the mRNA expression of cps III in the liver of fish during the induction phase (days 3 and 6) of aestivation in air. Aestivation in hypoxia or in mud had a delayed effect on the increase in the mRNA expression of cps III, which extended beyond the induction phase of aestivation, reiterating the importance of differentiating effects that are intrinsic to aestivation from those intrinsic to hypoxia. Furthermore, results from this study confirmed that environmental ammonia exposure led to a significant increase in the mRNA expression of cps III in the liver of P. annectens, alluding to the important functional role of urea not only as a product of ammonia detoxification but also as a putative internal cue for aestivation.     

Differential gene expression in the liver of the African lungfish, <Emphasis Type="Italic">Protopterus annectens</Emphasis>, after 6 days of estivation in air

This study aimed to identify estivation-specific gene clusters through the determination of differential gene expressions in the liver of Protopterus annectens after 6 days of estivation in a mucus cocoon in air (normoxia) using suppression subtractive hybridization polymerase chain reaction. Our results demonstrated that 6 days of estivation in normoxia led to up-regulation of mRNA expressions of several genes related to urea synthesis, including carbamoyl phosphate synthetase (Cps), argininosuccinate synthetase and glutamine synthetase. They indicate that increased urea synthesis, despite being energy-intensive, is an important adaptive response of estivation. They also offer indirect support to the proposition that urea synthesis in this lungfish involved a Cps that uses glutamine as a substrate. In addition, up- or down-regulation of several gene clusters occurred in the liver of P. annectens after 6 days of estivation in normoxia. These estivation-specific genes were involved in the prevention of clot formation, activation of the lectin pathway for complement activation, conservation of minerals (e.g. iron and copper) and increased production of hemoglobin beta. Since there were up- and down-regulation of mRNA expressions of genes related to ribosomal proteins and translational elongation factors, there could be simultaneous increases in protein degradation and protein synthesis during the first 6 days (the induction phase) of estivation, confirming the importance of reconstruction of protein structures in preparation for the maintenance phase of estivation.     

The NOS/NO system in an example of extreme adaptation: The African lungfish

African dipnoi (lungfish) are aestivating fish and obligate air breathers that, throughout their complex life cycle, undergo remarkable morpho-functional organ readjustment from biochemical to morphological level. In the present review we summarize the changes of the NOS/NO (Nitric Oxide Synthase/Nitric Oxide) system occurring in lungs, gills, kidney, heart, and myotomal muscle of African lungfish of the genus Protopterus (P. dolloi and P. annectens), in relation to the switch from freshwater to aestivation, and vice-versa. In particular, the expression and localization patterns of NOS, and its protein partners Akt, Hsp-90 and HIF-1α, have been discussed, together with the apoptosis rate, evaluated by TUNEL technique.We hypothesize that all these molecular components are crucial in signalling transduction/integration networks induced by environmental challenges (temperature, dehydration, inactivity)experienced at the beginning, during, and at the end of the dry season.     

Increased urea synthesis and/or suppressed ammonia production in the African lungfish, Protopterus annectens, during aestivation in air or mud

The objective of this study was to elucidate how the African lungfish, Protopterus annectens, ameliorated ammonia toxicity during 12 or 46 days of aestivation in air or in mud. Twelve days of aestivation in air led to significant increases in contents of urea, but not ammonia, in tissues of P. annectens. The estimated rate of urea synthesis increased 2.7-fold despite the lack of changes in the activities of hepatic ornithine–urea cycle enzymes, but there was only a minor change in the estimated rate of ammonia production. After 46 days of aestivation in air, the ammonia content in the liver decreased significantly and contents of urea in all tissues studied increased significantly, indicating that the fish shifted to a combination of increased urea synthesis (1.4-fold of the day 0 value) and decreased ammonia production (56% of the day 0 value) to defend against ammonia toxicity. By contrast, 12 days of aestivation in mud produced only minor increases in tissue urea contents, with ammonia contents remained unchanged. This was apparently achieved through decreases in urea synthesis and ammonia production (40 and 15%, respectively, of the corresponding day 0 value). Surprisingly, 46 days of aestivation in mud resulted in no changes in tissue urea contents, indicating that profound suppressions of urea synthesis and ammonia production (2.6 and 1.2%, respectively, of the corresponding day 0 value) had occurred. This is the first report on such a phenomenon, and the reduction in ammonia production was so profound that it could be the greatest reduction known among animals. Since fish aestivated in mud had relatively low blood pO₂ and muscle ATP content, they could have been exposed to hypoxia, which induced reductions in metabolic rate and ammonia production. Consequently, fish aestivating in mud had a lower dependency on increased urea synthesis to detoxify ammonia, which is energy intensive, than fish aestivating in air.     

