The effects of mating system and genetic variability on susceptibility to trematode parasites in a freshwater snail, Lymnaea stagnalis

The amount and distribution of genetic variability in host populations can have significant effects on the outcome of host-parasite interactions. We studied the effect of mating system and genetic variability on susceptibility of Lymnaea stagnalis snails to trematode parasites. Mating system of snails from eight populations differing in the amount of genetic variability was manipulated, and self- and cross-fertilized offspring were exposed to naturally occurring trematode parasites in a controlled lake experiment. Susceptibility of snails varied between populations, but mating-system treatment did not have a significant effect. Heterozygosity of snails was negatively correlated with the probability of trematode infection, however, suggesting that parasitic diseases may pose a serious threat to populations lacking genetic variability.     

Calpains promote α2β1 integrin turnover in nonrecycling integrin pathway

Collagen receptor integrins recycle between the plasma membrane and endosomes and facilitate formation and turnover of focal adhesions. In contrast, clustering of α2β1 integrin with antibodies or the human pathogen echovirus 1 (EV1) causes redistribution of α2 integrin to perinuclear multivesicular bodies, α2-MVBs. We show here that the internalized clustered α2 integrin remains in α2-MVBs and is not recycled back to the plasma membrane. Instead, receptor clustering and internalization lead to an accelerated down-regulation of α2β1 integrin compared to the slow turnover of unclustered α2 integrin. EV1 infection or integrin degradation is not associated with proteasomal or autophagosomal processes and shows no significant association with lysosomal pathway. In contrast, degradation is dependent on calpains, such that it is blocked by calpain inhibitors. We show that active calpain is present in α2-MVBs, internalized clustered α2β1 integrin coprecipitates with calpain-1, and calpain enzymes can degrade α2β1 integrin. In conclusion, we identified a novel virus- and clustering-specific pathway that diverts α2β1 integrin from its normal endo/exocytic traffic to a nonrecycling, calpain-dependent degradative endosomal route.     

Apple proteins that interact with DspA/E, a pathogenicity effector of Erwinia amylovora, the fire blight pathogen

The disease-specific (dsp) gene dspA/E of Erwinia amylovora encodes an essential pathogenicity effector of 198 kDa, which is critical to the development of the devastating plant disease fire blight. A yeast two-hybrid assay and in vitro protein pull-down assay demonstrated that DspA/E interacts physically and specifically with four similar putative leucine-rich repeat (LRR) receptor-like serine/threonine kinases (RLK) from apple, an important host of E. amylovora. The genes encoding these four DspA/E-interacting proteins of Malus xdomestica (DIPM1 to 4) are conserved in all genera of hosts of E. amylovora tested. They also are conserved in all cultivars of apple tested that range in susceptibility to fire blight from highly susceptible to highly resistant. The four DIPMs have been characterized, and they are expressed constitutively in host plants. In silico analysis indicated that the DIPMs have similar sequence structure and resemble LRR RLKs from other organisms. Evidence is presented for direct physical interaction between DspA/E and the apple proteins encoded by the four identified clones, which may act as susceptibility factors and be essential to disease development. Knowledge of DIPMs and the interaction with DspA/E thus may facilitate understanding of fire blight development and lead to new approaches to control of disease.     

Substitution of a conserved cysteine-996 in a cysteine-rich motif of the laminin alpha2-chain in congenital muscular dystrophy with partial deficiency of the protein.

Congenital muscular dystrophies (CMDs) are autosomal recessive muscle disorders of early onset. Approximately half of CMD patients present laminin alpha2-chain (merosin) deficiency in muscle biopsies, and the disease locus has been mapped to the region of the LAMA2 gene (6q22-23) in several families. Recently, two nonsense mutations in the laminin alpha2-chain gene were identified in CMD patients exhibiting complete deficiency of the laminin alpha2-chain in muscle biopsies. However, a subset of CMD patients with linkage to LAMA2 show only partial absence of the laminin alpha2-chain around muscle fibers, by immunocytochemical analysis. In the present study we have identified a homozygous missense mutation in the alpha2-chain gene of a consanguineous Turkish family with partial laminin alpha2-chain deficiency. The T-->C transition at position 3035 in the cDNA sequence results in a Cys996-->Arg substitution. The mutation that affects one of the conserved cysteine-rich repeats in the short arm of the laminin alpha2-chain should result in normal synthesis of the chain and in formation and secretion of a heterotrimeric laminin molecule. Muscular dysfunction is possibly caused either by abnormal disulfide cross-links and folding of the laminin repeat, leading to the disturbance of an as yet unknown binding function of the laminin alpha2-chain and to shorter half-life of the muscle-specific laminin-2 and laminin-4 isoforms, or by increased proteolytic sensitivity, leading to truncation of the short arm.     

