Database-assisted screening of autism spectrum disorder related gene set.

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by social and communication difficulties, along with repetitive behaviors. While genetic factors play a significant role in ASD, the precise genetic landscape remains complex and not fully understood, particularly in non-syndromic cases. The study performed an in silico comparison of three genetic databases. ClinVar, SFARI Gene, and AutDB were utilized to identify relevant gene subset and genetic variations associated with non-syndromic ASD. Gene set enrichment analysis (GSEA) and protein-protein interaction (PPI) network analysis were conducted to elucidate the biological significance of the identified genes. The integrity of ASD-related gene subset and the distribution of their variations were statistically assessed. A subset of twenty overlapping genes potentially specific for non-syndromic ASD was identified. GSEA revealed enrichment of biological processes related to neuronal development and differentiation, synaptic function, and social skills, highlighting their importance in ASD pathogenesis. PPI network analysis demonstrated functional relationships among the identified genes. Analysis of genetic variations showed predominance of rare variants and database-specific distribution patterns. The results provide valuable insights into the genetic landscape of ASD and outline the genes and biological processes involved in the condition, while taking into account that the study relied exclusively on in silico analyses, which may be subject to biases inherent to database methodologies. Further research incorporating multi-omics data and experimental validation is warranted to enhance our understanding of non-syndromic ASD genetics and facilitate the development of targeted research, interventions and therapies.

Allele-specific effect of various dietary fatty acids and ETS1 transcription factor on SCD1 expression.

Overnutrition and genetic predisposition are major risk factors for various metabolic disorders. Stearoyl-CoA desaturase-1 (SCD1) plays a key role in these conditions by synthesizing unsaturated fatty acids (FAs), thereby promoting fat storage and alleviating lipotoxicity. Expression of SCD1 is influenced by various saturated and cis-unsaturated FAs, but the possible role of dietary trans FAs (TFAs) and SCD1 promoter polymorphisms in its regulations has not been addressed. Therefore, we aimed to investigate the impact of the two main TFAs, vaccenate and elaidate, and four common promoter polymorphisms (rs1054411, rs670213, rs2275657, rs2275656) on SCD1 expression in HEK293T and HepG2 cell cultures using luciferase reporter assay, qPCR and immunoblotting. We found that SCD1 protein and mRNA levels as well as SCD1 promoter activity are markedly elevated by elaidate, but not altered by vaccenate. The promoter polymorphisms did not affect the basal transcriptional activity of SCD1. However, the minor allele of rs1054411 increased SCD1 expression in the presence of various FAs. Moreover, this variant was predicted in silico and verified in vitro to reduce the binding of ETS1 transcription factor to SCD1 promoter. Although we could not confirm an association with type 2 diabetes mellitus, the FA-dependent and ETS1-mediated effect of rs1054411 polymorphism deserves further investigation as it may modulate the development of lipid metabolism-related conditions.

Diversity and Classification of Genetic Variations in Autism Spectrum Disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental condition with symptoms that affect the whole personality and all aspects of life. Although there is a high degree of heterogeneity in both its etiology and its characteristic behavioral patterns, the disorder is well-captured along the autistic triad. Currently, ASD status can be confirmed following an assessment of behavioral features, but there is a growing emphasis on conceptualizing autism as a spectrum, which allows for establishing a diagnosis based on the level of support need, free of discrete categories. Since ASD has a high genetic predominance, the number of genetic variations identified in the background of the condition is increasing exponentially as genetic testing methods are rapidly evolving. However, due to the huge amount of data to be analyzed, grouping the different DNA variations is still challenging. Therefore, in the present review, a multidimensional classification scheme was developed to accommodate most of the currently known genetic variants associated with autism. Genetic variations have been grouped according to six criteria (extent, time of onset, information content, frequency, number of genes involved, inheritance pattern), which are themselves not discrete categories, but form a coherent continuum in line with the autism spectrum approach.

Molecular Basis of Unequal Alternative Splicing of Human SCD5 and Its Alteration by Natural Genetic Variations.

