Recombinant uracil phosphoribosyltransferase from the thermophile Bacillus caldolyticus: expression, purification, and partial characterization

The hydrophobic surfactant protein C (SP-C) is known to modulate the biophysical properties of surfactant phospholipid. Although SP-C mRNA has been demonstrated in human fetal lung, there is limited information regarding developmental expression and processing of proSP-C protein. Two epitope-specific human proSP-C antisera, anti-hCPROSP-C (His59-Ser72) and anti-hCTERMSP-C (Gly162-Gly175), were generated to complement previously produced anti-NPROSP-C (Met10-Gln23) for the study of proSP-C expression in human fetal lung. Western blotting and immunocytochemistry detected expression of proSP-C protein by 12-16 wk of gestation. ProSP-C immunoreactivity of preculture lung, limited to expression of proSP-C21 in airway epithelial cells, was markedly enhanced by culture of lung explants in dexamethasone. To examine synthesis of proSP-C, homogenates from explants were labeled with 35S-Met/Cys for 0.5-4 h. Immunoprecipitation with anti-NPROSP-C detected 35S-proSP-C21 by 30 min and, after 2 h of labeling, there was a 15-fold increase in 35S-proSP-C21 in dexamethasone-treated lungs versus controls. Synthesis of proSP-C21 was followed by the appearance of a 24-kD form and smaller processing intermediates including 6-10-kD forms. Posttranslational processing of proSP-C21 was not observed in control explants. SP-C(6-10) were not recognized by either anti-CPROSP-C or anti-hCTERMSP-C. These results indicate that low level expression of proSP-C protein first occurs in epithelial cells early in the second trimester and that expression can be enhanced by dexamethasone. Initial posttranslational processing of human proSP-C involves modification of proSP-C21 to SP-C24 and subsequent proteolysis of C-terminal propeptide domains. We speculate that absence of low Mr intermediates in unstimulated second trimester fetal lung tissue reflects developmental and glucocorticoid dependent regulation of proSP-C21 synthesis and posttranslational processing.     

Elevated free fatty acids induce uncoupling protein 3 expression in muscle: a potential explanation for the effect of fasting

The newly described uncoupling protein 3 (UCP3) may make an important contribution to thermogenesis in humans because of its high level of expression in skeletal muscle. Contrary to expectations, fasting, a condition that reduces resting energy expenditure, has been reported to increase UCP3 expression in muscle. We have confirmed that a 10-fold increase in UCP3 mRNA levels occurs in rat quadriceps muscle between 12 and 24 h of food removal. A less consistent twofold increase in muscle UCP2 mRNA levels was observed in animals fasted for up to 72 h. Administration of recombinant leptin to prevent a fall in circulating leptin levels did not eliminate the fasting-induced increase in quadriceps UCP3 expression. Administration of a high dose of glucocorticoid to fed animals to mimic the increase in corticosterone induced by fasting did not reproduce the increase in UCP3 expression observed in fasted animals. In contrast, elevation of circulating free fatty acid levels in fed animals by Intralipid plus heparin infusion caused significant increases in the UCP3/actin mRNA ratio compared with saline-infused fed controls in both extensor digitorum longus (2.01 +/- 0.34 vs. 0.68 +/- 0.11, P = 0.002) and soleus muscles (0.31 +/- 0.07 vs. 0.09 +/- 0.02, P = 0.014). We conclude that free fatty acids are a potential mediator of the increase in muscle UCP3 expression that occurs during fasting. This seemingly paradoxical induction of UCP3 may be linked to the use of free fatty acid as a fuel rather than an increased need of the organism to dissipate energy.     

Pharmacological inhibitors of mammalian fatty acid synthase suppress DNA replication and induce apoptosis in tumor cell lines

Pharmacological inhibitors of the anabolic enzyme, fatty acid synthase (FAS), including the natural product cerulenin and the novel compound c75, are selectively cytotoxic to cancer cells via induction of apoptosis, apparently related to the tumor cell phenotype of abnormally elevated fatty acid synthetic metabolism. As part of a larger effort to understand the immediate downstream effect of FAS inhibition that leads to apoptosis, the effects of these inhibitors on cell cycle progression were examined. Both FAS inhibitors produce rapid, profound inhibition of DNA replication and S phase progression in human cancer cells. The dose responses for fatty acid synthesis inhibition and DNA synthesis inhibition are similar. The kinetics of both effects are rapid, with fatty acid synthesis inhibition occurring within 30 min and DNA synthesis inhibition occurring within 90 min of drug exposure. Meanwhile, apoptotic changes are not detected until 6 h or later after inhibitor exposure. Fatty acid synthetic pathway activity and the magnitude of DNA synthesis inhibition by FAS inhibitors are increased in parallel by withdrawal of lipid-containing serum from the cultures. The mechanism of DNA synthesis inhibition by cerulenin is indirect, because expression of certain viral oncogenes rescues DNA synthesis/S phase progression in cerulenin-exposed cells. The data suggest a direct linkage at a regulatory level, between fatty acid synthesis and DNA synthesis in proliferating tumor cells.     

