Effect of prostaglandins on α-aminoisobutylic acid uptake in cultured fibroblasts

Effect of various prostaglandins on the uptake of α-aminoisobutylic acid by cultured fibroblasts was studied. All the prostaglandins having an OH functional group in an intramolecular 5-membered ring showed an inhibitory effect on the amino acid uptake. The active compounds can be ranked in potency according to the values for the inhibition of the amino acid uptake per cent of control: prostaglandin F_2α(53 %) >F_1α(54 %) >D₂(56 %) >E₂(62 %) >thromboxane B₂ (66 %). Thus, prostaglandin F_2α was found to be the most potent inhibitor to membrane permeability and the inhibitory effect was dose dependent. The inhibition was maximal after 1 hour of exposure to prostaglandin F_2α, persisted at least up to 6 hours in the presence of prostaglandin F_2α.     

Phytanic acid α-oxidation in human cultured skin fibroblasts

We studied the oxidation of [1-¹⁴C]phytanic acid, 3-methyl substituted fatty acid, to pristanic acid and ¹⁴CO₂ in human skin fibroblasts. The specific activity for α-oxidation of phytanic acid in peroxisomes was 29- and 124-fold higher than mitochondria and endoplasmic reticulum. This finding demonstrates for the first time the presence of fatty acid α-oxidation enzyme system in peroxisomes.     

Properties of α-l-iduronidase in cultured skin fibroblasts from α-l-iduronidase-deficient patients

Summary On DEAE cellulose column chromatography, -l-iduronidase in cultured skin fibroblasts was resolved into two distinct components, forms A and B. They had similar K_m values for 4-methylumbelliferyl--l-iduronide, but differed in pH optima and thermal stability. Form B was more heat-stable than form A.Residual -l-iduronidase activity in Hurler fibroblasts was heat-stable, while that in Scheie fibroblasts was heat-labile, and moreover, that in Hurler-Scheie compound fibroblasts lay intermediate between Hurler and Scheie syndromes. These findings demonstrated that Hurler syndrome, Scheie syndrome and Hurler-Scheie compound were enzymatically distinguishable.     

Metabolism of lysine in α-aminoadipic semialdehyde dehydrogenase-deficient fibroblasts: Evidence for an alternative pathway of pipecolic acid formation

The mammalian degradation of lysine is believed to proceed via two distinct routes, the saccharopine and the pipecolic acid routes, that ultimately converge at the level of α-aminoadipic semialdehyde (α-AASA). α-AASA dehydrogenase-deficient fibroblasts were grown in cell culture medium supplemented with either l-[α-¹⁵N]lysine or l-[ε-¹⁵N]lysine to explore the exact route of lysine degradation. l-[α-¹⁵N]lysine was catabolised into [¹⁵N]saccharopine, [¹⁵N]α-AASA, [¹⁵N]Δ¹-piperideine-6-carboxylate, and surprisingly in [¹⁵N]pipecolic acid, whereas l-[ε-¹⁵N]lysine resulted only in the formation of [¹⁵N]saccharopine. These results imply that lysine is exclusively degraded in fibroblasts via the saccharopine branch, and pipecolic acid originates from an alternative precursor. We hypothesize that pipecolic acid derives from Δ¹-piperideine-6-carboxylate by the action of Δ¹-pyrroline-5-carboxylic acid reductase, an enzyme involved in proline metabolism.     

Aggregated α-synuclein and complex I deficiency: exploration of their relationship in differentiated neurons