Alternative splicing (AS) is a major means of post-transcriptional control of gene expression, and provides a dynamic versatility of protein isoforms. Cancer-related AS disorders have diagnostic, prognostic and therapeutic values. Changes in the expression and AS of human stearoyl-CoA desaturase-5 (SCD5) are promising specific tumor markers, although the transcript variants (TVs) of the gene have not yet been confirmed. Our in silico, in vitro and in vivo study focuses on the distribution of SCD5 TVs (A and B) in human tissues, the functionality of the relevant splice sites, and their modulation by certain single-nucleotide variations (SNVs). An order of magnitude higher SCD5A expression was found compared with SCD5B. This unequal splicing is attributed to a weaker recognition of the SCD5B-specific splicing acceptor site, based on predictions confirmed by an optimized minigene assay. The pronounced dominance of SCD5A was largely modified (rs1430176385_A, rs1011850309_A) or even inverted (rs1011850309_C) by natural SNVs at the TV-specific splice sites. Our results provide long missing data on the proportion of SCD5 TVs in human tissues and reveal mutation-driven changes in SCD5 AS, potentially affecting tumor-associated reprogramming of lipid metabolism, thus having prognostic significance, which may be utilized for novel and personalized therapeutic approaches.

High fat diet and PCSK9 knockout modulates lipid profile of the liver and changes the expression of lipid homeostasis related genes.

BACKGROUND: High fat diet (HFD) increases the likelihood of dyslipidemia, which can be a serious risk factor for atherosclerosis, diabetes or hepatosteatosis. Although changes in different blood lipid levels were broadly investigated, such alterations in the liver tissue have not been studied before. The aim of the current study was to investigate the effect of HFD on hepatic triglyceride (TG), diglyceride (DG) and ceramide (CER) levels and on the expression of four key genes involved in lipid homeostasis (Pcsk9, Ldlr, Cd36 and Anxa2) in the liver. In addition, the potential role of PCSK9 in the observed changes was further investigated by using PCSK9 deficient mice. METHODS: We used two in vivo models: mice kept on HFD for 20 weeks and PCSK9-/- mice. The amount of the major TGs, DGs and CERs was measured by using HPLC-MS/MS analysis. The expression profiles of four lipid related genes, namely Pcsk9, Ldlr, Cd36 and Anxa2 were assessed. Co-localization studies were performed by confocal microscopy. RESULTS: In HFD mice, hepatic PCSK9 expression was decreased and ANXA2 expression was increased both on mRNA and protein levels, and the amount of LDLR and CD36 receptor proteins was increased. While LDLR protein level was also elevated in the livers of PCSK9-/- mice, there was no significant change in the expression of ANXA2 and CD36 in these animals. HFD induced a significant elevation in the hepatic levels of all measured TG and DG but not of CER types, and increased the proportion of monounsaturated vs. saturated TGs and DGs. Similar changes were detected in the hepatic lipid profiles of HFD and PCSK9-/- mice. Co-localization of PCSK9 with LDLR, CD36 and ANXA2 was verified in HepG2 cells. CONCLUSIONS: Our results show that obesogenic HFD downregulates PCSK9 expression in the liver and causes alterations in the hepatic lipid accumulation, which resemble those observed in PCSK9 deficiency. These findings suggest that PCSK9-mediated modulation of LDLR and CD36 expression might contribute to the HFD-induced changes in lipid homeostasis.

A Single Nucleotide Polymorphism (rs3811792) Affecting Human SCD5 Promoter Activity Is Associated with Diabetes Mellitus.

The combined prevalence of type 1 (T1DM) and type 2 (T2DM) diabetes mellitus is 10.5% worldwide and this is constantly increasing. The pathophysiology of the diseases include disturbances of the lipid metabolism, in which acyl-CoA desaturases play a central role as they synthesize unsaturated fatty acids, thereby providing protection against lipotoxicity. The stearoyl-CoA desaturase-5 (SCD5) isoform has received little scientific attention. We aimed to investigate the SCD5 promoter and its polymorphisms in vitro, in silico and in a case-control study. The SCD5 promoter region was determined by a luciferase reporter system in HepG2, HEK293T and SK-N-FI cells and it was proved to be cell type-specific, but it was insensitive to different fatty acids. The effect of the SCD5 promoter polymorphisms rs6841081 and rs3811792 was tested in the transfected cells. The T allele of rs3811792 single nucleotide polymorphism (SNP) significantly reduced the activity of the SCD5 promoter in vitro and modified several transcription factor binding sites in silico. A statistically significant association of rs3811792 SNP with T1DM and T2DM was also found, thus supporting the medical relevance of this variation and the complexity of the molecular mechanisms in the development of metabolic disorders. In conclusion, the minor allele of rs3811792 polymorphism might contribute to the development of diabetes by influencing the SCD5 promoter activity.