Purified soluble guanylyl cyclase expressed in a baculovirus/Sf9 system: stimulation by YC-1, nitric oxide, and carbon monoxide

Neonatal respiratory function depends on the development of a well-formed pulmonary capillary bed. Vascular endothelial growth factor (VEGF) is a potent inducer of endothelial cell growth and angiogenesis. High levels of VEGF protein and messenger RNA (mRNA) have been detected in the developing lung, suggesting that VEGF plays a role in the development of the pulmonary capillary bed. To begin to understand the role of VEGF in human lung development, we explored the regulation of VEGF gene expression and the localization of VEGF protein and mRNA in a model of the developing human lung. VEGF protein and mRNA were detected in midtrimester human fetal lung tissue, and their levels increased with time in explant culture. VEGF protein and mRNA were increased by the maintenance of human fetal lung explants in 2% O2 environments compared with 20% O2 environments. VEGF mRNA levels were found to be increased by cyclic adenosine monophosphate (cAMP) in explants that were incubated in 20% O2, but not in those incubated in 2% O2. Immunostaining for VEGF protein demonstrated localization primarily in airway epithelial cells in midtrimester human fetal lung tissue. Immunostaining for VEGF increased with incubation of human fetal lung explants in 2% and 20% O2. Interestingly, VEGF protein was localized primarily in the basement membrane subjacent to airway epithelial cells after 4 d of incubation in 20% O2. Incubation of tissues in the presence of dibutyryl cAMP resulted in an increase in immunostaining for VEGF, primarily in the basement membranes of prealveolar ducts in 20% O2-treated tissues. In situ hybridization studies indicated that VEGF mRNA was present in both mesenchymal cells and airway epithelial cells. These data suggest that VEGF gene expression is regulated by both oxygen and cAMP in the developing human lung. The detection of VEGF mRNA and protein in distal airway epithelial cells and the detection of VEGF protein in the basement membrane subjacent to the airway epithelial cells suggest that translocation of VEGF protein occurs after its synthesis in the epithelium. Localization of VEGF to the basement membrane of airway epithelial cells may be important for directing capillary development in the human lung.     

Always single and single again women: a qualitative study

This study reports the effects of a novel polyunsaturated 3-thia fatty acid, methyl 3-thiaoctadeca-6,9,12,15-tetraenoate on serum lipids and key enzymes in hepatic fatty acid metabolism compared to a saturated 3-thia fatty acid, tetradecylthioacetic acid. Palmitic acid treated rats served as controls. Fatty acids were administered by gavage in daily doses of 150 mg/kg body weight for 10 days. The aim of the present study was: (a) To investigate the effect of a polyunsaturated 3-thia fatty acid ester, methyl 3-thiaoctadeca-6,9,12,15-tetraenoate on plasma lipids in normolipidemic rats: (b) to verify whether the lipid-lowering effect could be consistent with enhanced fatty acid oxidation: and (c) to study whether decreased activity of esterifying enzymes and diversion to phospholipid synthesis is a concerted mechanism in limiting the availability of free fatty acid as a substrate for hepatic triglyceride formation. Repeated administration of the polyunsaturated 3-thia fatty acid ester for 10 days resulted in a reduction of plasma triglycerides (40%), cholesterol (33%) and phospholipids (20%) compared to controls. Administration of polyunsaturated and saturated 3-thia fatty acids (daily doses of 150 mg/kg body weight) reduced levels of lipids to a similar extent and followed about the same time-course. Both mitochondrial and peroxisomal fatty acid oxidation increased (1.4-fold- and 4.2-fold, respectively) and significantly increased activities of carnitine palmitoyltransferase (CPT) (1.6-fold), 2,4-dienoyl-CoA reductase (1.2-fold) and fatty acyl-CoA oxidase (3.0-fold) were observed in polyunsaturated 3-thia fatty acid treated animals. This was accompanied by increased CPT-II mRNA (1.7-fold). 2,4-dienoyl-CoA reductase mRNA (2.9-fold) and fatty acyl-CoA oxidase mRNA (1.7-fold). Compared to controls, the hepatic triglyceride biosynthesis was retarded as indicated by a decrease in liver triglyceride content (40%). The activities of glycerophosphate acyltransferase, acyl-CoA: 1,2-diacylglycerol acyltransferase and CTP:phosphocholine cytidylyltransferase were increased. The cholesterol lowering effect was accompanied by a reduction in HMG-CoA reductase activity (80%) and acyl-CoA:cholesterol acyltransferase activity (33%). In hepatocytes treated with methyl 3-thiaoctadeca-6,9,12,15-tetraenoate, fatty acid oxidation was increased 1.8-fold compared to controls. The results suggest that treatment with methyl 3-thiaoctadeca-6,9,12,15-tetraenoate reduces plasma triglycerides by a decrease in the availability of fatty acid substrate for triglyceride biosynthesis via enhanced fatty acid oxidation, most likely attributed to the mitochondrial fatty acid oxidation. It is hypothesized that decreased phosphatidate phosphohydrolase activity may be an additive mechanism which contribute whereby 3-thia fatty acids reduce triglyceride formation in the liver. The cholesterol-lowering effect of the polyunsaturated 3-thia fatty acid ester may be due to changes in cholesterol/cholesterol ester synthesis as 60% of this acid was observed in the hepatic cholesterol ester fraction.     

