Disruption of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsulinemia

In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and site-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a beta-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.     

Importance of topography and soil texture in the spatial distribution of two sympatric dipterocarp trees in a Bornean rainforest

Relationships between spatial distributions and site conditions, namely topography and soil texture, were analyzed for two congeneric emergent trees, Dryobalanops aromatica and Dryobalanops lanceolata (Dipterocarpaceae), in a tropical rainforest in Sarawak, East Malaysia. A 52-ha permanent plot was divided into 1300 quadrats measuring 20m×20m; for each Dryobalanops species, the number and total basal area of trees 1cm in d.b.h. were compared among groups of quadrats with different site conditions. Because spatial distributions of both Dryobalanops and site-condition variables were aggregated, Monte-Carlo permutation tests were applied to analyze the relationships. Both single and multifactor statistical tests showed that the density and basal area distributions of the two species were significantly non-random in relation to soil texture and topographic variables. D.aromatica was significantly more abundant at higher elevations, in sandy soils, and on convex and steep slopes. In contrast, D.lanceolata preferred lower elevations and less sandy soils. In the study plot, there were very few sites (3 of 1150 quadrats tested) where the models of Hayashis method predicted the co-occurrence of the two species. These results suggest that between-species differences in habitat preferences are so large that they alone explain the spatially segregated distributions of these two species within the 52-ha study plot.     

White spot syndrome virus (WSSV) in cultured Penaeus monodon in the Philippines

The prevalence and geographic distribution of white spot syndrome virus (WSSV) infection among cultured penaeid shrimp in the Philippines was determined from January to May, 1999, using PCR (polymerase chain reaction) protocol and Western blot assays. A total of 71 samples consisting of 18 post-larvae (PL) and 53 juvenile/adult shrimp samples (56 to 150 days-of-culture, DOC) were screened for WSSV. Of the 71 samples tested, 51 (72%) were found positive for WSSV by PCR: 61% (31/51) after 1-step PCR and 39% (20/51) after 2-step, non-nested PCR. Of the PL and juvenile/adult shrimp samples tested, 50 and 79% were positive for WSSV, respectively. By Western blot, only 6 of the 51 (12%) PCR-positive samples tested positive for WSSV. Of the 20 samples negative for WSSV by PCR, all tested negative for WSSV by Western blot assay. This is the first report of the occurrence of WSSV in the Philippines.     

Growth kinetics of Pseudomonas putida in cometabolism of phenol and 4-chlorophenol in the presence of a conventional carbon source

Growth kinetics of Pseudomonas putida (ATCC 49451) in cometabolism of phenol and 4-chlorophenol (4-cp) in the presence of sodium glutamate (SG) were studied. In the ternary substrate mixture, phenol and SG are growth substrates while 4-cp is a nongrowth substrate. Cell growth on phenol was found to follow Andrews kinetics and cells displayed substrate inhibition pattern on sodium glutamate in the range of 0-4 g L(-1) as well. A cell growth model for the ternary substrate system was established based on a simplified cell growth mechanism and subsequently modified by experimental results. Model analysis over a wide range of substrate concentrations shows that the inhibition of SG is much larger than phenol at low phenol concentrations (/=600 mg L(-1)). The nongrowth substrate, 4-cp, inhibits cell growth mainly through inactivation of cells (cell decay) and competitive inhibition to cell growth on phenol. In the absence of SG, 4-cp retards cell growth severely and cells cannot grow at 250 mg L(-1) 4-cp. Addition of sodium glutamate, however, greatly attenuates the toxicity of 4-cp and supports cell growth at 4-cp concentration higher than 250 mg L(-1). By using the proposed cell growth model, we were able to optimize the amount of SG needed to enhance cell growth rate and validate model predictions against experimental data.     

Amplified fragment length polymorphism fingerprinting of 16 banana cultivars (Musa cvs.)

Banana is one of the most important subtropical crops. The genetic system, however, is relatively unknown and is complicated by specific interhybridization, heterozygosity, and polyploidy, which are common in most clones. These factors make identification of closely related banana cultivars difficult, particularly when sterile. Amplified fragment length polymorphism (AFLP) analysis using eight primer combinations was carried out on 16 banana cultivars. Results showed that AFLP could be used to distinguish the different cultivars by their unique banding patterns. Unique AFLP molecular markers were detected for 12 banana cultivars, which can be used to develop specific probes for identification purposes. The cluster analysis also revealed the need for a link between genotype studies using molecular techniques and the current system of classification of Musa cultivars based purely on morphological traits.     

The SNARE Machinery Is Involved in Apical Plasma Membrane Trafficking in MDCK Cells

We have investigated the controversial involvement of components of the SNARE (soluble N-ethyl maleimide–sensitive factor [NSF] attachment protein [SNAP] receptor) machinery in membrane traffic to the apical plasma membrane of polarized epithelial (MDCK) cells. Overexpression of syntaxin 3, but not of syntaxins 2 or 4, caused an inhibition of TGN to apical transport and apical recycling, and leads to an accumulation of small vesicles underneath the apical plasma membrane. All other tested transport steps were unaffected by syntaxin 3 overexpression. Botulinum neurotoxin E, which cleaves SNAP-23, and antibodies against α-SNAP inhibit both TGN to apical and basolateral transport in a reconstituted in vitro system. In contrast, we find no evidence for an involvement of N-ethyl maleimide–sensitive factor in TGN to apical transport, whereas basolateral transport is NSF-dependent. We conclude that syntaxin 3, SNAP-23, and α-SNAP are involved in apical membrane fusion. These results demonstrate that vesicle fusion with the apical plasma membrane does not use a mechanism that is entirely unrelated to other cellular membrane fusion events, but uses isoforms of components of the SNARE machinery, which suggests that they play a role in providing specificity to polarized membrane traffic.     

Genomic organization and refined mapping of the mouse β-dystrobrevin gene

β-Dystrobrevin, a dystrophin-related protein that is expressed in non-muscle tissues, is highly homologous to α-dystrobrevin, a member of the dystrophin-associated protein complex (DPC). β-Dystrobrevin associates with Dp71 and syntrophin and is believed to have a role in non-muscle DPCs. Here we report the characterization and mapping of the mouse β-dystrobrevin gene. The mouse β-dystrobrevin gene is organized into 21 exons spanning over 130 kb of DNA. We provide evidence that this gene is transcribed from at least two promoter regions but appears to utilize a common translation initiation site. We show that the similarity between β-dystrobrevin and α-dystrobrevin is reflected in the conservation of their exon-intron junctions. β-Dystrobrevin has been localized to proximal mouse Chromosome (Chr) 12 by backcross mapping. A database search revealed that two mouse genetic diseases involving tissues expressing β-dystrobrevin have been mapped to this region, namely, congenital polycystic kidneys (cpk) and fatty liver dystrophy (fld). However, refined mapping analysis has excluded β-dystrobrevin as a candidate gene for either disease. Received: 1 June 1998 / Accepted: 16 July 1998