Different Metabolism and Toxicity of TRANS Fatty Acids, Elaidate and Vaccenate Compared to Cis-Oleate in HepG2 Cells.

Trans fatty acids (TFAs) are not synthesized in the human body but are generally ingested in substantial amounts. The widespread view that TFAs, particularly those of industrial origin, are unhealthy and contribute to obesity, cardiovascular diseases and diabetes is based mostly on in vivo studies, and the underlying molecular mechanisms remain to be elucidated. Here, we used a hepatoma model of palmitate-induced lipotoxicity to compare the metabolism and effects of the representative industrial and ruminant TFAs, elaidate and vaccenate, respectively, with those of cis-oleate. Cellular FAs, triacylglycerols, diacylglycerols and ceramides were quantitated using chromatography, markers of stress and apoptosis were assessed at mRNA and protein levels, ultrastructural changes were examined by electron microscopy and viability was evaluated by MTT assay. While TFAs were just slightly more damaging than oleate when applied alone, they were remarkably less protective against palmitate toxicity in cotreatments. These differences correlated with their diverse incorporation into the accumulating diacylglycerols and ceramides. Our results provide in vitro evidence for the unfavorable metabolic features and potent stress-inducing character of TFAs in comparison with oleate. These findings strengthen the reasoning against dietary trans fat intake, and they can also help us better understand the molecular mechanisms of lipotoxicity.

Molecular Mechanisms Underlying the Elevated Expression of a Potentially Type 2 Diabetes Mellitus Associated SCD1 Variant.

Disturbances in lipid metabolism related to excessive food intake and sedentary lifestyle are among major risk of various metabolic disorders. Stearoyl-CoA desaturase-1 (SCD1) has an essential role in these diseases, as it catalyzes the synthesis of unsaturated fatty acids, both supplying for fat storage and contributing to cellular defense against saturated fatty acid toxicity. Recent studies show that increased activity or over-expression of SCD1 is one of the contributing factors for type 2 diabetes mellitus (T2DM). We aimed to investigate the impact of the common missense rs2234970 (M224L) polymorphism on SCD1 function in transfected cells. We found a higher expression of the minor Leu224 variant, which can be attributed to a combination of mRNA and protein stabilization. The latter was further enhanced by various fatty acids. The increased level of Leu224 variant resulted in an elevated unsaturated: saturated fatty acid ratio, due to higher oleate and palmitoleate contents. Accumulation of Leu224 variant was found in a T2DM patient group, however, the difference was statistically not significant. In conclusion, the minor variant of rs2234970 polymorphism might contribute to the development of obesity-related metabolic disorders, including T2DM, through an increased intracellular level of SCD1.

Effect of cis- and trans-Monounsaturated Fatty Acids on Palmitate Toxicity and on Palmitate-induced Accumulation of Ceramides and Diglycerides.

Dietary trans fatty acids (TFAs) have been implicated in serious health risks, yet little is known about their cellular effects and metabolism. We aim to undertake an in vitro comparison of two representative TFAs (elaidate and vaccenate) to the best-characterized endogenous cis-unsaturated FA (oleate). The present study addresses the possible protective action of TFAs on palmitate-treated RINm5F insulinoma cells with special regards to apoptosis, endoplasmic reticulum stress and the underlying ceramide and diglyceride (DG) accumulation. Both TFAs significantly improved cell viability and reduced apoptosis in palmitate-treated cells. They mildly attenuated palmitate-induced XBP-1 mRNA cleavage and phosphorylation of eukaryotic initiation factor 2α (eIF2α) and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), but they were markedly less potent than oleate. Accordingly, all the three unsaturated FAs markedly reduced cellular palmitate incorporation and prevented harmful ceramide and DG accumulation. However, more elaidate or vaccenate than oleate was inserted into ceramides and DGs. Our results revealed a protective effect of TFAs in short-term palmitate toxicity, yet they also provide important in vitro evidence and even a potential mechanism for unfavorable long-term health effects of TFAs compared to oleate.

Investigation of the putative rate-limiting role of electron transfer in fatty acid desaturation using transfected HEK293T cells.