α-Synuclein becomes misfolded and aggregated upon damage by various factors, for example, by reactive oxygen species. These aggregated forms have been proposed to have differential toxicities and their interaction with mitochondria may cause dysfunction within this organelle that contributes to the pathogenesis of Parkinson''s disease (PD). In particular, the association of α-synuclein with mitochondria occurs through interaction with mitochondrial complex I and importantly defects of this protein have been linked to the pathogenesis of PD. Therefore, we investigated the relationship between aggregated α-synuclein and mitochondrial dysfunction, and the consequences of this interaction on cell survival. To do this, we studied the effects of α-synuclein on cybrid cell lines harbouring mutations in either mitochondrial complex I or IV. We found that aggregated α-synuclein inhibited mitochondrial complex I in control and complex IV-deficient cells. However, when aggregated α-synuclein was applied to complex I-deficient cells, there was no additional inhibition of mitochondrial function or increase in cell death. This would suggest that as complex I-deficient cells have already adapted to their mitochondrial defect, the subsequent toxic effects of α-synuclein are reduced.The pathological hallmark of Parkinson''s disease (PD) is the presence of α-synuclein aggregates, particularly within the substantia nigra (SN). These aggregations take the form of intracellular Lewy bodies, and also neuritic aggregations. However, both the effect of these inclusions on neuronal survival and the toxicity of different forms of α-synuclein are still debated. To aggregate α-synuclein must undergo a conformational change, however, the mechanism behind this change and subsequent aggregation in PD remains to be determined.Mutations within the α-synuclein gene (SNCA (MIM 163890)) were the first to be associated with autosomal dominant PD, while more recently genome-wide association studies have suggested that single-nucleotide polymorphisms in this gene are important for sporadic PD. A widely expressed protein α-synuclein is important for synaptic vesicle recycling and the modulation of dopamine transmission within SN neurons.^{1, 2, 3, 4, 5, 6, 7, 8} It interacts with curved cellular membranes including those of mitochondria suggesting a possible mode of its toxicity,^{9, 10, 11} and can be imported into mitochondria in an energy-dependent manner.⁹ The accumulation of α-synuclein within mitochondria leads to complex I impairment, decreased mitochondrial membrane potential (ΔΨ_m) and increased reactive oxygen species (ROS) production. The occurrence of these changes is also dependent on calcium homoeostasis.^{9, 12, 13}Mitochondrial dysfunction has also been heavily implicated in the pathogenesis of PD. Early studies showed a decrease in mitochondrial complex I in the SN of PD patients and studies involving the inhibition of this complex replicate many of the features of this disease. In addition, SN neurons show high levels of mitochondrial DNA deletions in old age,^{14, 15} which lead to respiratory deficiency, and the environment of the SN is believed to be particularly oxidative due to a number of processes, including the metabolism of dopamine. More recently a number of genes known to cause autosomal recessive forms of PD have been shown to encode proteins with functions associated with mitochondrial turnover (Parkin/Pink1 (MIM 602544, MIM 608309)) or oxidative stress (DJ-1 (MIM 602533)). However, the link between these two processes and the loss of dopaminergic neurons in PD remains to be elucidated.Several hypotheses have been suggested for what might cause α-synuclein to undergo the conformational change into more aggregate prone forms, from oxidative stress to gene mutations. Furthermore, the accumulation of mitochondrial DNA (mtDNA) mutations and dysfunctional mitochondria with advancing age are likely to have an effect on oxidative stress levels within the SN, which might contribute further to the misfolding and accumulation of this protein. Numerous studies have used rotenone and other toxins to induce mitochondrial dysfunction and monitor the accumulation of α-synuclein, despite the wealth of information that these studies provide they often do not reflect the subtleties of the slow accumulation of mitochondrial dysfunction within ageing SN neurons.Therefore, we investigated the relationship between mitochondria and aggregated α-synuclein, focussing on how these forms affect neurons with and without mitochondrial dysfunction. We wanted to understand how aggregated α-synuclein impacted on the survival of cells with mitochondrial dysfunction, to enable a deeper understanding of the effect of these two processes on neuronal survival. To investigate this we used cells with mutations in and partial inhibition of complexes I and IV.     

β-Galactosidase-neuraminidase deficiency in adults: Deficiency of a freeze-labile neuraminidase in leukocytes and fibroblasts

Summary 4-Methylumbelliferyl neuraminidase activity was studied in fibroblasts, leukocytes, and frozen tissues from adult patients with -galactosidase-neuraminidase deficiency and specific clinical manifestations. This enzyme was almost completely deficient in fibroblasts, but the residual activity was relatively high (20% of the control mean) in the leukocytes from the patients. The frozen liver from one patient showed the enzyme activity as high as controls.This enzyme consisted of two components, freeze-labile and freeze-stable, and it was demonstrated that only the labile enzyme was deficient in fibroblasts and leukocytes. The apparently normal activity of neuraminidase in frozen autopsy tissues of a patient may be explained by the loss of the labile component in control tissues after a long-term freezing. The neuraminidase activity was variable in parents and no definite conclusion was drawn on the hereditary nature of the disease.     

