Phylogenetics of Asphodelaceae (Asparagales): An Analysis of Plastid rbcL and trnL-F DNA Sequences

Phylogenetic relationships of Asphodelaceae were investigatedby parsimony analysis of 57 monocotrbcL nucleotide sequences,including 17 genera that have at some time been assigned tothe family. All genera of Asphodelaceae except for three (Hemiphylacus,Paradisea and Simethis) form a strongly supported monophyleticgroup with Hemerocallidaceae and Xanthorrhoeaceae as their immediatesister taxa. In a second analysis, we added 34 plastid trnL-Fsequences (an intron and a spacer between two transfer RNA genes)for the Asphodelaceae clade and nearest outgroup families (Doryanthaceae,Hemerocallidaceae, Iridaceae, Ixioliriaceae, Tecophilaeaceaeand Xanthorrhoeaceae) in an attempt to improve resolution andlevels of internal support. The results from the separate analysesproduced highly similar although not identical results. No stronglysupported incongruent groups occurred, and we combined bothsequence regions in one analysis, which demonstrated improvedresults. Strong support exists for a monophyletic subfamilyAlooideae, but this leaves a paraphyletic subfamily Asphodeloideaebecause Bulbine/Jodrellia alone are strongly supported as thesister group of Alooideae. Characters that have been used toseparate Alooideae as a distinct group (either as here a subfamilyor as a separate family by other authors), such as secondarygrowth and bimodal karyotypes, are found in at least some membersof Asphodeloideae, particularly in Bulbine and Jodrellia forthe karyotypes, making Alooideae less easily recognized. Copyright2000 Annals of Botany Company Alooideae, Asphodeloideae, Asphodelaceae, Asparagales, phylogenetic analysis, rbcL, trnL-F, molecular systematics     

Branched-chain amino acid-enriched diet: effects on insulin secretion and cellular immune aggression

Several reports have demonstrated that high-protein diets may have beneficial effects on experimental models of diabetes and have raised the possibility that branched-chain amino acids could play a role in these protective effects. We investigated the effect of a normoproteic, branched-chain amino acid-enriched diet (experimental diet) on insulin secretion from C57BL/6N mice transferred with splenocytes from diabetic syngeneic donors. Mice previously fed with the experimental or control diet received three intraperitoneal injections, every other day, of 5 x 107 viable mononuclear splenocytes obtained from control or diabetic donors. Results showed that mice fed with the experimental diet and transferred with "diabetic" splenocytes presented: i) normoglycemia, and (ii) significantly higher levels in both phases of glucose-induced insulin secretion and normal values of arginine-glucose-induced insulin secretion. To evaluate the in vitro cellular immune aggression, dispersed mouse islet cells were co-cultured with splenocytes from syngeneic diabetic mice. First, dispersed islet cells from mice on the experimental or control diet were co-cultured with splenocytes from control or diabetic mice on a commercial diet. In the presence of "diabetic splenocytes, dispersed islet cells from mice on the experimental diet presented a significantly lower in vitro cellular immune aggression. On the other hand, "diabetic" splenocytes from mice fed with the experimental diet produced a significantly reduced cellular immune aggression on dispersed islet cells. Our results showed that feeding branched-chain amino acids increased the capacity of beta cells to withstand a functional assault and diminished the extent of in vitro cellular immune aggression.     

Prevention of systemic lupus erythematosus in MRL/lpr mice by administration of an immunoglobulin-binding peptide

Systemic lupus erythematosus (SLE) is a multisystem chronic inflammatory disease of unknown etiology that affects many organs, including the kidney. The presence of multiple autoantibodies and other immunological abnormalities point to basic defects in immunoregulatory controls that normally maintain self-tolerance. The deposition on kidney tissue of autoantibodies as immune complexes (ICs) through the interaction with Fc-receptor gamma-chains is thought to trigger an inflammatory response typical of SLE, leading to glomerulonephritis. Using combinatorial chemistry approaches, we have identified a peptide able to bind to immunoglobulins and to interfere with Fcgamma-receptor recognition. Administration of this peptide to MRL/lpr mice, an animal model used to study SLE, resulted in a remarkable enhancement of the survival rate (80%) compared to placebo-treated animals (10%). Consistent with this was a significant reduction of proteinuria, a clinical sign of SLE. Kidney histological examination of treated animals confirmed the preservation of tissue integrity and a remarkable reduction in IC deposition. These results support the role of Fcgamma receptors in SLE pathogenesis and open new avenues for the development of drugs to treat autoimmune disorders.     

Biophysical studies of a transmembrane peptide derived from the T cell antigen receptor

Summary Core peptide (CP) is a unique peptide derived from the transmembrane sequence of T cell antigen receptor (TCR)-alpha chain and is capable of inhibiting the immune response both invitro and in animal models of T cell mediated inflammation. The structure of CP, with sequence GLRILLLKV, is similar to the amphipathic region of many peptides. Unlike antimicrobial peptides, however, which damage cell membranes, electron microscopy and propidium iodide exclusion assays on cell membranes suggest that CP does not create pores and may act by interfering with signal transduction at the membrane level. To investigate this effect further we report the results of³¹P and²H solid-state NMR spectroscopy of CP on model membranes. As predicted, even at high concentrations of CP, the structure of model membranes was not significantly perturbed. Only at the very high peptide-to-lipid molar ratio of 1∶10 significant effects on the model membranes were observed. We conclude that CP does not destroy the integrity of the lipid bilayer.     

