Endocytosis of Secreted Carboxyl Ester Lipase in a Syndrome of Diabetes and Pancreatic Exocrine Dysfunction

Maturity-onset diabetes of the young, type 8 (MODY8) is characterized by a syndrome of autosomal dominantly inherited diabetes and exocrine pancreatic dysfunction. It is caused by deletion mutations in the last exon of the carboxyl ester lipase (CEL) gene, resulting in a CEL protein with increased tendency to aggregate. In this study we investigated the intracellular distribution of the wild type (WT) and mutant (MUT) CEL proteins in cellular models. We found that both CEL-WT and CEL-MUT were secreted via the endoplasmic reticulum and Golgi compartments. However, their subcellular distributions differed, as only CEL-MUT was observed as an aggregate at the cell surface and inside large cytoplasmic vacuoles. Many of the vacuoles were identified as components of the endosomal system, and after its secretion, the mutant CEL protein was re-internalized, transported to the lysosomes, and degraded. Internalization of CEL-MUT also led to reduced viability of pancreatic acinar and beta cells. These findings may have implications for the understanding of how the acinar-specific CEL-MUT protein causes both exocrine and endocrine pancreatic disease.     

Diabetes and pancreatic exocrine dysfunction due to mutations in the carboxyl ester lipase gene-maturity onset diabetes of the young (CEL-MODY): a protein misfolding disease

CEL-maturity onset diabetes of the young (MODY), diabetes with pancreatic lipomatosis and exocrine dysfunction, is due to dominant frameshift mutations in the acinar cell carboxyl ester lipase gene (CEL). As Cel knock-out mice do not express the phenotype and the mutant protein has an altered and intrinsically disordered tandem repeat domain, we hypothesized that the disease mechanism might involve a negative effect of the mutant protein. In silico analysis showed that the pI of the tandem repeat was markedly increased from pH 3.3 in wild-type (WT) to 11.8 in mutant (MUT) human CEL. By stably overexpressing CEL-WT and CEL-MUT in HEK293 cells, we found similar glycosylation, ubiquitination, constitutive secretion, and quality control of the two proteins. The CEL-MUT protein demonstrated, however, a high propensity to form aggregates found intracellularly and extracellularly. Different physicochemical properties of the intrinsically disordered tandem repeat domains of WT and MUT proteins may contribute to different short and long range interactions with the globular core domain and other macromolecules, including cell membranes. Thus, we propose that CEL-MODY is a protein misfolding disease caused by a negative gain-of-function effect of the mutant proteins in pancreatic tissues.     

Length of Variable Numbers of Tandem Repeats in the Carboxyl Ester Lipase (CEL) Gene May Confer Susceptibility to Alcoholic Liver Cirrhosis but Not Alcoholic Chronic Pancreatitis

BackgroundCarboxyl-ester lipase (CEL) contributes to fatty acid ethyl ester metabolism, which is implicated in alcoholic pancreatitis. The CEL gene harbours a variable number of tandem repeats (VNTR) region in exon 11. Variation in this VNTR has been linked to monogenic pancreatic disease, while conflicting results were reported for chronic pancreatitis (CP). Here, we aimed to investigate a potential association of CEL VNTR lengths with alcoholic CP.MethodsOverall, 395 alcoholic CP patients, 218 patients with alcoholic liver cirrhosis (ALC) serving as controls with a comparable amount of alcohol consumed, and 327 healthy controls from Germany and the United Kingdom (UK) were analysed by determination of fragment lengths by capillary electrophoresis. Allele frequencies and genotypes of different VNTR categories were compared between the groups.ResultsTwelve repeats were overrepresented in UK ACP patients (P = 0.04) compared to controls, whereas twelve repeats were enriched in German ALC compared to alcoholic CP patients (P = 0.03). Frequencies of CEL VNTR lengths of 14 and 15 repeats differed between German ALC patients and healthy controls (P = 0.03 and 0.008, respectively). However, in the genotype and pooled analysis of VNTR lengths no statistical significant association was depicted. Additionally, the 16–16 genotype as well as 16 repeats were more frequent in UK ALC than in alcoholic CP patients (P = 0.034 and 0.02, respectively). In all other calculations, including pooled German and UK data, allele frequencies and genotype distributions did not differ significantly between patients and controls or between alcoholic CP and ALC.ConclusionsWe did not obtain evidence that CEL VNTR lengths are associated with alcoholic CP. However, our results suggest that CEL VNTR lengths might associate with ALC, a finding that needs to be clarified in larger cohorts.     

NK1, NK2, NK3 and CGRP1 receptors identified in rat oral soft tissues,and in bone and dental hard tissue cells

The distribution of the tachykinin receptors neurokinin-1 (NK1), neurokinin-2 (NK2) and neurokinin-3 (NK3), and the calcitonin gene-related peptide-1 (CGRP1) receptor were examined in rat teeth and tooth-supporting tissues by immunohistochemical methods and light and confocal microscopy. Western blot analysis was performed to identify the NK1- and the CGRP1-receptor proteins in the dental pulp. The results showed that odontoblasts and ameloblasts, cementoblasts and cementocytes, osteoblasts and osteocytes are all supported with the tachykinin receptors NK1 and NK2, but a distinct, graded cellular labeling pattern was demonstrated. The ameloblasts were also positive for CGRP1 receptor. Blood vessels in oral tissues expressed the tachykinin receptors NK1, NK2 and NK3, and the CGRP1 receptor. Both gingival and Malassez epithelium were abundantly supplied by NK2 receptor. Pulpal and periodontal fibroblasts demonstrated NK1 and NK2 receptors. Western blot analysis identified both the NK1- and the CGRP1-receptor proteins in the dental pulp. These results clearly indicate that the neuropeptides substance P, neurokinin A, neurokinin B and CGRP, released from sensory axons upon stimulation, directly modulate the function of the different types of bone and dental hard tissue cells, and regulate functions of blood vessels, fibroblasts and epithelial cells in oral tissues.     