Characterization of subfractions of β2-glycoprotein I: Evidence for sialic acid microheterogeneity

• 1.1. β₂-Glycoprotein I is a sialic acid microheterogeneous protein and contains on the average 11 mol sialic acid/mol.
• 2.2. Linear correlation was found between sialic acid content and pI of isolated subfractions.
• 3.3. Asialo-β₂2-glycoprotein I consists of 2 isoforms. Each of which can originate from the same subfraction.
• 4.4. The isolated subfractions exhibited almost the same amino acid composition.

     

Sjögren-Larsson syndrome: accumulation of free fatty alcohols in cultured fibroblasts and plasma

Sj?gren-Larsson syndrome (SLS) is an inherited disorder associated with deficient oxidation of long-chain aliphatic alcohols. Previous studies have reported modest elevations in total (free + esterified) fatty alcohols in SLS, but free fatty alcohols have not been selectively measured, in part because of their low concentrations in most tissues and the presence of trace fatty alcohol contaminants in some solvents used for their analysis. We adapted methods to measure free fatty alcohols in cultured cells and plasma that minimize exogenous alcohol contamination. Fatty alcohols were analyzed as acetate derivatives, using capillary column gas chromatography. By this method, cultured skin fibroblasts from SLS patients were found to have 7- and 8-fold elevations in the mean content of hexadecanol (16:0-OH) and octadecanol (18:0-OH), respectively. The mean plasma 16:0-OH and 18:0-OH concentrations in SLS patients (n = 11) were 9- and 22-fold higher than in normal controls, respectively. In SLS fibroblasts, most of the fatty alcohol (59%) that accumulated was free rather than esterified alcohol, whereas free alcohol accounted for 23% of the total alcohol in normal cells. These results indicate that elevations in free fatty alcohols provide a sensitive marker for the enzymatic defect in SLS. The ability to measure free fatty alcohols in cultured cells and plasma should prove useful for investigations of normal fatty alcohol metabolism and the deranged metabolism in SLS.     

Metabolism of linoleic and α-linolenic acids in cultured cardiomyocytes: Effect of different N-6 and N-3 fatty acid supplementation

The metabolites of linoleic (LA) and -linolenic (ALA) acids are involved in coronary heart disease. Both n-6 and n-3 essential fatty acids (EFAs) are likely to be important in prevention of atherosclerosis since the common risk factors are associated with their reduced 6-desaturation. We previously demonstrated the ability of heart tissue to desaturate LA. In this study we examined the ability of cultured cardiomyocytes to metabolize both LA and ALA in vivo, in the absence and in the presence of gamma linolenic acid (GLA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) alone or combined together. In control conditions, about 25% of LA and about 90% of ALA were converted in PUFAs. GLA supplementation had no influence on LA conversion to more unsaturated fatty acids, while the addition of n-3 fatty acids, alone or combined together, significantly decreased the formation of interconversion products from LA. Using the combination of n-6 and n-3 PUFAs, GLA seemed to counterbalance partially the inhibitory effect of EPA and DHA on LA desaturation/elongation. The conversion of ALA to more unsaturated metabolites was greatly affected by GLA supplementation. Each supplemented fatty acid was incorporated to a significant extent into cardiomyocyte lipids, as revealed by gas chromatographic analysis. The n-6/n-3 fatty acid ratio was greatly influenced by the different supplementations; the ratio in GLA+EPA+DHA supplemented cardiomyocytes was the most similar to that recorded in control cardiomyocytes. Since important risk factors for coronary disease may be associated with reduced 6-desaturation of the parent EFAs, administration of n-6 or n-3 EFA metabolites alone could cause undesirable effects. Since they appear to have different and synergistic roles, only combined treatment with both n-6 and n-3 metabolites is likely to achieve optimum results.     

