Biosynthesis In Fibroblasts Research Articles

Differential regulation of heparan sulfate biosynthesis in fibroblasts cocultured with normal vs. cancerous prostate cells.

Heparan sulfate proteoglycans (HSPGs) regulate a wide range of biological activities in both physiological and pathological conditions. Altered expression or deregulated function of HSPGs and their heparan sulfate (HS) chains significantly contribute to carcinogenesis as well and crucially depends on the functioning of the complex system of HS biosynthetic/modifying enzymes termed as "GAGosome". Here, we aimed at investigating the expression profile of the system in a cell culture model of stroma-epithelial crosstalk and searching for transcription factors potentially related to the regulation of expression of the genes involved. Coculture of BjTERT-fibroblasts with normal PNT2 human prostate epithelial cells resulted in significant downregulation (2-4-fold) of transcriptional activity of HS metabolism-involved genes (EXT1/2, NDST1/2, GLCE, HS2ST1, HS3ST1/2, HS6ST1/2, SULF1/2, HPSE) in both cell types, whereas coculture with prostate cancer cells (LNCaP, PC3, DU145) demonstrated no significant interchanges. Human Transcription Factor RT2 Profiler PCR array and manual RT-PCR verification supposed FOS, MYC, E2F, SRF, NR3C1 as potential candidates for regulation and/or coordination of HS biosynthesis. Taken together, transcriptional activity of HS biosynthetic system in normal fibroblasts and prostate epithelial cells during their coculture might be controlled by their intercellular communication, reflecting of adaptation of these cells to each other. The regulation is attenuated or abrogated if normal fibroblasts interact with prostate cancer cells making the cancer cells independent of the limiting effects of fibroblasts, thus contributing to possibility of unlimited growth and progression. Overall, these data demonstrate an ability of cell-cell interactions to affect transcriptional activity of HS biosynthesis-involved genes.

ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6.

Excessive deposition of extracellular matrix, mainly collagen protein, is the hallmark of organ fibrosis. The molecular mechanisms regulating fibrotic protein biosynthesis are unclear. Here, we find that chemoattractant receptor homologous molecule expressed on TH2 cells (CRTH2), a plasma membrane receptor for prostaglandin D2, is trafficked to the endoplasmic reticulum (ER) membrane in fibroblasts in a caveolin-1-dependent manner. ER-anchored CRTH2 binds the collagen mRNA recognition motif of La ribonucleoprotein domain family member 6 (LARP6) and promotes the degradation of collagen mRNA in these cells. In line, CRTH2 deficiency increases collagen biosynthesis in fibroblasts and exacerbates injury-induced organ fibrosis in mice, which can be rescued by LARP6 depletion. Administration of CRTH2 N-terminal peptide reduces collagen production by binding to LARP6. Similar to CRTH2, bumetanide binds the LARP6 mRNA recognition motif, suppresses collagen biosynthesis, and alleviates bleomycin-triggered pulmonary fibrosis invivo. These findings reveal a novel anti-fibrotic function of CRTH2 in the ER membrane via the interaction with LARP6, which may represent a therapeutic target for fibrotic diseases.

Enalapril stimulates collagen biosynthesis through prolidase-dependent mechanism in cultured fibroblasts

The mechanism of a lower incidence of dermatological manifestations in patients treated with enalapril compared to patients treated with other ACE-inhibitors, e.g., captopril, is not known. The finding that prolidase plays an important role in collagen biosynthesis and that some angiotensin-converting enzyme inhibitors affect prolidase activity led us to evaluate its effect on collagen biosynthesis in cultured human skin fibroblasts. Since insulin-like growth factor (IGF-I) and transforming growth factor beta 1 (TGF-β1) are the most potent stimulators of both collagen biosynthesis and prolidase activity, and prolidase is regulated by β1 integrin signaling, the effect of enalapril and enalaprilat on IGF-IR, TGF-β1, and β1 integrin receptor expressions was evaluated. Cells were treated with milimolar concentrations (0.3 and 0.5 mM) of enalapril and enalaprilat for 24 h. The activity of prolidase was determined by colorimetic assay. Collagen biosynthesis was evaluated by radiometric assay. Expression of signaling proteins was evaluated using Western blot. It was found that enalapril- and enalaprilat-dependent increase in prolidase activity and expression was accompanied by parallel increase in collagen biosynthesis. The exposure of the cells to 0.5 mM enalapril and enalaprilat contributed to increase in IGF-IR and α2β1 integrin receptor as well as TGF-β1 and NF-κB p65 expressions. Enalapril- and enalaprilat-dependent increase of collagen biosynthesis in fibroblasts results from increase of prolidase activity and expression, which may undergo through activation of α2β1 integrin and IGF-IR signaling as well as upregulation of TGF-β1 and NF-κB p65, the inhibitor of collagen gene expression.

