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Telomeres play an important role in the maintenance of genomic stability/integrity and are synthesized by the RNA-dependent polymerase telomerase. Progressive telomere shortening contributes to both in vitro and in vivo aging, and telomere length dynamics and telomerase expression profile in human tissues during extrauterine life have been well characterized. However, little is known about these changes in the early stage of gestation. In the present study, we determined telomere length and the expression of telomerase core units (telomerase reverse transcriptase, hTERT, and telomerase RNA component, hTERC) in human fetus tissues from 6 to 11 weeks of gestational age. A sharp decline in telomere length occurred between 6 and 7 weeks of gestational age, and a relatively stable or slightly shortened telomere length was thereafter maintained until birth. The inverse correlation between TERT or TERC expression and gestational age was steadily observed in these fetus tissues. Taken together, there is a rapid reduction followed by a slow erosion of telomere length in human fetus from gestational age 6-11 weeks, while hTERT and hTERC expression decreases steadily during this period. The present findings not only contribute to better understandings of telomere/telomerase biology in human embryonic development, but also are implicated in telomere/telomerase-related diseases or problems., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a key metabolite in the glucose homeostasis. Four genes, Pfkfb1-4, have been characterized in mammals that code for several isoforms generated by alternative splicing through the control of several promoters and 5' non-coding exons. Here, we characterize in fetal rat liver new mRNA variants which are transcribed from a new Pfkfb1 gene promoter. The long variant codes to a new isoform (FL-PFK-2) that would be of relevant function to modulate the transition of fetal to adult liver metabolism., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Despite the central role of proteasomes in relevant physiological pathways and pathological processes, this topic is unexpectedly largely unexplored in human liver. Here we present data on the presence of proteasome and immunoproteasome in human livers from normal adults, fetuses and patients affected by major hepatic diseases such as cirrhosis and chronic active hepatitis. Immunohistochemistry for constitutive (alpha4 and beta1) and inducible (LMP2 and LMP7) proteasome subunits, and for the PA28alphabeta regulator, was performed in liver samples from 38 normal subjects, 6 fetuses, 2 pediatric cases, and 19 pathological cases (10 chronic active hepatitis and 9 cirrhosis). The immunohistochemical data have been validated and quantified by Western blotting analysis. The most striking result we found was the concomitant presence in hepatocyte cytoplasm of all healthy subjects, including the pediatric cases, of constitutive proteasome and immunoproteasome subunits, as well as PA28alphabeta. At variance, immunoproteasome was not present in hepatocytes from fetuses, while a strong cytoplasmic and nuclear positivity for LMP2 and LMP7 was found in pathological samples, directly correlated to the histopathological grade of inflammation. At variance from other organs such as the brain, immunoproteasome is present in livers from normal adult and pediatric cases, in apparent absence of pathological processes, suggesting the presence of a peculiar regulation of the proteasome/immunoproteasome system, likely related to the physiological stimuli derived from the gut microbiota after birth. Other inflammatory stimuli contribute in inducing high levels of immunoproteasome in pathological conditions, where its role deserve further attention., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Leukotriene C(4) is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C(4) synthase (LTC(4)S) participate in its biosynthesis. We report evidence from insitu hybridization experiments that FLAP mRNA is abundantly expressed in fetal mouse liver from e11.5 until delivery. In contrast very little or no FLAP mRNA was detected in adult liver. The fetal expression in liver was not uniform but occurred in patches. Cells from e15.5 livers were fractionated by fluorescence activated cell sorting into hepatocytes and other CD45(-) cells and CD45(+) hematopoietic cells. The latter were further separated into immature (Lin(-)) and mature (Lin(+)) cells and analyzed for FLAP mRNA content by quantitative RT-PCR. FLAP mRNA expression was confined to CD45(+) cells and the mature cells had approximately 4-fold higher FLAP mRNA levels compared to the immature cells.

We recently reported the expression and activity of several fatty acid oxidation enzymes in human embryonic and fetal tissues including brain and spinal cord. Liver and heart showed expression of both very long-chain acyl-CoA dehydrogenase (VLCAD) and long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) mRNA. However, while mRNA expression of LCHAD could be clearly detected in the retina and spinal cord, expression of VLCAD mRNA was low to undetectable in these tissues. Nevertheless, abundant acyl-CoA dehydrogenase (ACAD) activity was detected with palmitoyl-CoA as substrate in fetal central nervous tissue. These conflicting data suggested the presence of a different long-chain ACAD in human embryonic and fetal brain. In this study, using in situ hybridization as well as enzymatic studies, we identified acyl-CoA dehydrogenase 9 (ACAD 9) as the long-chain ACAD in human embryonic and fetal central nervous tissue. Until now, no clinical signs and symptoms of central nervous system involvement have been reported in VLCAD deficiency. A novel long-chain FAO defect, i.e., ACAD 9 deficiency with only central nervous system involvement, could, if not lethal during intra uterine development, easily escape proper diagnosis, since probably no classical signs and symptoms of FAO deficiency will be observed. Screening for ACAD 9 deficiency in patients with undefined neurological symptoms and/or impairment in neurological development of unknown origin is necessary to establish if ACAD 9 deficiency exists as a separate disease entity.