Pulmonary surfactant concentration during transition from high frequency oscillation to conventional mechanical ventilation

OBJECTIVE: To test the hypothesis that conventional mechanical ventilation (CV) provides a greater stimulus to secretion of pulmonary surfactant than high frequency oscillatory ventilation (HFO). METHODOLOGY: Sequential examination of surfactant indices in lung lavage fluid in a group of six infants with severe lung disease (group 1), ventilated with HFO and then converted back to CV as their lung disease recovered. A similar group of 10 infants (group 2) ventilated conventionally throughout the course of their illness were studied for comparison. In groups 1 and 2, two sequential tracheal aspirate samples were taken, the first once lung disease was noted to be improving, and the second 48-72 h later. Group 1 infants had converted from HFO to CV during this time. RESULTS: A marked increase in concentration of total surfactant phospholipid (PL) and disaturated phosphatidylcholine (DSPC) was seen in group 1 after transition from HFO to CV; the magnitude of this increase was significantly greater than that sequentially observed in group II (total PL: 9.4-fold increase in group 1 vs 1.8-fold in group 2, P = 0.006; DSPC: group 1 6.4-fold increase vs. group 2 1.7-fold, P = 0.02). CONCLUSION: These findings suggest that intermittent lung inflation during CV produces more secretion of surfactant phospholipid than continuous alveolar distension on HFO, and raise the possibility that conservation and additional maturation of surfactant elements may occur when the injured lung is ventilated with HFO.     

The Saccharomyces cerevisiae FAT1 gene encodes an acyl-CoA synthetase that is required for maintenance of very long chain fatty acid levels

The Saccharomyces cerevisiae FAT1 gene appears to encode an acyl-CoA synthetase that is involved in the regulation of very long chain (C20-C26) fatty acids. Fat1p, has homology to a rat peroxisomal very long chain fatty acyl-CoA synthetase. Very long chain acyl-CoA synthetase activity is reduced in strains containing a disrupted FAT1 gene and is increased when FAT1 is expressed in insect cells under control of a baculovirus promoter. Fat1p accounts for approximately 90% of the C24-specific acyl-CoA synthetase activity in glucose-grown cells and approximately 66% of the total activity in cells grown under peroxisomal induction conditions. Localization of functional Fat1p:green fluorescent protein gene fusions and subcellular fractionation of C24 acyl-CoA synthetase activities indicate that the majority of Fat1p is located in internal cellular locations. Disruption of the FAT1 gene results in the accumulation of very long chain fatty acids in the sphingolipid and phospholipid fractions. This includes a 10-fold increase in C24 acids and a 6-fold increase in C22 acids. These abnormal accumulations are further increased by perturbation of very long chain fatty acid synthesis. Overexpression of Elo2p, a component of the fatty acid elongation system, in fat1Delta cells causes C20-C26 levels to rise to approximately 20% of the total fatty acids. These data suggest that Fat1p is involved in the maintenance of cellular very long chain fatty acid levels, apparently by facilitating beta-oxidation of excess intermediate length (C20-C24) species. Although fat1Delta cells were reported to grow poorly in oleic acid-supplemented medium when fatty acid synthase activity is inactivated by cerulenin, fatty acid import is not significantly affected in cells containing disrupted alleles of FAT1 and FAS2 (a subunit of fatty acid synthase). These results suggest that the primary cause of the growth-defective phenotype is a failure to metabolize the incorporated fatty acid rather than a defect in fatty acid transport. Certain fatty acyl-CoA synthetase activities, however, do appear to be essential for bulk fatty acid transport in Saccharomyces. Simultaneous disruption of FAA1 and FAA4, which encode long chain (C14-C18) fatty acyl-CoA synthetases, effectively blocks the import of long chain saturated and unsaturated fatty acids.     