Phosphoenolpyruvate-dependent inhibition of collagen biosynthesis, α2β1 integrin and IGF-I receptor signaling in cultured fibroblasts

The mechanism of collagen biosynthesis regulation is not fully understood. The finding that prolidase plays an important role in collagen biosynthesis and phosphoenolpyruvate inhibits prolidase activity "in vitro" led to evaluate its effect on collagen biosynthesis in cultured human skin fibroblasts. Confluent fibroblasts were treated with millimolar concentrations (1-4 mM) of phosphoenolpyruvate monopotassium salt (PEP) for 24 h. It was found that PEP-dependent decrease in prolidase activity and expression was accompanied by parallel decrease in collagen biosynthesis. However, the experiments with inhibitor of PEP production, 3-mercaptopicolinate revealed no direct correlation between collagen biosynthesis and prolidase activity and expression. Since insulin-like growth factor (IGF-I) is the most potent stimulator of both collagen biosynthesis and prolidase activity, and prolidase is regulated by beta(1) integrin signaling, the effect of PEP on IGF-I receptor (IGF-IR) and beta(1) integrin receptor expressions were evaluated. It was found that the exposure of the cells to 4 mM PEP contributed to a decrease in IGF-IR and beta(1) integrin receptor expressions. The data suggest that PEP-dependent decrease of collagen biosynthesis in cultured human skin fibroblasts may undergo through depression of alpha(2)beta(1) integrin and IGF-IR signaling. The hypothetical mechanism of the role of prolidase in IGF-IR, beta(1) integrin receptor expressions, and clinical significance of the process are discussed.     

Novel formation of α-amino acid from α-oxo acids and ammonia in an aqueous medium

In the course of a study of possible mechanisms for chemical evolution in the primeval sea, we found the novel formation of -amino acids and N-acylamino acids from -oxo acids and ammonia in an aqueous medium. Glyoxylic acid reacted with ammonia to form N-oxalylglycine, which gave glycine in a 5–39% yield after hydrolysis with 6N HCl. Pyruvic acid and ammonia reacted to give N-acetylalanine, which formed alanine in a 3–7% overall yield upon hydrolysis. The pH optima in these reactions were between pH 3 and 4. These reactions were further extended to the formation of other amino acids. Glutamic acid, phenylalanine and alanine were formed from -ketoglutaric acid, phenylpyruvic acid and oxaloacetic acid, respectively, under similar conditions. N-Succinylglutamic acid was obtained as an intermediate in glutamic acid synthesis. Phenylacetylphenyl-alanineamide was also isolated as an intermediate in phenylalanine synthesis. Alanine, rather than aspartic acid, was produced from oxaloacetic acid. These reactions provide a novel route for the prebiotic synthesis of amino acids. A mechanism for the reactions will be proposed.     

Incorporation and degradation of GM1 ganglioside and asialoGm1 ganglioside in cultured fibroblasts from normal individuals and patients with β-galactosidase deficiency

The uptake and degradation of G_M1 ganglioside (G_M1) and asialoG_M1 ganglioside (G_A1) were studied in cultured fibroblasts from normal individuals and patients with β-galactosidase deficiency, using the lipid-loading test. The glycolipids were incorporated from the media into the fibroblasts and the terminal galactose was hydrolyzed in normal cells. The hydrolysis rates of G_A1 were 80–86% of normal on the 3rd day after loading, while G_M1 was hydrolyzed slowly; 35–54% on the 14th day. In infantile G_M1 gangliosidosis and I-cell disease, little G_M1 and G_A1 was hydrolyzed on any day of culture, while fibroblasts from patients with adult G_M1 gangliosidosis, Morquio disease type B and galactosialidosis hydrolyzed the lipids at nearly normal rates. The intracellular accumulation of the glycolipids, on the basis of protein content, was abnormally high in the case of infantile G_M1 gangliosidosis and I-cell disease, but normal in the other disorders examined. These observations indicate that the in situ metabolism of G_M1 and G_A1 is probably normal in fibroblasts from patients with adult G_M1 gangliosidosis, Morquio disease type B and galactosialidosis, although in vitro β-galactosidase activities in these disorders are very low. The results are compatible with findings that G_M1 and G_A1 do not accumulate in the somatic organs of patients with adult G_M1 gangliosidosis and galactosialidosis. In I-cell disease, however, the results of the loading test did not agree with the finding that there is little accumulation of glycolipids in postmortem tissues.     

