Strain-specific effects of riboflavin supplementation on zymosan-induced peritonitis in C57BL/6J, BALB/c and CBA mice

AimsWe investigated the effects of riboflavin (vitamin B2) on the kinetics of zymosan-induced peritonitis in three strains of mice.Main methodsPeritonitis was induced in males of C57BL/6J, BALB/c and CBA mice by intraperitoneal injection of zymosan (40 mg/kg) or zymosan supplemented with riboflavin (50 mg/kg). During the first 45 min of inflammation the pain symptoms were scored. At the selected time points (4, 6, 8, 10, 24, and 30 h) the mice were sacrificed and peritoneal exudates were retrieved. Leukocytes, among them polymorphonuclear cells (PMNs) and macrophages (Mac3⁺ cells) were counted. Levels of inducible nitric oxide synthase (iNOS) were measured in cell pellets while supernatants were used for measurements of nitric oxide, cytokine/chemokines (IL-6, IL-10, MCP-1, IFNγ, TNF-α, and IL-12p70), and matrix metalloproteinase-9 (MMP-9).Key findingA riboflavin ip injection induced pain symptoms itself, but reduced zymosan-induced pain in C57BL/6J and CBA strains of mice when coinjected with zymosan. In comparison with the mice injected with zymosan only, riboflavin coinjection prolonged inflammation in C57BL/6J mice due to prolonged macrophage accumulation; inhibited peritoneal leukocytes (PTL) accumulation in BALB/c due to inhibited influx of macrophages and PMNs; and inhibited PTL accumulation in CBA mice due to delayed PMN influx. These effects corresponded with the delayed (C57BL/6J) or inhibited (BALB/c and CBA) expression of iNOS in PTL lysates, and with the prolonged (C57BL/6) or inhibited (BALB/c) intraperitoneal accumulation of MMP-9. Moreover, cytokine accumulation was affected in a strain-specific way.SignificanceRiboflavin is antinociceptive during yeast-induced peritonitis, but its anti-inflammatory effects are strain-specific.     

Salivary levels of tumor necrosis factor-alpha in oral lichen planus

OBJECTIVE: Oral lichen planus (OLP) is chronic inflammatory disease of the oral mucosa, presenting in various clinical forms. The etiology of OLP is still unknown but mounting evidence points to the immunologic basis of this disorder. AIM: Our study was undertaken to quantify the salivary levels of pro-inflammatory tumor necrosis factor-alpha (TNF-alpha) in the reticular and the erosive/atrophic forms of OLP, compared with age-matched healthy control volunteers. SUBJECTS AND METHODS: Whole saliva from 40 patients with active lesions of OLP, as well as from 20 healthy persons, was investigated for the presence of TNF-alpha by enzyme immunoassay. RESULTS: Salivary TNF-alpha levels were significantly increased in patients with OLP in comparison with healthy subjects. The presence of TNF-alpha showed positive correlation to clinical forms of OLP, being significantly higher in the erosive/atrophic type than in the reticular type of disease. CONCLUSION: Saliva provides an ideal medium for the detection of pro-inflammatory markers of the oral cavity. In patients with OLP, TNF-alpha levels in saliva are elevated, correlating with the severity of illness. Salivary TNF-alpha analysis may be a useful diagnostic tool and a potential prognostic marker in OLP.     

Immunohistochemical and electron-microscopic identification of neuroendocrine cells in the stomach of uremic rats

Many disturbances in electrolyte and hormonal balance in the body induced by functional impairment of renal parenchyma may affect the activity of amine precursor uptake and decarboxylation (APUD) cells, which constitute a very important link in the regulation of homeostasis. The aim of the present study was the morphological, immunohistochemical and ultrastructural estimation of enteroendocrine cells in the stomach of uremic rats. Fragments of gastric pylorus were collected 1, 2 and 4 weeks after nephrectomy. Paraffin embedded sections were stained with H + E and by silver impregnation. For identification of neuroendocrine cells, immunohistochemical reactions were performed using specific antibodies against somatostatin, synaptophysin, neuron-specific enolase and anti-calcitonin gene related peptide. The analysis showed an increased number of APUD cells in the stomach of uremic rats compared to control rats, which may be a morphological expression of their hyperfunction in the functional impairment of renal parenchyma. These results suggest that chronic renal failure can modulate the secretory processes of APUD cells.     