Hypothalamic/pituitary-axis of the spontaneous dwarf rat: autofeedback regulation of growth hormone (GH) includes suppression of GH releasing-hormone receptor messenger ribonucleic acid

In this study, the spontaneous dwarf rat (SDR) has been used to examine GHRH production and action in the selective absence of endogenous GH. This dwarf model is unique in that GH is not produced because of a point mutation in the GH gene. However, other pituitary hormones are not obviously compromised. Examination of the hypothalamic pituitary-axis of SDRs revealed that GHRH messenger RNA (mRNA) levels were increased, whereas somatostatin (SS) and neuropeptide Y (NPY) mRNA levels were decreased, compared with age- and sex-matched normal controls, as determined by Northern blot analysis (n = 5 animals/group; P < 0.05). The elevated levels of GHRH mRNA in the SDR hypothalamus were accompanied by a 56% increase in pituitary GHRH receptor (GHRH-R) mRNA, as determined by RT-PCR (P < 0.05). To investigate whether the up-regulation of GHRH-R mRNA resulted in an increase in GHRH-R function, SDR and control pituitary cell cultures were challenged with GHRH (0.001-10 nM; 15 min), and intracellular cAMP concentrations were measured by RIA. Interestingly, SDR pituitary cells were hyperresponsive to 1 and 10 nM GHRH, which induced a rise in intracellular cAMP concentrations 50% greater than that observed in control cultures (n = 3 separate experiments; P < 0.05 and P < 0.01, respectively). Replacement of GH, by osmotic minipump (10 microg/h for 72 h), resulted in the suppression of GHRH mRNA levels (P < 0.01), whereas SS and NPY mRNA levels were increased (P < 0.05), compared with vehicle-treated controls (n = 5 animals/treatment group). Consonant with the fall in hypothalamic GHRH mRNA was a decrease in pituitary GHRH-R mRNA levels. Although replacement of insulin-like growth factor-I (IGF-I), by osmotic pump (5 microg/h for 72 h), resulted in a rise in circulating IGF-I concentrations comparable with that observed after GH replacement, IGF-I treatment was ineffective in modulating GHRH, SS, or NPY mRNA levels. However, IGF-I treatment did reduce pituitary GHRH-R mRNA levels, compared with vehicle-treated controls (P < 0.05). These results further validate the role of GH as a negative regulator of hypothalamic GHRH expression, and they suggest that SS and NPY act as intermediaries in GH-induced suppression of hypothalamic GHRH synthesis. These data also demonstrate that increases in circulating IGF-I are not responsible for changes in hypothalamic function observed after GH treatment. Finally, this report establishes modulation of GHRH-R synthesis as a component of GH autofeedback regulation.     

Thrombolytic and antiplatelet action of xanthinol nicotinate (Sadamin): possible mechanisms

Primiparous Holstein cows received recombinant bovine growth hormone (bGH), bovine growth hormone-releasing factor (bGRF), or no treatment from 118 to 181 +/- 1 d. Milk yield was significantly increased with no change in milk fat percentage or composition. The mRNA and protein abundance of the key lipogenic enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) were measured in the mammary gland and adipose tissue. We hypothesized that bGH and bGRF treatment would increase the mRNA and protein abundance of ACC and FAS in the mammary gland, with an associated decrease in adipose tissue. Analysis of ACC mRNA and protein abundance in the mammary gland revealed that there was no significant influence of either bGH or bGRF treatment. Analysis of FAS mRNA in mammary gland revealed that both bGH and bGRF significantly increased the abundance. However, quantitation of FAS protein in the mammary gland revealed that neither treatment resulted in increased abundance. In adipose tissue, the mRNA and protein abundance of both ACC and FAS were significantly reduced. The increased substrate required for increased milk fatty acid yield may be provided through redirection of nutrients to the mammary gland away from adipose tissue and through overall increased metabolism of the mammary gland.     

