PARP-1 deficiency blocks IL-5 expression through calpain-dependent degradation of STAT-6 in a murine asthma model.

BACKGROUND: We recently showed that poly(ADP-ribose)polymerase-1 (PARP-1) may play a role in allergen (ovalbumin)-induced airway eosinophilia, potentially through a specific effect on IL-5 production. We also reported that while IL-5 replenishment promotes reversal of eosinophilia in lungs of PARP-1(-/-) mice, IL-4 or Immunoglobulin E replenishment do not, suggesting a potentially significant regulatory relationship between PARP-1 and IL-5. OBJECTIVE: To explore the mechanism by which PARP-1 regulates IL-5 production and to determine how PARP-1 inhibition blocks allergen-induced eosinophilia. METHODS: This study was conducted using a murine model of allergic airway inflammation and primary splenocytes. RESULTS: PARP-1 knockout-associated reduction in IL-5 upon allergen exposure occurs at the mRNA level. Such an effect appears to take place after IL-4 receptor activation as PARP-1 inhibition exerted no effect on JAK1/JAK3 activation. Signal transducer and activator of transcription-6 (STAT-6) protein was severely downregulated in spleens of PARP-1(-/-) mice without any effect on mRNA levels, suggesting an effect on protein integrity rather than gene transcription. Interestingly, the degradation of STAT-6 in PARP-1(-/-) mice required allergen stimulation. Additionally, PARP-1 enzymatic activity appears to be required for STAT-6 integrity. The downregulation of STAT-6 coincided with mRNA and protein reduction of GATA-binding protein-3 and occupancy of its binding site on the IL-5 gene promoter. IL-4 was sufficient to induce STAT-6 downregulation in both PARP-1(-/-) mice and isolated splenocytes. Such degradation may be mediated by calpain, but not by proteasomes. CONCLUSION: These results demonstrate a novel function of PARP-1 in regulating IL-5 expression during allergen-induced inflammation and explain the underlying mechanism by which PARP-1 inhibition results in IL-5 reduction.

Two dimensional non equilibrium pH gel electrophoresis mapping of cytosolic protein changes caused by dietary protein depletion in mouse liver.

Two-dimensional non-equilibrium pH gel electrophoresis (2D-NEPHGE) analysis was used to evaluate the effects of dietary protein depletion on the protein composition of mouse liver cytosol. Analysing the cytosol from both normal and protein depleted liver, the position in gels of more than three hundred protein spots was determined. After 5 days of protein depletion, about 20% of the spots either increased or decreased more than 2 fold. Five spots of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recognised by specific antibodies. The glutathione S-transferase (GSTs) subunits Ybl, Yc and Yf were identified by the simultaneous analysis of both glutathione-binding cytosolic proteins and the corresponding standards. As estimated by internal optical density (IOD) of spots, the changes caused by protein depletion in GAPDH and GST subunit contents were similar to those obtained by other methods. By means of mass spectrometric analysis of tryptic peptides generated from spots and/or comparison of two-dimensional gel electrophoretic patterns, carbonic anhydrase III (CAIII), Cu, Zn superoxide dismutase (CuZnSOD) and a cytochrome P450 cytosolic protein (cyt P450) were identified. These three proteins, as well as GSTs, are related with intracellular detoxification and free radical scavenging systems. Their contents were regulated by dietary protein restriction in a manner indicative of diminished liver defence against oxidising agents.

Floral genes expressed in tomato hypocotyl explants in liquid culture.

This paper confirms, at molecular level, previous data showing that small explants of many plants do form a floral meristem and express specific floral genes after only few days in culture. After 15-20 days of culture, small tomato hypocotyl explants develop differentiated structures often resembling primitive ancestral reproductive organs. Other specific reproductive functions such as chromosomal segregation (somatic meiosis) were also present and demonstrated by means of a cytological and histological analysis. By reverse transcriptase-PCR and in situ hybridization it was found that these structures are indeed able to express flower-specific genes. The TM8 gene, a tomato gene that is expressed very early during floral development, is detectable on the proliferating hypocotyl explants during the first week of culture. The MON9612 gene, which in vivo is expressed only by tomato pistils and ovules, is detectable on the ovulelike structures developed after 20 days of culture. The construction of transgenic tomato plants expressing the GUS gene under the control of the MON9612 promoter allowed us to follow the induction and the expression of this gene during explant proliferation and development of the flowerlike structures. These data confirm the hypothesis that a floral reprogramming can be induced in plant explants as a consequence of wounding and growth factors action. It appears to be an effort to survive stress by means of an unscheduled reproductive program.

