We investigated the biochemical properties and cellular expression of the c.346C>T (p.R116C) human cationic trypsinogen (PRSS1) mutant, which we identified in a German family with autosomal dominant hereditary pancreatitis. This mutation leads to an unpaired Cys residue with the potential to interfere with protein folding via incorrect disulfide bond formation. Recombinantly expressed p.R116C trypsinogen exhibited a tendency for misfolding in vitro. Biochemical analysis of the correctly folded, purified p.R116C mutant revealed unchanged activation and degradation characteristics compared to wild type trypsinogen. Secretion of mutant p.R116C from transfected 293T cells was reduced to ~20% of wild type. A similar secretion defect was observed with another rare PRSS1 variant, p.C139S, whereas mutants p.A16V, p.N29I, p.N29T, p.E79K, p.R122C, and p.R122H were secreted normally. All mutants were detected in cell extracts at comparable levels but a large portion of mutant p.R116C was present in an insoluble, protease-sensitive form. Consistent with intracellular retention of misfolded trypsinogen, the endoplasmic reticulum (ER) stress markers BiP and XBP1s were elevated in cells expressing mutant p.R116C. The results indicate that mutation induced misfolding and intracellular retention of human cationic trypsinogen causes hereditary pancreatitis in carriers of the p.R116C mutation. ER stress triggered by trypsinogen misfolding represents a new potential disease mechanism for chronic pancreatitis.

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. Key words: Saturated fatty acid; Lipotoxicity; Steatosis; Lipoapoptosis; Endoplasmic reticulum stress Core tip: Surplus of free fatty acids contributes to hepatic injuries in obesity and type 2 diabetes. Intracellular accumulation of fatty acyl-CoA causes oxidative and endoplasmic reticulum (ER) stress, which lead to cell death, inflammation and fibrosis. Steatohepatosis is the consequence of an intensive fat synthesis, aiming to reduce the metabolic burden. The higher toxicity of saturated vs unsaturated fatty acids is partly due to a limited capacity of the liver cells to insert them into triglycerides. Moreover, increased membrane saturation triggers the ER stress response though a unique mechanism, which aggravates the metabolic derangements and liver injuries.

Methylphenidate is the most frequently prescribed drug in the treatment of attention deficit hyperactivity disorder (ADHD) but it is not effective in every case. Therefore, identifying genetic and/or biological markers predicting drug-response is increasingly important. Here we present a case-control study and pharmacogenetic association analyses in ADHD investigating three dopaminergic polymorphisms. Previous studies suggested variable number of tandem repeats (VNTR) in the dopamine D4 receptor (DRD4) and the dopamine transporter (DAT1) genes as genetic risk factors for ADHD and as possible markers of methylphenidate response. Our results did not indicate substantial involvement of these two VNTRs in ADHD, however, both the case-control and the pharmacogenetic analyses showed significant role of the high activity Val-allele of cathecol-O-methyltransferase (COMT) Val158Met polymorphism in our ADHD population. The Val-allele was more frequent in the ADHD group (n = 173) compared to the healthy population (P = 0.016). The categorical analysis of 90 responders versus 32 non-responders showed an association between the Val-allele or Val/Val genotype and good methylphenidate response (P = 0.009 and P = 0.034, respectively). Analyzing symptom severity as a continuous trait, significant interaction of COMT genotype and methylphenidate was found on the Hyperactivity-Impulsivity scale (P = 0.044). Symptom severity scores of all three genotype groups decreased following methylphenidate administration (P < 0.001), however Val/Val homozygote children had significantly less severe symptoms than those with Met/Met genotype after treatment (P = 0.015). This interaction might reflect the regulatory effect of COMT dominated prefrontal dopamine transmission on subcortical dopamine systems, which are the actual site of methylphenidate action.

BACKGROUND AND AIMS-Two common haplotypes of the serine protease inhibitor Kazal type 1 (SPINK1) gene have been shown to increase the risk for chronic pancreatitis. A haplotype comprising the c.101A>G (p.N34S) missense variant and four intronic alterations has been found worldwide, whereas a second haplotype consisting of the c. −215G>A promoter variant and the c. 194+2T>C intronic alteration has been observed frequently in Japan.

Mutations in the activation peptide of human cationic trypsinogen have been found in patients with chronic pancreatitis. Previous biochemical studies demonstrated that mutations p.D19A, p.D22G, and p.K23R strongly stimulate trypsinogen autoactivation. In the present study, we characterized the cell biological effects of these mutants using human embryonic kidney 293T and AR42J rat acinar cells. We found that relative to wild-type trypsinogen, secretion of the mutants from transfected cells was markedly decreased. This apparent secretion defect was completely rescued by inhibition of autoactivation via (1)

Abnormalities of the dopamine neurotransmission have been hypothesized to play an important role in the pathophysiology of attention deficit hyperactivity disorder (ADHD). Promoter variants of the dopamine D4 receptor gene (DRD4) have attracted particular interest due to their possible role in regulation of gene transcription. Here we describe the haplotype analysis of the 120 base pair duplication (120-bp dup) and three SNPs (-616C/G, -615A/G, -521C/T) in the 5' region of the DRD4 gene among children with ADHD. We observed a trend (chi(2) = 14.905, df = 9, P = 0.093) in the four-locus haplotype distribution between ADHD probands (N = 173) and controls (N = 284). The homozygote genotype of the 1-repeat form of the 120-bp dup (1-1) had a significantly higher frequency among ADHD children than in controls (8.1% vs. 3.2%, chi(2) = 5.526, df = 1, P = 0.019, Odds Ratio = 2.71). In addition, a novel, 4-repeat allele was identified among ADHD patients. This particular allele has been cloned to the luciferase expression vector and its transcriptional activity has been compared to the 1- and 2-repeat allele. The number of repeats of the 120-bp dup was found to have an effect on transcriptional activity in both neuroblastoma and retinoblastoma cell lines in a dose-dependent manner (1-repeat > 2-repeat > 4-repeat). These results suggest that the 1-repeat form of the 120-bp dup might be a risk factor of ADHD, especially in the homozygous form and/or in the context of certain haplotypes.

Genetic polymorphisms in the human dopamine D4 receptor (DRD4) gene, especially the exon 3 variable number of tandem repeats (VNTR), have been related to several psychiatric disorders and personality traits. A homologous exon 3 VNTR has been described in dogs, and we previously showed an association between the DRD4 exon 3 polymorphism and activity/impulsivity trait in German shepherds. In this study, we present a detailed analysis of the intron 2 VNTR of the DRD4 gene. A short and a long form of the intronic variation were identified in 678 unrelated dogs from five breeds and in 22 wolves. For molecular analysis, the intron 2 region was cloned into a promoterless luciferase reporter vector that led to an elevation in transcriptional activity. Moreover, an allelic difference in promoter activity was detected, and a repressive effect of the long allele was observed. Behavioral analysis of 96 unrelated German shepherds showed a significant association between the social impulsivity endophenotype of the Greeting Test and both the exonic (P 5 0.002) and the intronic (P 5 0.003) VNTRs of the DRD4 gene. Moreover, an additive effect of the two polymorphisms was also shown (Spearman's rho 5 0.356, P 5 0.0004). In conclusion, these results give further support to our previous findings that the DRD4 gene is associated with dog behavior. We also present molecular evidence for the functional role of the intron 2 VNTR in the canine DRD4 gene.

BackgroundThere 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.ResultsFollowing 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.ConclusionsAccording 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.