Chiral discrimination by ligand-exchange chromatography: A comparison between phenylalaninamide-based stationary and mobile phases

The copper(II) complexes of two new diastereomeric ligands, N²-(R)- and N²-(S)-2′-hydroxypropyl-(S)-phenylalaninamide [(R, S)-1 and (S, S)-1], have been used as additives to the eluent in high-performance liquid chromatography (HPLC) reversed phase for the chiral separation of DNS-amino acids. The aim was that of comparing the separation process obtained by the chiral eluent with that obtained by an analogous bonded stationary phase containing (S)-phenylalaninamide, previously studied [CSP-(S)-Phe-NH₂]. The affinity of the ternary complexes for the C₁₈ column was determined by adsorption experiments in HPLC. It was shown that the two systems (chiral eluent, chiral stationary phase) work according to different mechanisms. Ternary complex formation in solution was studied by fluorescence spectroscopy. It was shown that chiral separation with the Cu(II) complexes added to the eluent was determined by the relative affinities of the ternary complexes for the column-stationary phase rather than by their stabilities in solution. With CSP-(S)-Phe-NH₂ the separation is accounted for by the relative stabilities of the ternary complexes, which depends mainly on the “allowed” geometry of the complex and on the steric repulsion of the amino acid side chain with the spacer. © 1996 Wiley-Liss, Inc.     

NK cells and monocytes modulate primary HTLV-1 infection

We investigated the impact of monocytes, NK cells, and CD8⁺ T-cells in primary HTLV-1 infection by depleting cell subsets and exposing macaques to either HTLV-1 wild type (HTLV-1_WT) or to the HTLV-1_p12KO mutant unable to infect replete animals due to a single point mutation in orf-I that inhibits its expression. The orf-I encoded p8/p12 proteins counteract cytotoxic NK and CD8⁺ T-cells and favor viral DNA persistence in monocytes. Double NK and CD8⁺ T-cells or CD8 depletion alone accelerated seroconversion in all animals exposed to HTLV-1_WT. In contrast, HTLV-1_p12KO infectivity was fully restored only when NK cells were also depleted, demonstrating a critical role of NK cells in primary infection. Monocyte/macrophage depletion resulted in accelerated seroconversion in all animals exposed to HTLV-1_WT, but antibody titers to the virus were low and not sustained. Seroconversion did not occur in most animals exposed to HTLV-1_p12KO. In vitro experiments in human primary monocytes or THP-1 cells comparing HTLV-1_WT and HTLV-1_p12KO demonstrated that orf-I expression is associated with inhibition of inflammasome activation in primary cells, with increased CD47 “don’t-eat-me” signal surface expression in virus infected cells and decreased monocyte engulfment of infected cells. Collectively, our data demonstrate a critical role for innate NK cells in primary infection and suggest a dual role of monocytes in primary infection. On one hand, orf-I expression increases the chances of viral transmission by sparing infected cells from efferocytosis, and on the other may protect the engulfed infected cells by modulating inflammasome activation. These data also suggest that, once infection is established, the stoichiometry of orf-I expression may contribute to the chronic inflammation observed in HTLV-1 infection by modulating monocyte efferocytosis.     

Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg · L⁻¹ of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment. Received: 12 June 1997 / Accepted: 24 October 1997     

Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment

Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor syntaxin 3, caused inhibition of FcepsilonRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment.     

Retinoic acid-induced cartilage degradation is caused by cartilage cells

Summary Cartilage cubes, prepared from the proximal epiphyses of neonatal rat humeri and consisting of cartilage tissue only, were cultured in the presence of retinoic acid. The retinoid induced the loss of metachromatic staining with toluidine blue, which correlates with the loss of proteoglycan, followed by tissue degradation processes resulting in a distinct reduction of the cartilage mass. Histologically, fibroblast-like cells appeared within chondrones, indicating a transformation of chondroblasts. Focal tissue degradation was observed after only 2 days. Electron microscopically, the clustered cells within the zone of tissue degradation were rich in various lysosomal structures indicating their lytic activity. Cycloheximide and EDTA completely blocked the retinoic acid effects suggesting that protein synthesis was required and that metalloproteinases may be involved in the degradation processes. In conclusion, with the new test system described here we demonstrated that cartilage cells themselves performed the tissue degradation induced by retinoic acid.     