Human mutations in methylenetetrahydrofolate dehydrogenase 1 impair nuclear de novo thymidylate biosynthesis

An inborn error of metabolism associated with mutations in the human methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene has been identified. The proband presented with SCID, megaloblastic anemia, and neurologic abnormalities, but the causal metabolic impairment is unknown. SCID has been associated with impaired purine nucleotide metabolism, whereas megaloblastic anemia has been associated with impaired de novo thymidylate (dTMP) biosynthesis. MTHFD1 functions to condense formate with tetrahydrofolate and serves as the primary entry point of single carbons into folate-dependent one-carbon metabolism in the cytosol. In this study, we examined the impact of MTHFD1 loss of function on folate-dependent purine, dTMP, and methionine biosynthesis in fibroblasts from the proband with MTHFD1 deficiency. The flux of formate incorporation into methionine and dTMP was decreased by 90% and 50%, respectively, whereas formate flux through de novo purine biosynthesis was unaffected. Patient fibroblasts exhibited enriched MTHFD1 in the nucleus, elevated uracil in DNA, lower rates of de novo dTMP synthesis, and increased salvage pathway dTMP biosynthesis relative to control fibroblasts. These results provide evidence that impaired nuclear de novo dTMP biosynthesis can lead to both megaloblastic anemia and SCID in MTHFD1 deficiency.

Synthesis of lipoic acid–peptide conjugates and their effect on collagen and melanogenesis

We report new examples of lipoic acid (LA)–peptide conjugates, their potential as codrugs having anti-melanogenic and anti-aging properties was evaluated. These multifunctional molecules were prepared by linking lipophilic moiety (LA) to the pentapeptide KTTKS. The inhibitory effect of LA–peptide conjugates on melanin synthesis and tyrosinase activity is stronger than that of LA or the pentapeptide alone. Importantly, the conjugates display no cytotoxicity at a high concentration. LA–KTTKS and LA–PEG–KTTKS also inhibit UV-induced matrix metalloproteinase-1 expression up to 49.5% and 69.5% at 0.5 mM, respectively. LA–peptide conjugates stimulate collagen biosynthesis in fibroblasts more efficiently than their parent molecules do. These data suggest that LA–peptide conjugates may have cosmeceutical application as anti-melanogenic and anti-aging agents.

Characterization of CoQ10 biosynthesis in fibroblasts of patients with primary and secondary CoQ10 deficiency

Primary coenzyme Q₁₀ (CoQ₁₀) deficiencies are associated with mutations in genes encoding enzymes important for its biosynthesis and patients are responsive to CoQ₁₀ supplementation. Early treatment allows better prognosis of the disease and therefore, early diagnosis is desirable. The complex phenotype and genotype and the frequent secondary CoQ₁₀ deficiencies make it difficult to achieve a definitive diagnosis by direct quantification of CoQ₁₀. We developed a non-radioactive methodology for the quantification of CoQ₁₀ biosynthesis in fibroblasts that allows the identification of primary deficiencies. Fibroblasts were incubated 72 h with 28 μmol/L (2)H₃-mevalonate or 1.65 mmol/L (13)C₆-p-hydroxybenzoate. The newly synthesized (2)H₃- and (13)C₆- labelled CoQ₁₀ were analysed by high performance liquid chromatography-tandem mass spectrometry. The mean and the reference range for (13)C₆-CoQ₁₀ and (2)H₃-CoQ₁₀ biosynthesis were 0.97 (0.83-1.1) and 0.13 (0.09-0.17) nmol/Unit of citrate synthase, respectively. We validated the methodology through the study of one patient with COQ2 mutations and six patients with CoQ₁₀ deficiency secondary to other inborn errors of metabolism. Afterwards we investigated 16 patients' fibroblasts and nine showed decreased CoQ₁₀ biosynthesis. Therefore, the next step is to study the COQ genes in order to reach a definitive diagnosis in these nine patients. In the patients with normal rates the deficiency is probably secondary. In conclusion, we have developed a non-invasive non-radioactive method suitable for the detection of defects in CoQ₁₀ biosynthesis, which offers a good tool for the stratification of patients with these treatable mitochondrial diseases.