Hydrophobic channels in crystals of an α-aminoisobutyric acid pentapeptide

The crystal structure of the pentapeptide p-toluene-sulfonyl-(α-aminoisobutyryl)₅-methyl ester (Tosyl-(Aib)₅-OMe) has been determined in the space group P$\text{I}$. Pentapeptide molecules are folded in the 3₁₀ helical conformation and packed together, so as to yield a hydrophobic channel with a minimim diameter of 5.2 Å. The channel contains crystallographically disordered material. This structure provides a model for channel formation by hydrophobic peptide aggregates and should prove useful in studies of alamethicin, suzukacillin and related Aib containing membrane channels. Triclinic (P$\text{I}$) crystals of the pentapeptide are obtained in the presence of LiClO₄ in aqueous methanol, whereas crystallization from methanol alone yields crystals in the space group Pbca. The conformations of the peptide in the two crystal forms are very similar and only the molecular packing is dramatically different.     

Relationship between transport and metabolism of α-naphthaleneacetic acid, β-naphthaleneacetic acid and α-decalylacetic acid in segments of Coleus

Summary Transportand metabolism of -naphthaleneacetic acid -naphthaleneacetic acid, and -decalylacetic acid, all labelled with ¹⁴C in the carboxyl, group, were studied. Only -naphthaleneacetic acid is transported in a polar way. Most of the radioactivity in the tissue is in a low molecular form, either free or as immobilization products. The immobilization of -naphthaleneacetic acid is similar to that of -naphthaleneacetic acid. Immobilization of -decalylacetic acid is typically different. Bioassays showed -naphthaleneacetic acid as the sole biologically active component. It is concluded that stereo requirements necessary for biological activity are also required for polar auxin transport. It is further concluded that the observed specificity of the transport system is not related to the formation of immobilization products.     

The effect of α2,6-linked sialic acid on anti-IgM antibody-induced apoptosis in Ramos cells

Apoptosis in B cells is induced through the B cell antigen receptor (BCR) and affects the sialic acid recognition molecules on B cells. We investigated the effects of ₁-acid glycoprotein (AGP), which mainly contains 2,6-linked sialic acid, on anti-IgM antibody (Ab)-induced apoptosis in Ramos cells, which are derived from Burkitt's lymphoma. When Ramos cells were incubated with anti-IgM-Ab in plates coated with AGP, neuraminidase-digested AGP (asAGP) or 2,3-sialylated AGP (2,3AGP), apoptosis was suppressed only in those coated with AGP. We also studied the effects of CD22, which is expressed on the surface of mature B cells and binds to sugar chains containing 2,6-linked sialic acid, with anti-CD22 monoclonal antibody (mAb). Anti-CD22mAb enhanced anti-IgM Ab-induced apoptosis in Ramos cells. These contradictory results suggested that the recognition molecules for 2,6-linked sialic acid on AGP, which inhibits B-cell apoptosis, is distinct from CD22, or that different binding domains of CD22 between 2,6-linked sialic acid and anti-CD22 mAb exert opposite functions of suppression or enhancement to anti-IgM Ab-induced B cells.     

Effect of α-naphthaleneacetic acid and sucrose levels on the development of cultured embryos of coconut

Mature coconut embryos were germinated in a modified Murashige and Skoog medium and then cultured on BMY₃ medium incorporating sucrose in the range of 4 to 8%. -Naphthaleneacetic acid (NAA) was added into the medium at concentrations ranging from 0 to 800 M for periods of 4 to 24 weeks. Application of NAA for 4 weeks stimulated shoot growth, whereas application periods greater than 4 weeks had no significant effect. NAA in the range of 100–300 M stimulated elongation of the primary root and the optimum concentration increased with increases in sucrose levels. Production of adventitious roots was stimulated by the addition of NAA with levels of 200 M and above being the most effective. Increasing the sucrose concentration from 4% through to 8% stimulated root elongation in the absence of NAA and inhibited shoot growth whether NAA was present or absent.Abbreviations IAA indole-acetic acid - NAA -naphthaleneacetic acid     