Differential distribution of aldolase A and C in the human central nervous system

We have analyzed the distribution of aldolase A and C mRNAs and proteins in various areas of the human brain using Northern blot analyses and immunohistochemistry. Aldolase A mRNA expression was higher than aldolase C mRNA expression in all areas of the brain examined. Aldolase C mRNA expression was highest in the cerebellum. Aldolase C protein was present in well-delimited regions of the CNS, and was distributed in stripes in the Purkinje cell layer of the cerebellum, in the inferior olives and in the sensory neurons of the posterior horn of the spinal cord. The novel finding of aldolase C in well-delimited cell compartments of the human cerebellum and in several other areas of the CNS lends weight to the hypothesis that this protein exerts other functions (e.g. sensory transmission) besides those characteristic of a glycolytic enzyme.     

Down-regulation of Wild-type p53-induced Phosphatase 1 (Wip1) Plays a Critical Role in Regulating Several p53-dependent Functions in Premature Senescent Tumor Cells

Premature or drug-induced senescence is a major cellular response to chemotherapy in solid tumors. The senescent phenotype develops slowly and is associated with chronic DNA damage response. We found that expression of wild-type p53-induced phosphatase 1 (Wip1) is markedly down-regulated during persistent DNA damage and after drug release during the acquisition of the senescent phenotype in carcinoma cells. We demonstrate that down-regulation of Wip1 is required for maintenance of permanent G₂ arrest. In fact, we show that forced expression of Wip1 in premature senescent tumor cells induces inappropriate re-initiation of mitosis, uncontrolled polyploid progression, and cell death by mitotic failure. Most of the effects of Wip1 may be attributed to its ability to dephosphorylate p53 at Ser¹⁵ and to inhibit DNA damage response. However, we also uncover a regulatory pathway whereby suppression of p53 Ser¹⁵ phosphorylation is associated with enhanced phosphorylation at Ser⁴⁶, increased p53 protein levels, and induction of Noxa expression. On the whole, our data indicate that down-regulation of Wip1 expression during premature senescence plays a pivotal role in regulating several p53-dependent aspects of the senescent phenotype.     

Correlation of ghrelin concentration and ghrelin,ghrelin-O-acetyltransferase (GOAT) and growth hormone secretagogue receptor 1a mRNAs expression in the proventriculus and brain of the growing chicken

To determine mechanisms for age-related decrease of GHS-R1a expression in the chicken proventriculus, changes in mRNA expression of ghrelin and ghrelin-O-acetyltransferase (GOAT) as well as ghrelin concentrations in the proventriculus and plasma were examined in growing chickens. Changes in expression levels of ghrelin, GOAT and GHS-R1a mRNAs were also examined in different brain regions (pituitary, hypothalamus, thalamus, cerebellum, cerebral cortex, olfactory bulb, midbrain and medulla oblongata). Ghrelin concentrations in the proventriculus and plasma increased with aging and reached plateaus at 30–50 days after hatching. High level of ghrelin mRNA decreased at 3 days after hatching, and it became stable at half of the initial level. Expression levels of GHS-R1a and GOAT decreased 3 or 5 days after hatching and became stable at low levels. Significant negative correlations were found between plasma ghrelin and mRNA levels of GOAT and GHS-R1a. Expression levels of ghrelin mRNA were different in the brain regions, but a significant change was not seen with aging. GHS-R1a expression was detected in all brain regions, and age-dependent changes were observed in the pituitary and cerebellum. Different from the proventriculus, the expression of GOAT in the brain increased or did not change with aging. These results suggest that decreased GHS-R1a and GOAT mRNA expression in the proventriculus is due to endogenous ghrelin-induced down-regulation. Expression levels of ghrelin, GOAT and GHS-R1a in the brain were independently regulated from that in the proventriculus, and age-related and region-dependent regulation pattern suggests a local effect of ghrelin system in chicken brain.     