In an E. coli expression system for human cytochrome P450 3A7 (CYP3A7), holo-CYP3A7 was not expressed as judged by CO-difference spectra, although apo-CYP3A7 was clearly detected by Western blot analysis. Unlike CYP3A7, CYP3A4 was expressed efficiently as a hemoprotein in E. coli transformed with a CYP3A4 expression plasmid. To achieve the high yield of the holo-CYP3A7 in E. coli, we examined a causal residue(s) preventing the expression of the holo-CYP3A7 using the chimeric gene of CYP3A4 with CYP3A7. It was found that the region between residues 405 and 503 of CYP3A7 was responsible for the prevention of the holo-CYP3A7 expression in E. coli. Among amino acids examined, substitution of Thr at position 485 in CYP3A7 with Pro, which is at the corresponding position of CYP3A4, resulted in an increase in the amount of holo-CYP3A7. The Thr residue was adjacent to the heme-binding region of CYP3A7. Thus, it appeared that the incorporation of heme into CYP3A7 was possibly affected by this particular amino acid residue. Moreover, holo-CYP3A7 was expressed efficiently when CYP3A7 was co-expressed with molecular chaperone GroEL, known to assist the correct folding of unfolded proteins. Dehydroepiandrosterone 16alpha-hydroxylation was catalyzed by CYP3A7 expressed in the presence of GroEL., (Copyright 2000 Academic Press.)

PCR amplifications with two sets of degenerate primers that were targeted to CYP26-specific regions were performed with cDNAs from human fetal liver and brain as templates. PCR products were purified, cloned, sequenced and analyzed with the BLAST program. Our results revealed expression of CYP26 in both human fetal liver and brain. Furthermore, human fetal CYP26 cDNA exhibited 99.2%-100% nucleotide sequence identity to its adult counterpart. Novel isoforms, that would have indicated additional CYP26 genes, were not found. A Northern blot containing poly(A+)RNAs from 43 human adult and 7 human fetal tissues was tested for CYP26 expression. We were able to detect CYP26 message in most tissues but hybridization signals varied in intensity. Highest levels of transcription were in adult liver, heart, pituitary gland, adrenal gland, placenta and regions of the brain. CYP26 expression in fetal tissues was strongest in the brain and comparable with message levels in adult tissues., (Copyright 1998 Academic Press.)

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in signal transduction. In the present study we examined developmental and aging changes in three PLC isozymes (beta 1, gamma 1, and delta 1) in the rat brain. Enzyme assays and immunoblot analyses after gel filtration chromatography of brain extracts from embryonic day 19 and postnatal 4- and 48-week rats indicated that gamma-specific activity was highest in fetal brain and decreased with aging, that beta 1-specific activity was high at 4 weeks but essentially undetected in fetal brain, and that delta 1-specific activity was high at both 4 and 48 weeks with faint detection in fetal brain. Our results suggest that the gamma 1 isozyme may be particularly involved in cell division and growth during the histo-genesis of the central nervous system, while beta 1 and delta 1 isozymes may take part in processes of its maturation and maintenance.

Androgens play a key role in human fetal development. Therefore, it is important to understand the distribution of the isozymes of 5 alpha-reductase, the enzyme that converts testosterone to the more potent androgen, dihydrotestosterone. The expression of the two isozymes of 5 alpha-reductase was studied in human fetal skin and fetal prostate by measuring the in vitro enzyme activity in crude preparations. Low levels of the type 1 5 alpha-reductase were found in fetal scalp and back skin. Studies with fetal prostate samples confirmed that the type 2 enzyme is expressed in levels similar to those found in adult tissues. These results provide the first evidence that both isozymes of 5 alpha-reductase are expressed during human fetal development.

The overt activity of hepatic carnitine palmitoyltransferase (CPT1) increased during the last day of gestation in the foetus and after prolonged starvation in the newborn kept at 37 degrees C. Its sensitivity to inhibition by malonyl-CoA decreased during the perinatal period studied. Brown fat CPT1 increased under the same experimental conditions. However, its sensitivity to malonyl-CoA remains unchanged. Hypothermia at 24 degrees C decreased in the liver and increased in brown adipose tissue CPT1 activity in response to fasting. Glucose injection at birth decreased CPT1 activity in the liver but did not have any effect in the presence of mannoheptulose. This effect of glucose was non-significant in brown adipose tissue.

3 specific cDNA clones for human aldolase A were isolated from a human muscle library. One of them was subcloned in M 13 phage, then used as a probe to investigate the patterns and the levels of aldolase A mRNA in various human tissues. Two mRNA species differing in length were observed. The lighter one -1550 bases- was found specific to skeletal muscle; its amount increased during muscle development. The heavier aldolase A mRNA -1650 bases- accounted for foetal and ubiquitous presence of aldolase A isozyme. The resurgence of aldolase A in hepatomas occurred through this latter mRNA species.