Effects of cell surface α2-3 sialic acid on osteogenesis

A cell culture model of osteoblast differentiation was applied in our study of the effect of sialic acid on the osteogenesis by using the pre-osteoblast of MC3T3-E1 subclone 14 cells. Following the treatment of different concentrations of α2,3-neuraminidase, which specifically removed the α2-3 sialic acid from cell surface, a significant decrease of α2-3 sialic acid was detected with fluorescein isothiocyanate (FITC)-labeled Maackia amurensis lectin (MAL-II) by flow cytometry analysis. von Kossa staining showed that the bone mineralization decreased in MC3T3-E1 subclone 14 cells after the treatment of α2,3-neuraminidase for 2 weeks. However α2,3-neuraminidase did not affect the formation of osteoblasts in MC3T3-E1 subclone 14 cells, which was demonstrated by positive alkaline phosphatase (ALP)-staining. Characteristic biological markers and osteoblast-like cell-related factors of osteoblastic cells were also examined. Both RT-PCR and Western blot analysis demonstrated that the expression of bone sialoprotein (BSP), osteoprotegerin (OPG), and vitamin D receptor (VDR) were significantly decreased when α2-3 sialic acid expression decreased on the cell surface, while the expression of osteocalcin (OC) and osteopontin (OPN) remained unchanged. We propose a hypothesis that α2-3 sialic acid affects bone mineralization but not osteogenic differentiation.     

Expression of core-binding factor α1 and osteocalcin in fluoride-treated fibroblasts and osteoblasts

To study the effects and importance of fluoride on FBs in the development of extraperiosteal calcification and the ossification of skeletal fluorosis, the presence of the osteogenic phenotype, which is indicated by the expression of core-binding factor α1 (Cbfa1) and osteocalcin (OCN), in an FB cell line (L929) and in osteoblasts (OBs) exposed to fluoride was determined. Fibroblasts and osteoblasts were exposed to different concentrations of fluoride (0, 0.0001, 0.001, 0.1, 1.0, 10.0 and 20.0 mg/L F⁻). By using RT-PCR and ELISA, the mRNA levels of Cbfa1 and OCN were measured at 48 h, and the protein levels of Cbfa1 and OCN were measured at 2, 4, 24, 48 and 72 h. The data demonstrated the following: (1) The Cbfa1 protein level in fluoride-treated fibroblasts clearly increased at 48 h in the groups treated with 0.0001, 0.001, 0.1, 1.0 and 20.0 mg/L F⁻. The Cbfa1 protein level of the group treated with 10 mg/L F⁻ at 72 h was higher than that of the control group. The level of Cbfa1 mRNA in the fibroblasts was much higher at 48 h in the group treated with 10.0 mg/L F⁻ than in the control group. (2) The OCN protein level in fluoride-treated fibroblasts was significantly higher than that of the control group in the 0.0001, 0.1, 1.0, 10.0 and 20.0 mg/L F⁻ groups at 2 h, and in the 0.001 and 0.1 F⁻ groups at 4 h. A slightly higher level of OCN mRNA in fluoride-treated fibroblasts was also found in the 1.0 and 20.0 mg/L F⁻ groups compared to the control group. (3) The expressions of Cbfa1 and OCN in osteoblasts treated with the same experimental conditions as the fibroblasts were up-regulated by fluoride following the same trend as in the fibroblasts. Our results showed an increase in the expression of Cbfa1 and OCN in fibroblasts and osteoblasts exposed to fluoride and suggested that the osteogenic function of fibroblasts induced by fluoride could play an important role in the development of extraperiosteal ossification during skeletal fluorosis.     

Detection of an alteration of the α2 gene in a patient with chronic lung disease and serum α2 deficiency

Summary ₂-Macrpglobulin (A2M) is a major human plasma protease inhibitor capable of inhibiting most endopeptidases tested so far. In the case of the other major plasma protease inhibitor, ₁-antitrypsin, genetically determined deficiency states are known to increase the risk of chronic obstructive pulmonary disease (COPD) 20- to 30-fold in affected individuals. No defects of the A2M gene have been described as yet, but A2M may play a role in the regulation of protease activity in the lung, especially with respect to those proteases not inhibited by ₁-antitrypsin. We report here the molecular genetic detection of an alteration of the A2M gene in a patient with serum A2M deficiency and chronic lung disease since childhood. The alteration involves restriction sites detected with 10 different enzymes and is most probably caused by a major deletion or rearrangement of the gene. Nine of the restriction enzymes used detected no polymorphisms in 40 healthy control subjects and 39 COPD patients. The polymorphism detected in this patient with the enzyme PvuII was different from another described previously, and was found in this patient only. The patient is heterozygous for an alteration in the A2M gene; this may be responsible for his serum A2M deficiency and may be relevant to the early onset of pulmonary disease in his case.