Effect of subinhibitory levels of selected antibiotics on susceptibility of Staphylococcus aureus strains to phagocytosis and killing by rabbit granulocytes

The aim of the study was to assess the of chosen antibiotics in subinhibitory concentrations (sub-MIC) on the sensitive of Staphylococcus aureus cells to phagocytosis and killing by rabbit granulocytes. The following antibiotics were used: cloxacillin, cefadroxil, cefuroxim, cefotaxim, gentamicin, netilmicin, lincomicin, doxycycline and riphamicin. A total of 144 S. aureus strains with varied sensitivity to these antibiotics were selected for the study. The experiment used granulocytes isolated from rabbit blood and S. aureus strains incubated for 18 h in TSB broth containing antibiotics in the concentrations of 0.1 MIC, 0.2 MIC and 0.5 MIC, and in the antibiotics-free medium. Phagocytosis was assessed by the method of differential staining with acridine orange and crystal violet, allowing simultaneous determination of phagocytised and killed S. aureus cell counts. The findings revealed that the culture of S. aureus in the presence of all the antibiotics used in subinhibitory concentrations increased significantly the susceptibility of most S. aureus strains to phagocytosis and killing by granulocytes. The above effect usually occurred in the concentrations of 0.1 MIC (54.2%), more seldom in 0.2 MIC (13%) and 0.5 MIC (15% of strains). Each group of S. aureus contained some which showed no change in susceptibility following culture with the chemotherapeutic agents in subinhibitory concentrations (26.3%). Insensitive strains to the subinhibitory effects were equally common among susceptible (27%), intermediate (23%) and resistant (26%) strains of S. aureus to the antibiotics used. No statistically significant reduction was noted in phagocytosis or killing by rabbit granulocytes. No correlation was observed between the susceptibility to the subinhibitory effects of the antibiotics involved and their biochemical mechanisms.     

An unusual halotolerant alpha-type carbonic anhydrase from the alga Dunaliella salina functionally expressed in Escherichia coli

A 60-kDa, salt-inducible, internally duplicated alpha-type carbonic anhydrase (Dca) is associated with the plasma membrane of the extremely salt-tolerant, unicellular, green alga Dunaliella salina. Unlike other carbonic anhydrases, Dca remains active over a very broad range of salinities (0-4M NaCl), thus representing a novel type of extremely halotolerant enzyme. To elucidate the structural principles of halotolerance, structure-function investigations of Dca have been initiated. Such studies require considerable amounts of the enzyme, and hence, large-scale algal cultivation. Furthermore, the purified enzyme is often contaminated with other, co-purifying algal carbonic anhydrases. Expression in heterologous systems offers a means to produce, and subsequently purify, sufficiently large amounts of Dca required for activity and structural studies. Attempts to over-express Dca in the Escherichia coli BL21(DE3)pLysS strain, after optimizing various expression parameters, produced soluble, but weakly active protein, composed of fully reduced and variably -S-S- cross-linked chains (each of the Dca repeats contains a pair of cysteine residues, presumably forming a disulfide bond). However, when the E. coli Origami B(DE3)pLysS strain was used as a host, a functionally active enzyme with proper disulfide bonds was formed in good yield. Affinity-purified recombinant Dca resembled the native enzyme from D. salina in activity and salt tolerance. Hence, this expression system offers a means of pursuing detailed studies of this extraordinary protein using biochemical, biophysical, and crystallographic approaches.     