Cinnamon Extract Promotes Type I Collagen Biosynthesis via Activation of IGF-I Signaling in Human Dermal Fibroblasts

The breakdown of collagenous networks with aging results in hypoactive changes in the skin. Accordingly, reviving stagnant collagen synthesis can help protect dermal homeostasis against aging. We searched for type I collagen biosynthesis-inducing substances in various foods using human dermal fibroblasts and found that cinnamon extract facilitates collagen biosynthesis. Cinnamon extract potently up-regulated both mRNA and protein expression levels of type I collagen without cytotoxicity. We identified cinnamaldehyde as a major active component promoting the expression of collagen by HPLC and NMR analysis. Since insulin-like growth factor-I (IGF-I) is the most potent stimulator of collagen biosynthesis in fibroblasts, we examined the effect of cinnamaldehyde on IGF-I signaling. Treatment with cinnamaldehyde significantly increased the phosphorylation levels of the IGF-I receptor and its downstream signaling molecules such as insulin receptor substrate-1 and Erk1/2 in an IGF-I-independent manner. These results suggested that cinnamon extract is useful in antiaging treatment of skin.

A severe human metabolic disease caused by deficiency of the endoplasmatic mannosyltransferase hALG11 leads to congenital disorder of glycosylation-Ip

A new type of congenital disorders of glycosylation, designated CDG-Ip, is caused by the deficiency of GDP-Man:Man3GlcNAc2-PP-dolichol-alpha1,2-mannosyltransferase, encoded by the human ortholog of ALG11 from yeast. The patient presented with a multisystemic disorder characterized by muscular hypotonia, seizures, developmental retardation and death at the age of 2 years. The isoelectric focusing pattern of the patient's serum transferrin showed the partial loss of complete N-glycan side chains, which is a characteristic sign for CDG-I. Analysis of dolichol-linked oligosaccharides in patient-derived fibroblasts revealed an accumulation of Man3GlcNAc2-PP-dolichol and Man4GlcNAc2-PP-dolichol. Determination of mannosyltransferase activities of early steps of lipid-linked oligosaccharide biosynthesis in fibroblasts indicated that the patient was deficient in elongating Man3GlcNAc2-PP-dolichol. These findings gave rise to genetic analysis of the hALG11 cDNA, in which homozygosity for mutation c.T257C (p.L86S) was identified. Verification of the mutation as a primary cause for the genetic defect was proved by retroviral expression of human wild-type and mutated ALG11 cDNA in patient-derived fibroblasts as well as using a yeast alg11 deletion strain as a heterologous expression system for hALG11 variants. Immunofluorescence examinations combined with western blotting showed no differences of intracellular localization or expression of ALG11 between control and patient fibroblasts, respectively, indicating no mislocalization or degradation of the mutated transferase.

Coordinate Regulation of Phospholipid Biosynthesis and Secretory Pathway Gene Expression in XBP-1(S)-induced Endoplasmic Reticulum Biogenesis