Insights into the molecular basis for the carbenicillinase activity of PSE-4 beta-lactamase from crystallographic and kinetic studies

PSE-4 is a class A beta-lactamase produced by strains of Pseudomonas aeruginosa and is highly active for the penicillin derivative carbenicillin. The crystal structure of the wild-type PSE-4 carbenicillinase has been determined to 1.95 A resolution by molecular replacement and represents the first structure of a carbenicillinase published to date. A superposition of the PSE-4 structure with that of TEM-1 shows a rms deviation of 1.3 A for 263 Calpha atoms. Most carbenicillinases are unique among class A beta-lactamases in that residue 234 is an arginine (ABL standard numbering scheme), while in all other class A enzymes this residue is a lysine. Kinetic characterization of a R234K PSE-4 mutant reveals a 50-fold reduction in k(cat)/K(m) and confirms the importance of Arg 234 for carbenicillinase activity. A comparison of the structure of the R234K mutant refined to 1.75 A resolution with the wild-type structure shows that Arg 234 stabilizes an alternate conformation of the Ser 130 side chain, not seen in other class A beta-lactamase structures. Our molecular modeling studies suggest that the position of a bound carbenicillin would be shifted relative to that of a bound benzylpenicillin in order to avoid a steric clash between the carbenicillin alpha-carboxylate group and the conserved side chain of Asn 170. The alternate conformation of the catalytic Ser 130 in wild-type PSE-4 may be involved in accommodating this shift in the bound substrate position.     

Deficient IL-12(p35) gene expression by dendritic cells derived from neonatal monocytes

To gain insight into the defects responsible for impaired Th1 responses in human newborns, we analyzed the production of cytokines by dendritic cells (DC) derived from cord blood monocytes. We observed that neonatal DC generated from adherent cord blood mononuclear cells cultured for 6 days in the presence of IL-4 and GM-CSF show a phenotype similar to adult DC generated from adherent PBMC, although they express lower levels of HLA-DR, CD80, and CD40. Measurement of cytokine levels produced by neonatal DC upon stimulation by LPS, CD40 ligation, or poly(I:C) indicated a selective defect in the synthesis of IL-12. Determination of IL-12(p40) and IL-12(p35) mRNA levels by real-time RT-PCR revealed that IL-12(p35) gene expression is highly repressed in stimulated neonatal DC whereas their IL-12(p40) gene expression is not altered. The addition of rIFN-gamma to LPS-stimulated newborn DC restored their expression of IL-12(p35) and their synthesis of IL-12 (p70) up to adult levels. Moreover, we observed that neonatal DC are less efficient than adult DC to induce IFN-gamma production by allogenic adult CD4(+) T cells. This defect was corrected by the addition of rIL-12. We conclude that neonatal DC are characterized by a severe defect in IL-12(p35) gene expression which is responsible for an impaired ability to elicit IFN-gamma production by T cells.     

Physico-chemical studies on DNA triplexes containing an alternate third strand with a non-nucleotide linker

Differential scanning calorimetric (DSC), circular dichroism (CD) and molecular mechanics studies have been performed on two triple helices of DNA. The target duplex consists of 16 base pairs in alternate sequence of the type 5′-(purine)_m(pyrimidine)_m-3′. In both the triplexes, the third oligopyrimidine strand crosses the major groove at the purine–pyrimidine junction, with a simultaneous binding of the adjacent purine tracts on alternate strands of the Watson–Crick duplex. The switch is ensured by a non-nucleotide linker, the 1,2,3 propanetriol residue, that joins two 3′–3′ phosphodiester ends. The third strands differ from each other for a nucleotide in the junction region. The resulting triple helices were termed 14-mer-PXP and 15-mer-PXP (where P=phosphate and X=1,2,3-propanetriol residue) according to the number of nucleotides that compose the third strand. DSC data show two independent processes: the first corresponding to the dissociation of the third strand from the target duplex, the second to the dissociation of the double helix in two single strands. The two triple helices show the same stability at pH 6.6. At pH 6.0, the 15-mer-PXP triplex is thermodynamically more stable than the 14-mer-PXP triplex. Thermodynamic data are discussed in relation to structural models. The results are useful when considering the design of oligonucleotides that can bind in an antigene approach to the DNA for therapeutic purposes.     

Impact of spray-drying on the pili of Lactobacillus rhamnosus GG

The preservation of the viability of microorganisms in probiotic formulations is the most important parameter ensuring the adequate concentration of live microorganisms at the time of administration. The formulation and processing techniques used to produce these probiotic formulations can influence the preservation of the microbial viability. However, it is also required that the bacteria maintain their key probiotic capacities during processing, formulation and shelf life. In this study, we investigated the impact of spray-drying on different cell wall properties of the model probiotic strain Lactobacillus rhamnosus GG, including its adherence to intestinal epithelial cells. The dltD gene knock-out mutant, L. rhamnosus GG CMPG5540, displaying modified cell wall lipoteichoic acids, showed significantly increased colony-forming units after spray-drying and subsequent storage under standard conditions compared to wild-type L. rhamnosus GG. In contrast, disruption of the biosynthesis of exopolysaccharides or pili expression did not impact survival. However, spray-drying did significantly affect the adherence capacity of L. rhamnosus GG. Scanning electron microscopy confirmed that the pili, key surface factors for adherence to intestinal cells and mucus, were sheared off during the spray-drying process. These data thus highlight that both the functionality and viability of probiotics should be assessed during the spray-drying process and subsequent storage.