Elevated fatty acid (FA) levels contribute to severe metabolic diseases. Unbalanced oversupply of saturated FAs is particularly damaging, which renders stearoyl-CoA desaturase (SCD1) activity an important factor of resistance. A SCD1-related oxidoreductase protects cells against palmitate toxicity, so we aimed to test whether desaturase activity is limited by SCD1 itself or by the associated electron supply. Unsaturated/saturated FA ratio was markedly elevated by SCD1 overexpression while it remained unaffected by the overexpression of SCD1-related electron transfer proteins in HEK293T cells. Electron supply was not rate-limiting either in palmitate-treated cells or in cells with enhanced SCD1 expression. Our findings indicate the rate-limiting role of SCD1 itself, and that FA desaturation cannot be facilitated by reinforcing the electron supply of the enzyme.

Cellular toxicity of dietary trans fatty acids and its correlation with ceramide and diglyceride accumulation.

High fatty acid (FA) levels are deleterious to pancreatic ß-cells, largely due to the accumulation of biosynthetic lipid intermediates, such as ceramides and diglycerides, which induce ER stress and apoptosis. Toxicity of palmitate (16:0) and oleate (18:1 cis-Δ9) has been widely investigated, while very little data is available on the cell damages caused by elaidate (18:1 trans-Δ9) and vaccenate (18:1 trans-Δ11), although the potential health effects of these dietary trans fatty acids (TFAs) received great publicity. We compared the effects of these four FAs on cell viability, apoptosis, ER stress, JNK phosphorylation and autophagy as well as on ceramide and diglyceride contents in RINm5F insulinoma cells. Similarly to oleate and unlike palmitate, TFAs reduced cell viability only at higher concentration, and they had mild effects on ER stress, apoptosis and autophagy. Palmitate increased ceramide and diglyceride levels far more than any of the unsaturated fatty acids; however, incorporation of TFAs in ceramides and diglycerides was strikingly more pronounced than that of oleate. This indicates a correlation between the accumulation of lipid intermediates and the severity of cell damage. Our findings reveal important metabolic characteristics of TFAs that might underlie a long term toxicity and hence deserve further investigation.

Microsomal pre-receptor cortisol production is inhibited by resveratrol and epigallocatechin gallate through different mechanisms.

Local activation of cortisol in hormone target tissues is a major determinant of glucocorticoid effect. Disorders in this peripheral cortisol metabolism play an important role in the development of metabolic diseases, such as obesity or type 2 diabetes mellitus. Hence, dietary factors influencing the activity of the involved enzymes can have major impacts on the risk of the above diseases. Resveratrol and epigallocatechin gallate (EGCG), two natural polyphenols found in several nutriments and in green tea, respectively, are well-known for their antiobesity and antidiabetic activities. EGCG has been shown to interfere with microsomal cortisol production through decreasing the luminal NADPH:NADP+ ratio. The aim of this study was to clarify if resveratrol also induces such a redox shift or causes any direct enzyme inhibition that influences local cortisol production. Cortisone-cortisol conversions and changes in NADPH levels were monitored in rat liver microsomal vesicles. Cortisol production was inhibited by resveratrol in a concentration dependent manner while the intrinsic reducing and oxidizing capacity as well as the NADPH level inside the ER-derived vesicles remained unaffected. Activity measurements performed in permeabilized microsomes confirmed that resveratrol, unlike EGCG, inhibits 11ß-hydroxysteroid dehydrogenase type 1 directly. Long-term moderation of pre-receptor cortisol production likely contributes to the beneficial health effects of both polyphenols. © 2018 BioFactors, 45(2):236-243, 2019.

Cytosolic localization of NADH cytochrome b5 oxidoreductase (Ncb5or).

Acyl-CoA desaturation in the endoplasmic reticulum (ER) membrane depends on cytosolic NADH or NADPH, whereas NADPH in the ER lumen is utilized by prereceptor glucocorticoid production. It was assumed that NADH cytochrome b5 oxidoreductase (Ncb5or) might connect Acyl-CoA desaturation to ER luminal redox. We aimed to clarify the ambiguous compartmentalization of Ncb5or and test the possible effect of stearoyl-CoA on microsomal NADPH level. Amino acid sequence analysis, fluorescence microscopy of GFP-tagged protein, immunocytochemistry, and western blot analysis of subcellular fractions unequivocally demonstrated that Ncb5or, either endogenous or exogenous, is localized in the cytoplasm and not in the ER lumen in cultured cells and liver tissue. Moreover, the involvement of ER-luminal reducing equivalents in stearoyl-CoA desaturation was excluded.