Development of the expansive endoplasmic reticulum (ER) present in specialized secretory cell types requires X-box-binding protein-1 (Xbp-1). Enforced expression of XBP-1(S), a transcriptional activator generated by unfolded protein response-mediated splicing of Xbp-1 mRNA, is sufficient to induce proliferation of rough ER. We previously showed that XBP-1(S)-induced ER biogenesis in fibroblasts correlates with increased production of phosphatidylcholine (PtdCho), the primary phospholipid of the ER membrane, and enhanced activities of the choline cytidylyltransferase (CCT) and cholinephosphotransferase enzymes in the cytidine diphosphocholine (CDP-choline) pathway of PtdCho biosynthesis. Here, we report that the level and synthesis of CCT, the rate-limiting enzyme in the CDP-choline pathway, is elevated in fibroblasts overexpressing XBP-1(S). Furthermore, overexpression experiments demonstrated that raising the activity of CCT, but not cholinephosphotransferase, is sufficient to augment PtdCho biosynthesis in fibroblasts, indicating that XBP-1(S) increases the output of the CDP-choline pathway primarily via its effects on CCT. Finally, fibroblasts overexpressing CCT up-regulated PtdCho synthesis to a level similar to that in XBP-1(S)-transduced cells but exhibited only a small increase in rough ER and no induction of secretory pathway genes. The more robust XBP-1(S)-induced ER expansion was accompanied by induction of a wide array of genes encoding proteins that function either in the ER or at other steps in the secretory pathway. We propose that XBP-1(S) regulates ER abundance by coordinately increasing the supply of membrane phospholipids and ER proteins, the key ingredients for ER biogenesis.

Cerebellar ataxia with coenzyme Q 10 deficiency: Diagnosis and follow-up after coenzyme Q 10 supplementation

Our aim was to report a new case with cerebellar ataxia associated with coenzyme Q 10 (CoQ) deficiency, the biochemical findings caused by this deficiency and the response to CoQ supplementation. Patient A 12-year-old girl presenting ataxia and cerebellar atrophy. Biochemical studies Coenzyme Q 10 in muscle was analysed by HPLC with electrochemical detection and mitochondrial respiratory chain (MRC) enzyme activities by spectrophotometric methods. CoQ biosynthesis in fibroblasts was assayed by studying the incorporation of radiolabeled 4-hydroxy[U- 14C] benzoic acid by HPLC with radiometric detection. Results Mitochondrial respiratory chain enzyme analysis showed a decrease in complex I + III and complex II + III activities. CoQ concentration in muscle was decreased (56 nmol/g of protein: reference values: 157–488 nmol/g protein). A reduced incorporation of radiolabeled 4-hydroxy[U- 14C] benzoic acid was observed in the patient (19% of incorporation respect to the median control values). After 16 months of CoQ supplementation, the patient is now able to walk unaided and cerebellar signs have disappeared. Conclusions Cerebellar ataxia associated with CoQ deficiency in our case might be allocated in the transprenylation pathway or in the metabolic steps after condensation of 4-hydroxybenzoate and the prenyl side chain of CoQ. Clinical improvement after CoQ supplementation was remarkable, supporting the importance of an early diagnosis of this kind of disorders.

Butyrate-induced collagen biosynthesis in cultured fibroblasts is independent on α2β1 integrin signalling and undergoes through IGF-I receptor cascade

The potential role of butyrate to modulate cellular metabolism through integrin receptor led to evaluation of its effect on collagen biosynthesis in cultured fibroblasts. Confluent human dermal fibroblasts were treated with 2 mM and 4 mM of sodium butyrate (NaB) for 48 h. It was found that butyrate induced collagen biosynthesis and prolidase activity independently of alpha2beta1 integrin signaling. The expressions of both alpha2 and beta1 integrin subunits as well as integrin-induced activation of focal adhesion kinase (FAK) were not affected in the cells treated with NaB. Since insulin-like growth factor-I (IGF-I) is the most potent stimulator of collagen biosynthesis in fibroblasts, the effect of butyrate on IGF-I receptor (IGF-IR) expression was evaluated. It was found that the exposure of the cells to 4 mM butyrate contributed to a distinct increase in IGF-IR. It was accompanied by a parallel increase in the expression of Sos protein and MAP-kinases (ERK1, ERK2). The data suggests that butyrate-dependent stimulation of collagen biosynthesis in cultured human skin fibroblasts undergoes through IGF-IR signaling.