Rat fetuses of 20 days gestational age were treated in utero with the inhibitor of DNA methylation, 5-azacytidine. The liver enzyme tyrosine aminotransferase, normally expressed at very low levels until several hours after birth, was increased by the drug in the fetal livers after a lag period of about 9 hours, reaching a level 70-fold above control levels 18 hours after treatment. The high levels attained after 5-azacytidine treatment are comparable to those of glucocorticoid-treated adult livers, and were not further increased by administration of hydrocortisone to dams carrying treated fetuses. Cytidine and two other analogs, cytosine arabinoside and 6-azacytidine, were essentially without effect.

Glycogen synthase first appears in significant levels in fetal rat liver on day 18 of gestation (22 day term), but in a mostly inactive, phospho- form. This was reflected in a decreased affinity for its allosteric activator (increased A0.5 for glucose-6-phosphate), and with limited glycogen synthesis and accumulation. Peak glycogen synthesis occurs on day 21 of gestation, and glycogen synthase is in a more dephospho-form with decreased A0.5 for G6P and increased -G6P/+G6P activity ratio. The data suggest that a significant change in phosphorylation state of synthase occurs in late gestation to facilitate the large increase in glycogen synthesis and accumulation.

The characteristic of arylhydrocarbon hydroxylase system in fetal liver microsomes of rat was investigated. NADH-synergistic effect on NADPH-dependent arylhydrocarbon hydroxylase was observed in fetal liver microsomes of rat but not in maternal liver microsomes. NADH-synergistic effect decreased in parallel with the decrease of the ratio of cytochrome b5/cytochrome P-450 in liver microsomes. The cytochrome P-450 in arylhydrocarbon hydroxylase system in fetal liver microsomes of rat seemed to be different from that in offspring liver microsomes in respect of its dependency on cytochrome b5 system for its maximum activity.

Fetal-rat liver explants cultured in a defined protein-free medium containing dexamethasone, corticosterone or cortisol (all 2 microM) exhibit precocious development of UDPglucuronyltransferase activity to o-aminophenol. Transferase activity in 14-day fetal livers cultured with the glucocorticoids for 3 days rises from virtually zero to 5 times the activity seen in fresh 17 day fetal liver. With 15-day fetal livers, precocity was also observed, but to a lesser degree. Precocity always required addition of glucocorticoids, though explants were viable without them. Protein synthesis, not activation, was probably involved, for assays were performed in a range of digitonin concentrations to ensure 'optimal' activation; also, precocious development of transferase activity and uptake of [14C]-leucine into protein exhibited parallel behaviour during inhibition by, and recovery from, cycloheximide-pulsing. This is the first demonstration of a protein-synthesis-dependent stimulation of fetal mammalian UDPglucuronyltransferase by known compounds of endogenous origin. Results with other substrates are discussed.

P-450 HFLa is a form of cytochrome P-450 purified from human fetal livers. The amounts of P-450 HFLa in several fetal tissues were determined immunochemically. Detectable amounts presented in livers, kidneys, adrenals, lungs and some other tissues of human fetuses. The amounts were the highest in livers. Activities of 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase in livers but not in adrenals were inhibited by the anti-P-450 HFLa antibodies, probably suggesting that distinct forms of cytochrome P-450 are responsible for the oxidations in livers and adrenals.

Phenylalanine hydroxylase from adult and foetal livers was purified by single step monoclonal antibody affinity chromatography. From adult and foetal livers, about 1280- and 1450-fold purified enzymes were obtained with 37% and 23% yield, respectively. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the resultant adult enzyme showed an essentially single band with an apparent molecular weight of 49K. On the other hand, two subunits (molecular weights 52K and 49K) were observed from the foetal enzyme. Molecular weights of the native adult and foetal enzymes as determined on Sepharose CL-6B column chromatogram were 150K and 160K, respectively. It was clear that adult and foetal liver phenylalanine hydroxylases were different proteins having different subunit molecular weights.

Two molecular forms of the catalytic subunit of the Na,K-ATPase can be isolated from brain (1). Only the alpha form was detected in rat embryo brain at 13 days of gestation (E13). The alpha (+) form, which is characteristic of myelinated axons, appeared at E15 before myelination begins. Hence its expression is not dependent on prior myelination. Axonal transport of the alpha (+) form was demonstrated in 4 day-old rats. The ratio of alpha (+): alpha was 1:1 in adult retina, cortex and cerebellum and 10:1 in brain stem. Although alpha (+) is characteristic of myelinated axons, this regional difference was present not only in enzyme extracted from crude microsomes, that contain myelinated axon fragments, but also in enzyme from isolated synaptosomes. Hence, the alpha (+): alpha ratio is an inherent characteristic of the neuron and does not depend on regional differences in myelination.