Postnatally disturbed pancreatic islet cell distribution in human islet amyloid polypeptide transgenic mice

OBJECTIVE: Islet amyloid polypeptide (IAPP)/amylin is produced by the pancreatic islet beta-cells, which also produce insulin. To study potential functions of IAPP, we have generated transgenic mice overexpressing human IAPP (hIAPP) in the beta-cells. These mice show a diabetic phenotype when challenged with an oral glucose load. In this study, we examined the islet cytoarchitecture in the hIAPP mice by examining islet cell distribution in the neonatal period, as well as 1, 3 and 6 months after birth. RESULTS: Neonatal transgenic mice exhibited normal islet cell distribution with beta-cells constituting the central islet portion, whereas glucagon and somatostatin-producing cells constituted the peripheral zone. In contrast, in hIAPP transgenic mice at the age of 1 month, the glucagon-immunoreactive (IR) cells were dispersed throughout the islets. Furthermore, at the age of 3 and 6 months, the islet organisation was similarly severely disturbed as at 1 month. Expression of both endogenous mouse IAPP and transgenic hIAPP was clearly higher in 6-month-old mice as compared to newborns, as revealed by mRNA in situ hybridisation. CONCLUSIONS: Mice transgenic for hIAPP have islets with disrupted islet cytoarchitecture in the postnatal period, particularly affecting the distribution of glucagon-IR cells. This islet cellular phenotype of hIAPP transgenic mice is similar to that of other mouse models of experimental diabetes and might contribute to the impaired glucose homeostasis.     

Identification of essential genes in the human fungal pathogen Aspergillus fumigatus by transposon mutagenesis

The opportunistic pathogen Aspergillus fumigatus is the most frequent cause of deadly airborne fungal infections in developed countries. In order to identify novel antifungal-drug targets, we investigated the genome of A. fumigatus for genes that are necessary for efficient fungal growth. An artificial A. fumigatus diploid strain with one copy of an engineered impala160 transposon from Fusarium oxysporum integrated into its genome was used to generate a library of diploid strains by random in vivo transposon mutagenesis. Among 2,386 heterozygous diploid strains screened by parasexual genetics, 1.2% had a copy of the transposable element integrated into a locus essential for A. fumigatus growth. Comparison of genomic sequences flanking impala160 in these mutants with that of the genome of A. fumigatus allowed the characterization of 20 previously uncharacterized A. fumigatus genes. Among these, homologues of genes essential for Saccharomyces cerevisiae growth have been identified, as well as genes that do not have homologues in other fungal species. These results confirm that heterologous transposition using the transposable element impala is a powerful tool for functional genomics in ascomycota, and they pave the way for defining the complete set of essential genes in A. fumigatus, the first step toward target-based development of new antifungal drugs.     

Dual benefit of reduced Cx43 on atherosclerosis in LDL receptor-deficient mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR(-/-)) mice were intercrossed with mice heterozygous for Cx43 (Cx43(+/-) mice). Male mice with normal (Cx43(+/+)LDLR(-/-)) or reduced (Cx43(+/-)LDLR(-/-)) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43(+/-)LDLR(-/-) mice compared with Cx43(+/+)LDLR(-/-) littermate controls. In addition, atheroma in Cx43(+/-)LDLR(-/-) mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

Evolvability and genetic constraint in Dalechampia blossoms: genetic correlations and conditional evolvability

The short-term evolvability of a character is closely related to its level of additive genetic variation. However, a large component of the variation in any one character may be pleiotropically linked to other characters under the influence of different selective factors. Therefore, the organization of the organism into quasi-independent modules may be an important prerequisite for evolvability. In this paper we propose to study character evolvability in terms of conditional genetic variation. By estimating the amount of genetic variation in a character, y, that is independent of other characters, x, we can assess the evolvability of y when there is stabilizing selection on x. We suggest that systematic use of conditioning may help build a picture of modular organization and quasi-independent evolvability. As an illustration, we use this approach to assess the evolvability of floral characters in Dalechampia scandens (Euphorbiaceae). Although our study population had relatively low levels of genetic variation at the outset, we find evidence that conditioning may lead to substantial further reduction in the genetic variation available for independent adaptation. This provides additional evidence that the D. scandens blossom is constrained in its short-term evolvability.     

Automated large-volume sample stacking procedure to detect labeled peptides at picomolar concentration using capillary electrophoresis and laser-induced fluorescence detection

We have developed an automated large-volume sample stacking (LVSS) procedure to detect fluorescein isothiocyanate-labeled peptides in the picomolar range. The injection duration is 10 min at 50 mbar to fill 62% of the capillary volume to the detection cell. The calculated limit of detection (S/N=3), filling 1% of the capillary volume, is 74 pM for bradykinin and 45 pM for L-enkephalin with samples diluted in water and analyzed in a 50 mM borate buffer, pH 9.2. With the automated LVSS system, the limits of detection are 7 pM for bradykinin, 3 pM for L-enkephalin and 2 pM for substance P. LVSS is shown to be quantitative from 500 to 10 pM.     

Proteomic analysis of Lactococcus lactis,a lactic acid bacterium

Lactococcus lactis is a Gram-positive bacteria, which belongs to the group of lactic acid bacteria among which several genera play an essential role in the manufacture of food products. Cytosolic proteins of L. lactis IL1403 cultivated in M17 broth have been resolved by two-dimensional gel electrophoresis using two pH gradients (pH 4-7, 4.5-5.5). More than 230 spots were identified by peptide mass fingerprints, corresponding to 25% of the predicted acid proteome. The present study made it possible to describe at the proteome level a significant number of cellular pathways (glycolysis, fermentation, nucleotide metabolism, proteolysis, fatty acid and peptidoglycan synthesis) related to important physiological processes and technological properties. It also indicated that the fermentative metabolism, which characterizes L. lactis is associated with a high expression of glycolytic enzymes. Thirty-four proteins were matched to open reading frames for which there is no assigned function. The comparison at the proteome level of two strains of L. lactis showed an important protein polymorphism. The comparison of the proteomes of glucose- and lactose-grown cells revealed an unexpected link between the nature of the carbon source and the metabolism of pyrimidine nucleotides.     

The MUR3 gene of Arabidopsis encodes a xyloglucan galactosyltransferase that is evolutionarily related to animal exostosins

Xyloglucans are the principal glycans that interlace cellulose microfibrils in most flowering plants. The mur3 mutant of Arabidopsis contains a severely altered structure of this polysaccharide because of the absence of a conserved alpha-L-fucosyl-(1-->2)-beta-D-galactosyl side chain and excessive galactosylation at an alternative xylose residue. Despite this severe structural alteration, mur3 plants were phenotypically normal and exhibited tensile strength in their inflorescence stems comparable to that of wild-type plants. The MUR3 gene was cloned positionally and shown to encode a xyloglucan galactosyltransferase that acts specifically on the third xylose residue within the XXXG core structure of xyloglucan. MUR3 belongs to a large family of type-II membrane proteins that is evolutionarily conserved among higher plants. The enzyme shows sequence similarities to the glucuronosyltransferase domain of exostosins, a class of animal glycosyltransferases that catalyze the synthesis of heparan sulfate, a glycosaminoglycan with numerous roles in cell differentiation and development. This finding suggests that components of the plant cell wall and of the animal extracellular matrix are synthesized by evolutionarily related enzymes even though the structures of the corresponding polysaccharides are entirely different from each other.