Efficacy of transfer factor in treating patients with recurrent ocular herpes infections

Recurrent ocular herpes is an insoluble problem for the clinician. As cellular immunity plays an important role in controlling herpes relapses, and other studies have shown the efficacy of HSV-specific transfer factor (TF) for the treatment of herpes patients, an open clinical trial was undertaken in 134 patients (71 keratitis, 29 kerato-uveitis, 34 uveitis) suffering from recurrent ocular herpetic infections. The mean duration of the treatment was 358 days, and the entire follow-up period 189121 before, and 64062 days after TF treatment. The cell-mediated immune response to the viral antigens, evaluated by the lymphocyte stimulation test (LST) and the leucocyte migration test (LMT) (P<0.001), was significantly increased by the TF treatment. The total number of relapses was decreased significantly during/after TF treatment, dropping from 832 before, to 89 after treatment, whereas the cumulative relapse index (RI) dropped, during the same period, from 13.2 to 4.17 (P<0.0001). No side effects were observed. It is concluded that patients with relapsing ocular herpes can benefit from treatment with HSV-specific TF.     

X-linked Alport syndrome: an SSCP-based mutation survey over all 51 exons of the COL4A5 gene.

The COL4A5 gene encodes the alpha5 (type IV) collagen chain and is defective in X-linked Alport syndrome (AS). Here, we report the first systematic analysis of all 51 exons of COL4A5 gene in a series of 201 Italian AS patients. We have previously reported nine major rearrangements, as well as 18 small mutations identified in the same patient series by SSCP analysis of several exons. After systematic analysis of all 51 exons of COL4A5, we have now identified 30 different mutations: 10 glycine substitutions in the triple helical domain of the protein, 9 frameshift mutations, 4 in-frame deletions, 1 start codon, 1 nonsense, and 5 splice-site mutations. These mutations were either unique or found in two unrelated families, thus excluding the presence of a common mutation in the coding part of the gene. Overall, mutations were detected in only 45% of individuals with a certain or likely diagnosis of X-linked AS. This finding suggests that mutations in noncoding segments of COL4A5 account for a high number of X-linked AS cases. An alternative hypothesis is the presence of locus heterogeneity, even within the X-linked form of the disease. A genotype/phenotype comparison enabled us to better substantiate a significant correlation between the degree of predicted disruption of the alpha5 chain and the severity of phenotype in affected male individuals. Our study has significant implications in the diagnosis and follow-up of AS patients.     

Pyrrolizidine alkaloid profiles of the Senecio cineraria group (Asteraceae)

Alkaloid profiles of five Senecio species (Asteraceae), including S. ambiguus subsp. ambiguus, S. ambiguus subsp. nebrodensis, S. gibbosus subsp. bicolor, S. gibbosus subsp. gibbosus, and S. gibbosus subsp. cineraria, were studied. Eleven pyrrolizidine alkaloids were identified and their content was evaluated by GLC-MS and GLC analysis. Otosenine and florosenine were found to be the major alkaloids in all studied species. It is interesting that only S. ambiguus subsp. nebrodensis was characterized by a high content of the alkaloids jacobine, jacoline, jaconine, and jacozine.     

Pathophysiology of ageing, longevity and age related diseases

On April 18, 2007 an international meeting on Pathophysiology of Ageing, Longevity and Age-Related Diseases was held in Palermo, Italy. Several interesting topics on Cancer, Immunosenescence, Age-related inflammatory diseases and longevity were discussed. In this report we summarize the most important issues. However, ageing must be considered an unavoidable end point of the life history of each individual, nevertheless the increasing knowledge on ageing mechanisms, allows envisaging many different strategies to cope with, and delay it. So, a better understanding of pathophysiology of ageing and age-related disease is essential for giving everybody a reasonable chance for living a long and enjoyable final part of the life.