Chitosan induces different L-arginine metabolic pathways in resting and inflammatory macrophages

Chitosan is a linear polymer of N-acetyl-D-glucosamine and deacetylated glucosamine widely used as a wound-healing accelerator in clinical and veterinary medicine. Chitosan enhances the functions of inflammatory cells such as macrophages (Mphi), inducing the production of cytokines as well as the expression of activation markers, Fc receptors and mannose receptor. In this work we studied the effects of chitosan on the arginine metabolic pathways of both resident and inflammatory (proteose-peptone elicited) rat Mphi. Our results show that low molecular weight (LMW) chitosan activated moderately both the inducible nitric oxide synthase (iNOS) and arginase pathways in resident Mphi. In inflammatory Mphi treated with chitosan instead, the arginase activity was strongly enhanced. Supernatants of chitosan-stimulated Mphi enhanced the proliferation of the rat cell line C6. These findings suggest that the healing activity of chitosan could rely on the enhanced arginase activity observed in a wound-associated inflammatory milieu.     

Novel plant-microbe rhizosphere interaction involving Streptomyces lydicus WYEC108 and the pea plant (Pisum sativum)

A previously undescribed plant-microbe interaction between a root-colonizing Streptomyces species, S. lydicus WYEC108, and the legume Pisum sativum is described. The interaction is potentially of great importance to the health and growth in nature of this nodulating legume. The root-colonizing soil actinomycete S. lydicus WYEC108 influences pea root nodulation by increasing root nodulation frequency, possibly at the level of infection by Rhizobium spp. S. lydicus also colonizes and then sporulates within the surface cell layers of the nodules. Colonization leads to an increase in the average size of the nodules that form and improves the vigor of bacteroids within the nodules by enhancing nodular assimilation of iron and possibly other soil nutrients. Bacteroid accumulation of the carbon storage polymer, poly-beta-hydroxybutyrate, is reduced in colonized nodules. Root nodules of peas taken from agricultural fields in the Palouse hills of northern Idaho were also found to be colonized by actinomycete hyphae. We hypothesize that root and nodule colonization is one of several mechanisms by which Streptomyces acts as a naturally occurring plant growth-promoting bacterium in pea and possibly other leguminous plants.     

The extreme C terminus of rat liver carnitine palmitoyltransferase I is not involved in malonyl-CoA sensitivity but in initial protein folding

We previously reported that the N-terminal domain (1-147 residues) of rat liver carnitine palmitoyltransferase I (L-CPTI) was essential for import into the outer mitochondrial membrane and for maintenance of a malonyl-CoA-sensitive conformation. Malonyl-CoA binding experiments using mitochondria of Saccharomyces cerevisiae strains expressing wild-type L-CPTI or previously constructed chimeric CPTs (Cohen, I., Kohl, C., McGarry, J.D., Girard, J., and Prip-Buus, C. (1998) J. Biol. Chem. 273, 29896-29904) indicated that the N-terminal domain was unable, independently of the C-terminal domain, to bind malonyl-CoA with a high affinity, suggesting that the modulation of malonyl-CoA sensitivity occurred through N/C intramolecular interactions. To assess the role of the C terminus in malonyl-CoA sensitivity, a series of C-terminal deletion mutants was generated. The kinetic properties of Delta772-773 and Delta767-773 deletion mutants were similar to those of L-CPTI, indicating that the last two highly conserved Lys residues in all known L-CPTI species were not functionally essential. By contrast, Delta743-773 deletion mutant was totally inactive and unfolded, as shown by its sensitivity to trypsin proteolysis. Because the C terminus of the native folded L-CPTI could be cleaved by trypsin without inducing protein unfolding, we concluded that the last 31 C-terminal residues constitute a secondary structural determinant essential for the initial protein folding of L-CPTI.     

In situ delivery of passive immunity by lactobacilli producing single-chain antibodies

Lactobacilli have previously been used to deliver vaccine components for active immunization in vivo. Vectors encoding a single-chain Fv (scFv) antibody fragment, which recognizes the streptococcal antigen I/II (SAI/II) adhesion molecule of Streptococcus mutans, were constructed and expressed in Lactobacillus zeae (American Type Culture Collection (ATCC) 393). The scFv antibody fragments secreted into the supernatant or expressed on the surface of the bacteria showed binding activity against SAI/II in enzyme-linked immunosorbent assay (ELISA), and surface scFv-expressing lactobacilli agglutinated SAI/II-expressing S. mutans in vitro without affecting the corresponding SAI/II knockout strain. Lactobacilli expressing the scFv fragment fused to an E-tag were visualized by scanning electron microscopy (SEM) using beads coated with a monoclonal anti-E-tag antibody, and they bound directly to beads coated with SAI/II. After administration of scFv-expressing bacteria to a rat model of dental caries development, S. mutans bacteria counts and caries scores were markedly reduced. As lactobacilli are generally regarded as safe (GRAS) microorganisms, this approach may be of considerable commercial interest for in vivo immunotherapy.     

Evidence for a founder effect for pseudoxanthoma elasticum in the Afrikaner population of South Africa

Pseudoxanthoma elasticum (PXE) is a heritable elastic tissue disorder recently shown to be attributable to mutations in the ABCC6 ( MRP6) gene. Whereas PXE has been identified in all ethnic groups studied to date, the prevalence of this disease in various populations is uncertain, although often assumed to be similar. A notable exception however is the prevalence of PXE among South African Afrikaners. A previous report has suggested that a founder effect may explain the higher prevalence of PXE in Afrikaners, a European-derived population that first settled in South Africa in the 17th century. To investigate this hypothesis, we performed haplotype and mutational analysis of DNA from 24 South African families of Afrikaner, British and Indian descent. Among the 17 Afrikaner families studied, three common haplotypes and six different disease-causing variants were identified. Three of these mutant alleles were missense variants, two were nonsense mutations and one was a single base-pair insertion. The most common variant accounted for 53% of the PXE alleles, whereas other mutant alleles appeared at lower frequencies ranging from 3% to 12%. Haplotype analysis of the Afrikaner families showed that the three most frequent mutations were identical-by-descent, indicating a founder origin of PXE in this population.     

Laser-based micromanipulation for separation and identification of individual Frankia vesicles

In studies of symbiotic efficiency it is of great importance to identify and separate individual Frankia strains from a nodule. Therefore, a new laser-based micromanipulation technique has been developed in which individual vesicles from root nodules of two Frankia-Alnus symbioses have been successfully cut loose and separated from clusters of vesicles in sterile conditions under light microscopy using a laser scalpel and optical tweezers. Vesicles from the Alnus incana-Frankia AvCI1 symbiosis were successfully isolated and grown in culture using this technique. The DNA from both Frankia sources was amplified by polymerase chain reaction (PCR). The work shows that a combination of laser-based manipulation techniques and PCR can be used for the separation and study of individual vesicles. This novel laser-based micromanipulation technique opens up various new possibilities, for instance, to study whether several Frankia strains can grow simultaneously in the same root nodule.