The effect of hyaluronic acid on interleukin-1-induced deregulation of collagen metabolism in cultured human skin fibroblasts

Although hyaluronic acid (HA) has been used in the treatment of osteoarthritis for 30 years, the mechanism of its protective action on collagen metabolism disturbances in tissues during inflammation is not known. The present study was undertaken to evaluate the mechanism of Interleukin-1 (IL-1)-induced deregulation of collagen metabolism in cultured human skin fibroblast and the effect of HA on the process. In normal fibroblasts IL-1 strongly induced inhibition of collagen biosynthesis, while HA counteracted the process. The mechanism of this phenomenon was independent of prolidase activity, an enzyme that plays an important role in collagen biosynthesis at the post-translational level. Instead, IL-1 was found to inhibit the expression of insulin-like growth factor-I receptor (IGF-IR) and MAP kinases—ERK1 and ERK2, while HA was shown to counteract this process. Since insulin-like growth factor-I (IGF-I) is a most potent stimulator of collagen biosynthesis in fibroblasts the mechanism of IL-1-dependent inhibition of collagen biosynthesis may be related to inhibition of IGF-IR expression and signaling. The data suggest that hyaluronic acid protects collagen against IL-1-induced inhibition of biosynthesis of this protein in cultured human skin fibroblasts at the level of IGF-IR signaling.

Combined Effects of Estrogen and Mechanical Loading on Anterior Cruciate Ligament Fibroblast Biosynthesis

For some time, estrogen has been suspected to play a negative role in anterior cruciate ligament (ACL) fibroblast biosynthesis; however, reports on this issue have been controversial. In a recent study, our group demonstrated a negative combined effect of estrogen and mechanical loading on the gene expression of major extracellular matrix component molecules in ACL fibroblasts.

Cytotoxicity and effect on collagen biosynthesis of proline analogue of melphalan as a prolidase-convertible prodrug in cultured human skin fibroblasts

Proline analogue of melphalan (MEL-PRO) was synthesised as a prodrug susceptible to the action of ubiquitously distributed, cytosolic imidodipeptidase—prolidase [E.C.3.4.13.9]. Conjugation of melphalan (MEL) with proline (PRO) through an imido-bond resulted in formation of a good substrate for prolidase. The susceptibility of MEL-PRO to the action of prolidase was found to be similar, compared to glycyl-proline—the most abundant, endogenous substrate for prolidase and about 6-fold higher compared to its substrate—glycyl-hydroxyproline. We have compared the transport of MEL and its prodrug through cell membrane, their antimitotic activity, cytotoxicity and effect on collagen biosynthesis in cultured, normal human skin fibroblasts. The prodrug was found to be more effectively transported into the cells than the free drug. Moreover, a lower cytotoxicity, antimitotic activity and inhibitory effect on collagen biosynthesis of the prodrug, compared to the free drug were observed after 24 h of incubation. MEL and MEL-PRO at concentrations of 12 μM led to the decrease in cell viability in confluent human skin fibroblasts by about 40 and 20%, respectively, during 24 h of incubation. IC 50 of MEL for DNA synthesis (measured by thymidine incorporation assay) was found at about 7 μM, while MEL-PRO used at this concentration produced about 35% reduction in thymidine incorporation. Similarly, MEL and MEL-PRO used at 7 μM concentrations inhibited collagen biosynthesis in fibroblasts cultured for 24 h to about 30 and 80% of control values, respectively. However, when the cells were cultured with the drugs for 72 h, similar effects of both drugs on DNA and collagen biosynthesis were observed. The data suggest that MEL-PRO may serve as a prolidase-convertible prodrug that evokes lower cytotoxicity, antimitotic activity, and lower inhibitory effect on collagen biosynthesis in fibroblast cultures, compared to the free drug.