Functional significance of SPINK1 promoter variants in chronic pancreatitis.

Chronic pancreatitis is a progressive inflammatory disorder of the pancreas, which often develops as a result of genetic predisposition. Some of the most frequently identified risk factors affect the serine protease inhibitor Kazal type 1 (SPINK1) gene, which encodes a trypsin inhibitor responsible for protecting the pancreas from premature trypsinogen activation. Recent genetic and functional studies indicated that promoter variants in the SPINK1 gene might contribute to disease risk in carriers. Here, we investigated the functional effects of 17 SPINK1 promoter variants using luciferase reporter gene expression assay in four different cell lines, including three pancreatic acinar cell lines (rat AR42J with or without dexamethasone-induced differentiation and mouse 266-6) and human embryonic kidney 293T cells. We found that most variants caused relatively small changes in promoter activity. Surprisingly, however, we observed significant variations in the effects of the promoter variants in the different cell lines. Only four variants exhibited consistently reduced promoter activity in all acinar cell lines, confirming previous reports that variants c.-108G>T, c.-142T>C, and c.-147A>G are risk factors for chronic pancreatitis and identifying c.-52G>T as a novel risk variant. In contrast, variant c.-215G>A, which is linked with the disease-associated splice-site mutation c.194 + 2T>C, caused increased promoter activity, which may mitigate the overall effect of the pathogenic haplotype. Our study lends further support to the notion that sequence evaluation of the SPINK1 promoter region in patients with chronic pancreatitis is justified as part of the etiological investigation.

Lipotoxicity in the liver.

Obesity due to excessive food intake and the lack of physical activity is becoming one of the most serious public health problems of the 21(st) century. With the increasing prevalence of obesity, non-alcoholic fatty liver disease is also emerging as a pandemic. While previously this pathophysiological condition was mainly attributed to triglyceride accumulation in hepatocytes, recent data show that the development of oxidative stress, lipid peroxidation, cell death, inflammation and fibrosis are mostly due to accumulation of fatty acids, and the altered composition of membrane phospholipids. In fact, triglyceride accumulation might play a protective role, and the higher toxicity of saturated or trans fatty acids seems to be the consequence of a blockade in triglyceride synthesis. Increased membrane saturation can profoundly disturb cellular homeostasis by impairing the function of membrane receptors, channels and transporters. However, it also induces endoplasmic reticulum stress via novel sensing mechanisms of the organelle's stress receptors. The triggered signaling pathways in turn largely contribute to the development of insulin resistance and apoptosis. These findings have substantiated the lipotoxic liver injury hypothesis for the pathomechanism of hepatosteatosis. This minireview focuses on the metabolic and redox aspects of lipotoxicity and lipoapoptosis, with special regards on the involvement of endoplasmic reticulum stress responses.

Inhibition of microsomal cortisol production by (-)-epigallocatechin-3-gallate through a redox shift in the endoplasmic reticulum--a potential new target for treating obesity-related diseases.

Conversion of cortisone to cortisol by 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) in the endoplasmic reticulum (ER) of the target cells is a major determinant of glucocorticoid action, and plays an important role in the development of obesity-related diseases. Inhibition of 11ßHSD1 activity is, therefore, considered as a promising novel strategy for the treatment of metabolic syndrome and diabetes. Tea flavanols and their major representative, epigallocatechin gallate are known as antiobesity and antidiabetic agents. Their impacts on blood glucose level, hepatic glucose production, and insulin responsiveness resemble those observed on inhibition or depletion of 11ßHSD1. We aimed to study the effect of epigallocatechin gallate on 11ßHSD1 activity in ER-derived rat liver microsomes by measuring cortisone and cortisol with HPLC. Cortisol production was efficiently suppressed in a concentration dependent manner in intact microsomal vesicles. However, this effect was abolished by membrane permeabilization; and the three proteins involved in the overall process (11ßHSD1, hexose 6-phosphate dehydrogenase, and glucose 6-phosphate transporter) were not or only mildly affected. Further investigation revealed the oxidation of luminal NADPH to NADP⁺, which attenuates cortisone reduction and favors cortisol oxidation in this compartment. Such a redox shift in the ER lumen might contribute to the beneficial health effects of tea flavanols and should be regarded as a promising strategy for the development of novel selective 11ßHSD1 inhibitors to treat obesity-related diseases.