Virus Attenuation after Deletion of the Cytomegalovirus Fc Receptor Gene Is Not due to Antibody Control

The murine cytomegalovirus (MCMV) fcr-1 gene codes for a glycoprotein located at the surface of infected cells which strongly binds the Fc fragment of murine immunoglobulin G. To determine the biological significance of the fcr-1 gene during viral infection, we constructed MCMV fcr-1 deletion mutants and revertants. The fcr-1 gene was disrupted by insertion of the Escherichia coli lacZ gene. In another mutant, the marker gene was also deleted, by recombinase cre. As expected for its hypothetical role in immunoevasion, the infection of mice with fcr-1 deletion mutants resulted in significantly restricted replication in comparison with wild-type MCMV and revertant virus. In mutant mice lacking antibodies, however, the fcr-1 deletion mutants also replicated poorly. This demonstrated that the cell surface-expressed viral glycoprotein with FcR activity strongly modulates the virus-host interaction but that this biological function is not caused by the immunoglobulin binding property.     

The SSR-based molecular profile of 1005 grapevine (Vitis vinifera L.) accessions uncovers new synonymy and parentages, and reveals a large admixture amongst varieties of different geographic origin

A collection of 1005 grapevine accessions was genotyped at 34 microsatellite loci (SSR) with the aim of analysing genetic diversity and exploring parentages. The comparison of molecular profiles revealed 200 groups of synonymy. The removal of perfect synonyms reduced the database to 745 unique genotypes, on which population genetic parameters were calculated. The analysis of kinship uncovered 74 complete pedigrees, with both parents identified. Many of these parentages were not previously known and are of considerable historical interest, e.g. Chenin blanc (Sauvignon × Traminer rot), Covè (Harslevelu selfed), Incrocio Manzoni 2–14 and 2–15 (Cabernet franc × Prosecco), Lagrein (Schiava gentile × Teroldego), Malvasia nera of Bolzano (Perera × Schiava gentile), Manzoni moscato (Raboso veronese × Moscato d’Amburgo), Moscato violetto (Moscato bianco × Duraguzza), Muscat of Alexandria (Muscat blanc à petit grain × Axina de tres bias) and others. Statistical robustness of unexpected pedigrees was reinforced with the analysis of an additional 7–30 SSRs. Grouping the accessions by profile resulted in a weak correlation with their geographical origin and/or current area of cultivation, revealing a large admixture of local varieties with those most widely cultivated, as a result of ancient commerce and population flow. The SSRs with tri- to penta-nucleotide repeats adopted for the present study showed a great capacity for discriminating amongst accessions, with probabilities of identity by chance as low as 1.45 × 10^?27 and 9.35 × 10^?12 for unrelated and full sib individuals, respectively. A database of allele frequencies and SSR profiles of 32 reference cultivars are provided.     

Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cells

p66Shc, a longevity adaptor protein, is demonstrated as a key regulator of reactive oxygen species (ROS) metabolism involved in aging and cardiovascular diseases. Vascular endothelial growth factor (VEGF) stimulates endothelial cell (EC) migration and proliferation primarily through the VEGF receptor-2 (VEGFR2). We have shown that ROS derived from Rac1-dependent NADPH oxidase are involved in VEGFR2 autophosphorylation and angiogenic-related responses in ECs. However, a role of p66Shc in VEGF signaling and physiological responses in ECs is unknown. Here we show that VEGF promotes p66Shc phosphorylation at Ser36 through the JNK/ERK or PKC pathway as well as Rac1 binding to a nonphosphorylated form of p66Shc in ECs. Depletion of endogenous p66Shc with short interfering RNA inhibits VEGF-induced Rac1 activity and ROS production. Fractionation of caveolin-enriched lipid raft demonstrates that p66Shc plays a critical role in VEGFR2 phosphorylation in caveolae/lipid rafts as well as downstream p38MAP kinase activation. This in turn stimulates VEGF-induced EC migration, proliferation, and capillary-like tube formation. These studies uncover a novel role of p66Shc as a positive regulator for ROS-dependent VEGFR2 signaling linked to angiogenesis in ECs and suggest p66Shc as a potential therapeutic target for various angiogenesis-dependent diseases.