Kinetics of Toll-like receptor-4 splice variants expression in lipopolysaccharide-stimulated antigen presenting cells of healthy donors and patients with cystic fibrosis

Toll-like receptors (TLR) are key components of innate immune system. As TLR activation could induce potentially harmful inflammatory response, activation of TLR signaling pathways has to be under tight control. Besides other control mechanisms, an inhibitory function of murine TLR4 splice variants was recently demonstrated. In this study we investigated expression of four TLR4 splice variants in human antigen presenting cells (APC). Furthermore, we studied modification in TLR4 splice variants expression in APC in cystic fibrosis (CF) patients chronically infected by Gram-negative bacteria. We developed a novel reliable real-time PCR detection system that allowed monitoring of individual TLR4 splice variants expression. In APC from healthy donors we detected a characteristic transient increase of two out of four splice variants after lipopolysaccharide (LPS) stimulation. Similarly to murine TLR4, one of these variants, NM 003266, might translate to a potentially inhibitory protein. In contrast to controls, CF monocytes had significantly changed LPS-induced expression of TLR4 gene and its variants including reduced ability to up-regulate the expression of the potentially inhibitory variant upon stimulation. In accordance with this observation, monocytes from CF patients produced significantly more tumor necrosis factor after LPS stimulation than healthy controls. Our results thus describe the kinetics of TLR4 splicing variants expression after LPS stimulation and indicate a possible alteration of its regulation in CF patients.     

Mutational analysis in podocin-associated hereditary nephrotic syndrome in Polish patients: founder effect in the Kashubian population

Hereditary nephrotic syndrome is caused by mutations in a number of different genes, the most common being NPHS2. The aim of the study was to identify the spectrum of NPHS2 mutations in Polish patients with the disease. A total of 141 children with steroid-resistant nephrotic syndrome (SRNS) were enrolled in the study. Mutational analysis included the entire coding sequence and intron boundaries of the NPHS2 gene. Restriction fragment length polymorphism (RFLP) and TaqMan genotyping assay were applied to detect selected NPHS2 sequence variants in 575 population-matched controls. Twenty patients (14 %) had homozygous or compound heterozygous NPHS2 mutations, the most frequent being c.1032delT found in 11 children and p.R138Q found in four patients. Carriers of the c.1032delT allele were exclusively found in the Pomeranian (Kashubian) region, suggesting a founder effect origin. The 14 % NPHS2 gene mutation detection rate is similar to that observed in other populations. The heterogeneity of mutations detected in the studied group confirms the requirement of genetic testing the entire NPHS2 coding sequence in Polish patients, with the exception of Kashubs, who should be initially screened for the c.1032delT deletion.     

Mechanical strain regulation of the chicken glypican-4 gene expression in the avian eggshell gland

Comparison of RNA fingerprinting of the avian eggshell gland (ESG) without and with an egg revealed upregulation of a 382-bp cDNA fragment that showed high homology to the mammalian glypican 4 (GPC-4). The gene sequence revealed a conserved glypican signature, a glycosyl phosphatidyl inositol-anchorage site, and cystein residues, most of which were conserved. GPC-4 was expressed in the ESG in a circadian fashion only during the period of eggshell calcification, when maximal mechanical strain was imposed. Removal of the egg just before to its entry into the ESG, with consequent elimination of the mechanical strain, caused reduction in the gene expression. Artificial application of the mechanical strain induced expression of the GPC-4 gene that was related to the level of the strain. GPC-4 expression was strain dependent in other parts of the oviduct. In the ESG, GPC-4 was expressed exclusively by the glandular epithelium and not by the pseudostratified epithelium facing the lumen. In summary, we cloned the avian homologue of GPC-4, established its pattern of expression in the avian ESG, and demonstrated for the first time that this gene is regulated by mechanical strain.     

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.     

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.