Transfection efficiency and cytotoxicity of cationic liposomes in primary cultures of rainbow trout (Oncorhynchus mykiss) gill cells

Immunisation of fish by immersion has been applied for inactivated, whole cell bacterins, where the gill epithelial cells are considered as one of the prime uptake sites. Antigen entry is a critical factor for delivery of vaccine antigens through the immersion route, also for DNA vaccines, and delivery systems like cationic liposomes may enhance uptake. In this study, the aim was to examine the efficiency of cationic liposomes as a means to transfect primary cultures of rainbow trout gill cells with plasmids encoding viral or reporter proteins. Furthermore, the effects of the concentration and composition of liposomes/lipoplex on the viability of the cells were evaluated. Transfection of the gill cells was possible with both plasmids following transfection with lipoplexes of a neutral charge. Low concentrations and neutral/negatively charged formulations were favourable with respect to the toxicity of the formulations. Given that the mucous barrier covering the gills is overcome, this system might be useful for the priming of the local immunity in the fish gills.     

Temporal relation between leukocyte accumulation in muscles and halted recovery 10-20 h after strength exercise.

Effects of normal strength exercise on leukocyte accumulation were examined in 10 well-trained male subjects (27.2 +/- 2.7 yr). The workout, consisting of five maximal sets of three repetitions of leg press exercise and five maximal sets of six repetitions of knee extension exercise, was performed with the dominant leg, and the other leg served as control. Repeated maximal isokinetic knee extensions at 60 degrees /s were performed to evaluate neuromuscular fatigue and recovery after the workout. Accumulation of leukocytes was assessed with 99mTc-labeled cells, and repeated images of the thighs were taken 1-24 h after the workout. Maximal force-generating capacity in the exercised leg was reduced by 17 +/- 2% (P < 0.01) after the workout. The course of recovery followed a biphasic pattern characterized by halted recovery 10-23 h after exercise. The presence of leukocytes was approximately 10% higher in the exercised than in the control thigh 10 h after exercise (P < 0.05). This difference increased to approximately 15% at 20 h after exercise (P < 0.05). The retarded recovery of maximal force-generating capacity 10-20 h after exercise, together with a significant infiltration of leukocytes in exercised muscle during the same time interval, shows a temporal relation between leukocyte infiltration and impaired recovery.     

DNA markers for Fusarium head blight resistance QTLs in two wheat populations

Genetic resistance to Fusarium head blight (FHB), caused by Fusarium graminearum, is necessary to reduce the wheat grain yield and quality losses caused by this disease. Development of resistant cultivars has been slowed by poorly adapted and incomplete resistance sources and confounding environmental effects that make screening of germplasm difficult. DNA markers for FHB resistance QTLs have been identified and may be used to speed the introgression of resistance genes into adapted germplasm. This study was conducted to identify and map additional DNA markers linked to genes controlling FHB resistance in two spring wheat recombinant inbred populations, both segregating for genes from the widely used resistance source ’Sumai 3’. The first population was from the cross of Sumai 3/Stoa in which we previously identified five resistance QTLs. The second population was from the cross of ND2603 (Sumai 3/Wheaton) (resistant)/ Butte 86 (moderately susceptible). Both populations were evaluated for reaction to inoculation with F. graminearum in two greenhouse experiments. A combination of 521 RFLP, AFLP, and SSR markers were mapped in the Sumai 3/Stoa population and all DNA markers associated with resistance were screened on the ND2603/Butte 86 population. Two new QTL on chromosomes 3AL and 6AS wer found in the ND2603/Butte 86 population, and AFLP and SSR markers were identified that explained a greater portion of the phenotypic variation compared to the previous RFLP markers. Both of the Sumai 3-derived QTL regions (on chromosomes 3BS, and 6BS) from the Sumai 3/Stoa population were associated with FHB resistance in the ND2603/Butte 86 population. Markers in the 3BS QTL region (Qfhs.ndsu-3BS) alone explain 41.6 and 24.8% of the resistance to FHB in the Sumai 3/Stoa and ND2603/Butte 86 populations, respectively. This region contains a major QTL for resistance to FHB and should be useful in marker-assisted selection. Received: 17 August 2000 / Accepted: 16 October 2000     

Identification of a novel putative signaling center,the tertiary enamel knot in the postnatal mouse molar tooth

The final shape of the molar tooth crown is thought to be regulated by the transient epithelial signaling centers in the cusp tips, the secondary enamel knots (SEKs), which are believed to disappear after initiation of the cusp growth. We investigated the developmental fate of the signaling center using the recently characterized Slit1 enamel knot marker as a lineage tracer during morphogenesis of the first molar and crown calcification in the mouse. In situ hybridization analysis showed that after Fgf4 downregulation in the SEK, Slit1 expression persisted in the deep compartment of the knot. After the histological disappearance of the SEK, Slit1 expression was evident in a novel epithelial cell cluster, which we call the tertiary enamel knot (TEK) next to the enamel-free area (EFA)-epithelium at the cusp tips. In embryonic tooth, Slit1 was also observed in the stratum intermedium (SI) and stellate reticulum cells between the parallel SEKs correlating to the area where the inner enamel epithelium cells do not proliferate. After birth, the expression of Slit1 persisted in the SI cells of the transverse connecting lophs of the parallel cusps above the EFA-cells. These results demonstrate the presence of a novel putative signaling center, the TEK, in the calcifying tooth. Moreover, our results suggest that Slit1 signaling may be involved in the regulation of molar tooth shape by regulating epithelial cell proliferation and formation of EFA of the crown.