Effects of maturation on the phospholipid and phospholipid fatty acid compositions in primary rat cortical astrocyte cell cultures

Phospholipid and phospholipid fatty acid compositional changes were studied in rat cortical astrocytes during dibutyryl cyclic adenosine monophosphate (dBcAMP, 0.25 mM) treatment starting after 14 days in culture (DIC). After 15 DIC, ethanolamine- and choline glycerophospholipid levels were increased 1.2- and 1.3-fold, respectively in treated compared to control cells. However, after 21 and 28 DIC, these levels were not significantly different between groups. Both groups had an increase in phosphatidylserine levels with increasing time in culture. Similarly, ethanolamine plasmalogen levels were transiently elevated after 21 DIC, but returned to previous levels after 28 DIC. The phospholipid fatty acid compositions for the acid stable and labile ethanolamine- and choline glycerophospholipids indicated that in dBcAMP treated cells, 20:4 n-6 and 22:6 n-3 proportions were elevated with increasing time in culture relative to control cells. As 20:4 n-6 proportions increased, there was a concomitant decrease in 20:3 n-9 proportions, suggesting an up regulation of n-6 series elongation and desaturation. In contrast, in control cells, the 20:4 n-6 proportions decreased with a corresponding increase in the 20:3 n-9 proportions. Thus, in treated cells, the cellular phospholipid fatty acid composition was dramatically different than control cells, suggesting that dBcAMP treatment may act to increase fatty acid elongation and desaturation.     

Lipid balance in HepG2 cells: active synthesis and impaired mobilization

The human hepatoma cell line HepG2 in culture medium synthesized fatty acids de novo (144 +/- 9 nmol fatty acid/mg protein per 24 h) at a rate similar to that observed in freshly prepared rat hepatocytes (192 +/- 8 nmol/mg per 24 h) and in primary cultures of rat hepatocytes (165.4 +/- 29.3 nmol/mg per 24 h). In HepG2 cells, fatty acid synthesis was inhibited by extracellular oleate (0.75 mM), and, to a lesser extent, by glucagon (10(-7) M). Insulin (7.8 x 10(-8) M) had a mild stimulatory effect. Fatty acid synthesis was not influenced by lipogenic precursors (lactate plus pyruvate), substances which, in rat hepatocytes, had pronounced stimulatory effects. Fatty acid synthesis rates were also unchanged in the presence of prostaglandin E2 (PGE2). In general, compared to rat hepatocytes, fatty acid synthesis in HepG2 cells was less sensitive to manipulation of the culture medium in vitro. HepG2 cells had a high capacity for triacylglycerol synthesis from extracellular oleate (469 +/- 43 nmol triacylglycerol/mg protein per 24 h) but phospholipid synthesis was relatively low (15.8 +/- 0.4% of total glycerolipids). Very little of the above newly synthesized triacylglycerol was secreted as lipoprotein (4.62 +/- 0.88 nmol triacylglycerol/mg protein per 24 h) resulting in a large intracellular accumulation of triacylglycerol. This was exacerbated by the absence of any detectable ketogenesis. The secretion of triacylglycerol produced from de novo synthesized fatty acids was also very low in HepG2 compared to that observed in primary cultures of rat hapatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiotensin-II stimulates an increase in cAMP and expression of 17 alpha-hydroxylase cytochrome P450 in fetal bovine adrenocortical cells

While known to be a potent activator of phosphoinositidase C, angiotensin II (A-II) also causes a small but significant increase in cAMP production through the type 1 A-II (AT1) receptor in bovine adrenocortical cells (Mol Cell Endocrinol 81:33-41, 1991). We have carried out studies on primary cultures of fetal bovine adrenocortical cells to examine the effects of A-II on the expression of cytochrome P450 17 alpha-hydroxylase (P450c17), which is known to be regulated in a cAMP-dependent fashion. Prolonged treatment (48 h) of cells with A-II (10(-7) M) did not give rise to a detectable increase in P450c17 as measured by immunoblotting, although both A-II and the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (TPA) attenuated the large increase in P450c17 induced by ACTH (10(-8) M). A-II alone (10(-7) M) however, caused a time-dependent increase in cAMP secretion, reaching 8-fold within 3 h. Prolonged treatment of cells with A-II also resulted in a 3-fold increase in P450c17 mRNA within 12 h (10(-7) M), and a dose-dependent increase in 17 alpha-hydroxylase activity within 48 h (16.4-fold max at 10(-7) M). The stimulatory actions of A-II alone (10(-7) M) on cAMP levels, P450c17 mRNA, and 17 alpha-hydroxylase activity were much smaller than in response to ACTH (10(-8) M), but were largely reproduced by TPA (10(-7) M), suggesting a role for protein kinase C in mediating these responses to A-II. These findings indirectly support the hypothesis that A-II alone can stimulate an increase in cAMP in adrenocortical cells. Such a stimulation of cAMP may then result in increased expression of steroidogenic enzymes, as we have shown is the case for P450c17 expression. However, A-II in the presence of ACTH appears to attenuate the ACTH-stimulated expression of P450c17.