Enzyme activity in the aestivating Green-striped burrowing frog (Cyclorana alboguttata)

Green-striped burrowing frogs (Cyclorana alboguttata) can depress their resting metabolism by more than 80% during aestivation. Previous studies have shown that this species is able to withstand long periods of immobilisation during aestivation while apparently maintaining whole muscle mass and contractile performance. The aim of this study was to determine the effect of prolonged aestivation on the levels of metabolic enzymes (CCO, LDH and CS) in functionally distinct skeletal muscles (cruralis, gastrocnemius, sartorius, iliofibularis and rectus abdominus) and liver of C. alboguttata. CS activity was significantly reduced in all tissues except for the cruralis, gastrocnemius and the liver. LDH activity was significantly reduced in the sartorius and rectus abdominus, but remained at control (active) levels in the other tissues. CCO activity was significantly reduced in the gastrocnemius and rectus abdominus, and unchanged in the remaining tissues. Muscle protein was significantly reduced in the sartorius and iliofibularis during aestivation, and unchanged in the remaining muscles. The results suggest that the energy pathways involved in the production and consumption of ATP are remodelled during prolonged aestivation but selective. Remodelling and subsequent down-regulation of metabolic activity seem to target the smaller non-jumping muscles, while the jumping muscles retain enzyme activities at control levels during aestivation. These results suggest a mechanism by which aestivating C. alboguttata are able to maintain metabolic depression while ensuring that the functional capacity of critical muscles is not compromised upon emergence from aestivation.     

Multilocus isozyme systems in African lungfish,Protopterus annectens: distribution,differential expression and variation in dipnoans

Distribution of ADH, ALP, FBALD, GAPDH, G3PDH, G6PDH, GPI, LDH, MDH, PGM, and SOD was identified in retina, heart, muscle, liver, kidney, gills, brain, gut, lung and ovary of the African lungfish. Data are compared with patterns previously described in dipnoans and other vertebrates. The number of loci expressed for all enzymes was found to be similar to those of diploid Actinopterygii. Differences in the number of loci expressed in Amphibia were found for ALP, sG3PDH, GPI, LDH, MDH and SOD. Differences in tissue distribution were noted in ALP due to the absence of an intestinal-specific form typical of teleostean fish, amphibians, reptiles and birds, and in GPI and MDH, due to the tissue expression, as in primitive fish. There were also differences in LDH, where a third locus (LDH-C*) was expressed in the gills of Protopterus annectens and not in the retina or liver tissues, as in teleosts. LDH-A₄ was most common in all the tissues. Major differences were noted in the tissue patterns of protein expression in the three dipnoans compared. As expected, the least divergence was found between the two species belonging to the same family (Lepidosirenidae). The highest index of divergence was observed between Neoceratodus forsteri and Lepidosiren paradoxa, belonging to the families Ceratontidae and Lepidosirenidae, respectively. The divergence is revealed by changes at the enzyme and morphological levels. These results suggest that P. annectens occupies an interesting systematic position, its biochemical characteristics distinguishing it from N. forsteri, L. paradoxa, the advanced fish and amphibians.     

Dual-origin plasmids containing an amplifiable ColE1 ori; temperature-controlled expression of cloned genes

Dual-origin plasmids comprising an inducible ColE1-derived origin of replication controlled by the λ p_R promoter, the c1857 temperature-sensitive represser gene and the pSC101 origin of replication and its associated par sequence, were constructed. Such plasmids carrying cloned genes were stably maintained at four copies per chromosome, and were readily amplifiable by thermal induction. Cloned gene expression increased with copy number, and accumulation values of > 20% total cellular protein were detected. These vectors should prove useful for the production of foreign protein on a large scale, since they provide for stable plasmid maintenance during the growth phase, and high-level gene expression without plasmid loss during the production phase.     

Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapα is the most predominant isoform. The Gfapδ isoform is expressed in proliferating neurogenic astrocytes of the developing human brain and in the adult human and mouse brain. Here we provide a characterization of mouse Gfapδ mRNA and Gfapδ protein. RT-qPCR analysis showed that Gfapδ mRNA and Gfapα mRNA expression is coordinately increased in the post-natal period. Immunohistochemical staining of developing mouse brain samples showed that Gfapδ is expressed in the sub-ventricular zones in accordance with the described localization in the developing and adult human brain. Immunofluorescence analysis verified incorporation of Gfapδ into the Gfap intermediate filament network and overlap in Gfapδ and Gfapα subcellular localization. Subcellular mRNA localization studies identified different localization patterns of Gfapδ and Gfapα mRNA in mouse primary astrocytes. A larger fraction of Gfapα mRNA showed mRNA localization to astrocyte protrusions compared to Gfapδ mRNA. The differential mRNA localization patterns were dependent on the different 3′-exon sequences included in Gfapδ and Gfapα mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential to participate in subcellular region-specific intermediate filament dynamics during brain development, maintenance and in disease.