Potential role of pyrroline 5-carboxylate in regulation of collagen biosynthesis in cultured human skin fibroblasts

Although insulin-like growth factor-I (IGF-I) is known as an important stimulator of collagen biosynthesis in collagen-producing cells, the mechanism and endpoints by which it regulate the process remain largely unknown. Serum of acutely fasted rats contained reduced amount of IGF-I (72±16 ng/ml) and showed about 75% reduced ability to stimulate collagen and DNA synthesis in confluent human skin fibroblasts in comparison to the effect of control rat serum (IGF-I, 168±19 ng/ml). An addition of IGF-I (at least 40 ng/ml) to fasted rat serum restored its mitogenic activity but could not restore its ability to stimulate collagen biosynthesis to control values during 24 h of incubation. However, when the cells were incubated in fasted rat serum supplemented with 40 ng/ml of IGF-I for 48 h, collagen biosynthesis was restored to control values. It suggests that the stimulatory role of IGF-I in collagen biosynthesis undergo indirectly. We considered pyrroline-5-carboxylate (P5C) as a candidate to play a direct role in this process. Since IGF-I and P5C are known to be decreased in serum of fasted rats it seems that the action of IGF-I on collagen biosynthesis may involve participation of P5C. We have found that serum of fasted rats (showing low level of P5C) supplemented with 1 mmol/l P5C induced collagen biosynthesis in confluent human skin fibroblasts during 24 h to control values. Supporting evidence comes from the experiment showing stimulatory action of P5C on collagen biosynthesis in fibroblasts cultured in serum-free medium. Our results postulate potential role of P5C in regulation of collagen biosynthesis and indicate participation of this molecule in the pathway of IGF-I action in this process.

Fibronectin gene expression differs in normal and abnormal human wound healing

The overproduction of fibronectin and type I collagen in keloids and hypertrophic scars implicates altered regulation of extracellular matrix components as an important aspect of these wound healing pathologies. However, little is known about the similarities and differences in extracellular matrix gene expression during normal and abnormal wound healing. This study compared the content of fibronectin messenger RNA and rates of fibronectin protein biosynthesis in fibroblasts derived from normal skin, normal scar, keloid, and hypertrophic scar. Fibronectin expression was enhanced in cells from both normal and abnormal wounds relative to cells from quiescent normal skin. Matched pairs of normal and keloid fibroblasts from the same individuals were also compared, and three of the four pairs showed higher fibronectin expression by the keloid cells at the levels of messenger RNA and protein synthesis. This was consistent with previous studies showing elevated steady state content of fibronectin in keloid cells relative to normal cells from the same individual. Fibronectin messenger RNA and protein content in the tissues from which these cells were derived was examined by in situ hybridization and immunohistochemistry. These studies revealed that in vivo, the steady state content of fibronectin messenger RNA and protein was highest in abnormal wounds, less in most normal scars, and lowest in normal skin. Thus, fibroblasts from keloids and hypertrophic scars overexpressed fibronectin in vivo relative to normal skin and normal scar and retain this characteristic in vitro relative to normal skin. Although normal scars contained little fibronectin protein and messenger RNA, cultured fibroblasts derived from these scars had contents of fibronectin messenger RNA and rates of biosynthesis in vitro similar to those of keloid fibroblasts. This indicates that the fibronectin regulatory pathway in scar fibroblasts is influenced by the tissue environment. These results are discussed with respect to the relationship of fibronectin expression in keloids, hypertrophic scars, and normal wounds in human beings.

The effect of reactive oxygen species on the biosynthesis of collagen and glycosaminoglycans in cultured human dermal fibroblasts.

The purpose of this study was to evaluate the possibility that the biological changes observed in connective tissue matrix components of photoaging skin may be induced by an alteration of biosynthesis in fibroblasts damaged by reactive oxygen species (ROS). We investigated the effect of ROS induced by xanthine and the xanthine oxidase system on the biosynthesis of connective tissue matrix components, collagen and glucosaminoglycans (GAGs) in cultured human dermal fibroblasts. ROS decreased collagen production and increased GAGs synthesis. Interestingly, these changes were consistent with the biological alterations of connective tissue matrix components observed in photoaging skin. Moreover, catalase and alpha-tocopherol completely prevented the ROS-induced alterations of collagen and GAGs biosynthesis, whereas superoxide dismutase had no effect on the ROS-induced changes. These results suggest that ROS may be one of the factors which cause the biological changes of connective tissue matrix components observed in photoaging skin.

Decreased cholesterol biosynthesis in fibroblasts from patients with Parkinson disease.