Natural mutations lead to enhanced proteasomal degradation of human Ncb5or, a novel flavoheme reductase.

NADH cytochrome b5 oxidoreductase (Ncb5or) protects ß-cells against oxidative stress and lipotoxicity. The predominant phenotype of lean Ncb5or-null mouse is insulin-dependent diabetes due to ß-cell death. This suggests the putative role of NCB5OR polymorphism in human diabetes. Therefore, we aimed to investigate the effect of natural missense mutations on the expression of human NCB5OR. Protein and mRNA levels of five non-synonymous coding variants were analyzed in transfected HEK293 and HepG2 cells. Although the mRNA levels were only slightly affected by the mutations, the amount of Ncb5or protein was largely reduced upon two Glu to Gly replacements in the third exon (p.E87G, p.E93G). These two mutations remarkably and synergistically shortened the half-life of Ncb5or and their effect could be attenuated by proteasome inhibitors. Our results strongly indicate that p.E87G, p.E93G mutations lead to enhanced proteasomal degradation due to manifest conformational alterations in the b5 domain. These data provide first evidence for natural mutations in NCB5OR gene resulting in decreased protein levels and hence having potential implications in human pathology.

Altered gene expression profiles in the hippocampus and prefrontal cortex of type 2 diabetic rats.

BACKGROUND: There has been an increasing body of epidemiologic and biochemical evidence implying the role of cerebral insulin resistance in Alzheimer-type dementia. For a better understanding of the insulin effect on the central nervous system, we performed microarray-based global gene expression profiling in the hippocampus, striatum and prefrontal cortex of streptozotocin-induced and spontaneously diabetic Goto-Kakizaki rats as model animals for type 1 and type 2 diabetes, respectively. RESULTS: Following pathway analysis and validation of gene lists by real-time polymerase chain reaction, 30 genes from the hippocampus, such as the inhibitory neuropeptide galanin, synuclein gamma and uncoupling protein 2, and 22 genes from the prefrontal cortex, e.g. galanin receptor 2, protein kinase C gamma and epsilon, ABCA1 (ATP-Binding Cassette A1), CD47 (Cluster of Differentiation 47) and the RET (Rearranged During Transfection) protooncogene, were found to exhibit altered expression levels in type 2 diabetic model animals in comparison to non-diabetic control animals. These gene lists proved to be partly overlapping and encompassed genes related to neurotransmission, lipid metabolism, neuronal development, insulin secretion, oxidative damage and DNA repair. On the other hand, no significant alterations were found in the transcriptomes of the corpus striatum in the same animals. Changes in the cerebral gene expression profiles seemed to be specific for the type 2 diabetic model, as no such alterations were found in streptozotocin-treated animals. CONCLUSIONS: According to our knowledge this is the first characterization of the whole-genome expression changes of specific brain regions in a diabetic model. Our findings shed light on the complex role of insulin signaling in fine-tuning brain functions, and provide further experimental evidence in support of the recently elaborated theory of type 3 diabetes.

The endoplasmic reticulum as the extracellular space inside the cell: role in protein folding and glycosylation.

SIGNIFICANCE: Proteins destined to secretion and exposure on the cell surface are synthesized and processed in the extracellular-like environment of the endoplasmic reticulum (ER) of higher eukaryotic cells. Compartmentation plays a crucial role in the post-translational modifications, such as oxidative folding and N-glycosylation in the ER lumen. Transport of the required intermediates across the ER membrane and maintenance of the luminal redox conditions and Ca(2+) ion concentration are indispensable for appropriate protein maturation. RECENT ADVANCES: Cooperation of enzymes and transporters to maintain a thiol-oxidizing milieu in the ER lumen has been recently elucidated. Ca(2+)-dependence of certain ER chaperones is a subject of intensive research. CRITICAL ISSUES: Mounting evidence supports the existence of a real barrier between the ER lumen and the cytosol. The unique set of enzymes, selection of metabolites, and characteristic ion and redox milieu of the luminal compartment strongly argue against the general permeability of the ER membrane. FUTURE DIRECTIONS: Alterations in the luminal environment can trigger the unfolded protein response, a common event in a variety of pathological conditions. Therefore, redox and calcium homeostasis and protein glycosylation in the ER provide novel drug-targets for medical treatment in a wide array of diseases.