Determination of catechol-O-methyltransferase activity in brain tissue by high-performance liquid chromatography with on-line radiochemical detection

A sensitive assay for catechol-O-methyltransferase (COMT) activity by high-performance liquid chromatography with on-line radiochemical detection was described. The method was based on the measurement of 3H-labeled 3-O- and 4-O-methylated products of the substrate, 3,4-dihydroxybenzoic acid, using S-adenosyl-L-[methyl-3H]methionine as the methyl donor, or the measurement of 14C-labeled 3-O- and 4-O-methylated products of the substrate, [7-14C]dopamine. The reaction products were determined from the incubation mixture after removal of protein by injecting an aliquot into the liquid chromatograph. The detection limit with counting efficiency of 40% was 0.45 pmol 3H-labeled product, and 0.04 pmol 14C-labeled product with 61% counting efficiency. The method is suitable for assaying membrane-bound and soluble COMT activities in the brain tissue and for calculation of meta/para product ratios.     

ACE gene and physical activity, blood pressure, and hypertension: a population study in Finland.

The study evaluated the association of the insertion/deletion polymorphism of the angiotensin-converting enzyme gene (ACE I/D) with self-reported moderate-intensity leisure time physical activity (MILTPA), arterial blood pressure (BP) and history of hypertension (HT). A representative population-based sample of 721 middle-aged adults (358 women) from two areas of Finland was genotyped for the ACE I/D. After exclusion criteria were applied, 455 subjects (288 women) were selected for the analysis. The distribution of the ACE I/D genotypes did not differ significantly among frequent vs. nonfrequent MILTPA groups (chi(2) = 2.556; df = 2; P value = 0.279). The main predictors of BP were male gender, age, body mass index, and arterial pulse. Additionally, tobacco smoking and alcohol consumption also had a significant main effect on diastolic BP. HT was significantly more frequent in subjects with obesity, family history of cardiovascular disease, or lower educational level. As for BP, neither ACE I/D nor MILTPA was associated with HT. The study confirmed recent reports from population-based studies of no association between ACE I/D and physical fitness. The study also confirmed a lack of association between ACE I/D and BP or HT.     

Purification and heterogeneity of human kininogen. Use of DEAE-chromatography, molecular sieving and antibody specific immunosorbents.

Various methods of preparing human kininogen were investigated with an aim to limit the immunoreactive contaminant proteins to permit purification by immunosorption. A five-step procedure is described giving 7.5% yield of highly purified kininogen (pharmacological purity 14--20) from pooled human plasma, and containing approximately 30% alpha-2HS-glycoprotein and 2.8% albumin. Alpha-2HS could not be removed by polyacrylamide gel electrophoresis or isoelectric focusing in column. Analysis of heterogeneity of kininogen after chromatography on DEAE-Sephadex using various linear gradients and gel filtration on Sephadex G-100 suggested that a minor component may be an aggregate, not included in the yield. It remains uncertain whether this component derives from an occasionally observed high molecular form of active kininogen in the primary purification steps in the 7-12 S sieve fractions from Sephadex G-200, and excluded from further purification by pooling. Purification with immunosorbents was investigated using batch operations with antibody specific polymers prepared with antisera insolubilized with ethylchloroformate. It was found that the adsorption-desorption procedure was favourable for immunization purposes in producing highly specific immunologically pure kininogen. The kininogen obtained by this method or by the removal of contaminant alpha-2HS and albumin with the corresponding antibody specific polymers gave similar heterogenous patterns by polyacrylamide gel electrophoresis, indicating a main band of kininogen and several faintly stained bands which responded only to anti-kininogen. With 200 mug of the kininogen protein purified by immunosorption using monospecific antiserum the kininogen precipitation titre was 1:8 after 6--8 weeks in rabbits. With a polymer prepared with 4 ml anti-kininogen serum (1:8) and incubated with 800 mug highly purified kininogen approximately half the protein was desorbed with 2 M and 3 M sodium iodide in the first adsorption-desorption procedure.     

Ontogeny of histamine-immunoreactive cells in rat stomach

Summary An antiserum against hemocyanin-conjugated histamine was used to study the cellular stores of histamine in the stomach, especially the oxyntic mucosa, of fetal and early postnatal rats. Tissues were fixed in 4% 1-ethyl-3(3-dimethyl-aminopropyl) carbodiimide (EDC-DI) and standard immunofluorescence technique was used. Histamine was first detected on the 16th embryonic (E16) day when a few histamine-immunoreactive (HA-ir) cells and nerve fibers were observed in the muscular layer of the stomach wall. On day E18, HA-ir cells were visualized for the first time in the oxyntic mucosa of the stomach, and from that day on the number of such cells increased slowly initially and after day E20 more rapidly. At birth many of the HA-ir cells in the oxyntic mucosa possessed processes giving them a paracrine-like appearance typical of enterochromaffin-like cells (ECL cells). Only a very small number of the HA-ir cells represented metachromatically stained mast cells and were located in the submucosa. After birth, the number of HA-ir ECL cells increased steadily, until day 21 when the distribution and number was very similar to that of the adult. The results suggest that histamine-containing neurons and ECL cells appear in the stomach wall before birth, and that there are histamine-containing ECL cells in the mucosa and mast cells in the submucosa of the stomach wall at birth.