The underlying cause of cellular degeneration in the substantia nigra of patients with Parkinson disease has not been clearly established. With the objective of investigating whether metabolic abnormalities would be detected in peripheral non-neuronal cells, we began assessing key metabolic parameters in skin fibroblasts of these patients. The present report focuses on the finding of a remarkably reduced cholesterol biosynthetic capability of fibroblasts from patients with Parkinson disease. 14C-Acetate incorporation into cholesterol of these fibroblasts was 27.8 +/- 9.4% that observed in normal fibroblasts, and the reduced cholesterol synthesis was confirmed by measuring the activity of the rate-limiting enzyme HMGCoA reductase which averaged 6.64 +/- 2.50 nmol/h/mg protein in the patient's fibroblasts compared to 14.70 +/- 0.69 nmol/h/mg protein in the control fibroblasts. Cholesterol esterifying activity, as cholesteryl oleate formed from 14C-oleate, of the fibroblasts from Parkinson patients, was reduced by an average 43%. Two hypotheses are put forward to link these findings with the current experimental evidences for both increased lipid peroxidation and defective mitochondrial respiratory chain complex I activity in a number of cell types from Parkinson patients. Considering that decreased cholesterol biosynthesis has been detected in all the Parkinson cell lines thus far investigated, it is suggested that this may be a hallmark of the disease.

Heterogeneity of Defects in Mitochondrial Acetoacetyl-CoA Thiolase Biosynthesis in Fibroblasts from Four Patients with 3-Ketothiolase Deficiency

Deficient mitochondrial acetoacetyl-CoA thiolase in fibroblasts from four patients with 3-ketothiolase deficiency was studied using immunochemical methods. We also examined fibroblasts from two heterozygotes, the mother and the brother of the case 1 patient, identified on the basis of the results of the enzyme activity measurements, using 2-methylacetoacetyl-CoA as substrate. The results were as follows: 1) in fibroblasts from all four patients, the thiolase activity using acetoacetyl-CoA was not activated by K+, although that of the controls and the heterozygotes was activated about 2-fold. 2) by immunoblot analyses, mitochondrial acetoacetyl-CoA thiolase was not detectable in fibroblasts from cases 2 and 3, although a very faint band was seen in tissues from cases 1 and 4. However, the band of mitochondrial 3-ketoacyl-CoA thiolase was clearly detected in all patients to the same extent as in the controls. 3) mitochondrial acetoacetyl-CoA thiolase was observed after pulse labeling for 1-h and a 72-h chase period in three cell lines (cases 1, 2, and 4), but was fainter compared to the controls. In another cell line (case 3), a fluorographic band at the same position was detected following a 1-h pulse, but disappeared following a 6-h chase. These results demonstrate heterogeneity in the enzyme defect resulting in a deficiency of mitochondrial acetoacetyl-CoA thiolase in fibroblasts from patients with 3-ketothiolase deficiency.

The cerebro-hepato-renal (Zellweger) syndrome: prenatal detection based on impaired biosynthesis of plasmalogens.

Prenatal diagnosis of the cerebro-hepato-renal (Zellweger) syndrome has been performed in 10 pregnancies at risk by measuring both the activity of acyl CoA: dihydroxyacetone-phosphate acyltransferase (DHAP-AT) and the de novo plasmalogen biosynthesis, either in cultured amniotic fluid cells or in fibroblasts cultured from a chorionic villus biopsy. In 7 of the pregnancies both tests indicated no abnormality. All 7 continued to term and normal infants were delivered. However, in amniotic fluid cells from 2 fetuses affected by Zellweger syndrome unequivocal differences from control values were found. The activity of DHAP-AT was clearly deficient and the de novo plasmalogen biosynthesis was impaired. In one pregnancy at risk prenatal diagnosis was performed during the first trimester by measuring both the DHAP-AT activity and the de novo plasmalogen biosynthesis in fibroblasts cultured from a chorionic villi biopsy. From the deficient DHAP-AT activity and the impaired de novo plasmalogen biosynthesis it was concluded that the fetus was affected. This was confirmed biochemically after induced abortion. It can be concluded that measurement of the DHAP-AT activity and the de novo plasmalogen biosynthesis provides convenient methods for the early prenatal detection of Zellweger syndrome.