[A new case of hepatic adenomatosis. A critical review of the literature]. / Un nuovo caso di adenomatosi epatica. Revisione critica della letteratura.

Liver adenomatosis is a rare disease--only 22 cases are previously described--characterized by multiple adenomas (more than ten) in a normal parenchyma. After a literature review the authors cannot exclude a link between the estroprogestatives use and the development of adenomatosis. Most important risks are bleeding and development of new adenomas, after the operation. On the basis of an extensive literature review, diagnosis features, treatment and follow-up are discussed. The aim of this report is to describe a case of multiple adenomas in a 38 years old housewife treated for seven years with oral contraceptives, admitted to hospital for spontaneous subcapsular haemorrhage due to rupture of adenomas, macroscopically considered suggestive of angiomas during the operation. Right lobectomy was performed and microscopic examination diagnosed multiple adenomas (liver adenomatosis). A repeated CT scan eight months later showed more than three new nodules in the liver. A new right lobectomy and tumorectomy of the nodules in III liver segment (remained after the first operation for the erroneous diagnosis) was performed and over a period of 20 months the patient remains in excellent condition.

[Inflammatory pseudotumor of the lung. Diagnostic-therapeutic effectiveness of its radical resection]. / Pseudotumore infiammatorio del polmone. Utilità diagnostico-terapeutica della resezione radicale.

Clinical, therapeutical observations and experience in 3 cases of pulmonary inflammatory pseudotumors (PIP) are presented. A retrospective analysis is made of cases with pulmonary "mass" suspected as malignant tumor, resected in a general surgery department between 1988 and 1995, and finally diagnosed as inflammatory pseudotumor. Three of the 10 cases originally diagnosed as malignant lung tumor were inflammatory pseudotumor (30%). Pulmonary inflammatory pseudotumors, may be a pitfall diagnosing a lung mass and implicate legal problems. Surgical resection leads to the final diagnosis in doubtful cases. A wide resection has a diagnostic aim and may preserve healthy parenchyma. Clinicians, pathologists and surgeons should accurately inform patients with doubtful diagnosis of pulmonary malignancy. Any decision should be kept altogether either choosing the simple observation or the timely surgical diagnostic and therapeutical approach.

[Hepatic cystadenocarcinoma]. / Cistoadenocarcinoma epatico.

The biliary cystadenocarcinoma is a rare liver neoplasm with a preoperative difficult diagnosis to make, but increased by ultrasonography. Is reported the case of a caucasian male 78 years old, operated 11 years previously for partial resection of a benign liver cyst. Recurrent cholangitis and obstructive jaundice required a reoperation for right hepatectomy. Microscopic examination showed a malignant transformation in cystadenocarcinoma.

Nested allele-specific PCR primers distinguish genetic groups of Uncinula necator.

Isolates of the obligately biotrophic fungus Uncinula necator cluster in three distinct genetic groups (groups I, II, and III). We designed PCR primers specific for these groups in order to monitor field populations of U. necator. We used the nucleotide sequences of the gene that encodes eburicol 14alpha-demethylase (CYP51) and of the ribosomal DNA internal transcribed spacer 1 (ITS1), ITS2, and 5. 8S regions. We identified four point mutations (three in CYP51 and one in ITS1) that distinguished groups I and II from group III based on a sample of 132 single-spore isolates originating from Europe, Tunisia, Israel, India, and Australia. We developed a nested allele-specific PCR assay in which the CYP51 point mutations were used to detect and distinguish groups I and II from group III in crude mildewed samples from vineyards. In a preliminary study performed with samples from French vineyards in which isolates belonging to genetic groups I and III were present, we found that a shift from a population composed primarily of group I isolates to a population composed primarily of group III isolates occurred during the grapevine growing season.

[The spontaneous rupture of a pyogenic liver abscess. A clinical case]. / Rottura spontanea di ascesso epatico piogenico. Caso clinico.

The authors report a case of a large monolocular liver abscess in a patient with gallbladder and choledocic stones and biliary stent, complicated by rupture into the peritoneal cavity, that required surgical treatment. Surgical exploration versus other approach results the choice treatment, as shown by resolution of the pathology.

On the occurrence of somatic meiosis in embryogenic carrot cell cultures.

During the establishment of an embryogenic cell line from a carrot hypocotyl explant, processes closely resembling meiotic divisions are seen. A microdensitometric analysis revealed that the amount of cellular DNA diminished in the majority of cells to the haploid level. However, the diploid level was re-established in a matter of a few days. The genetic consequences of this segregation were studied by analyzing restriction fragment length polymorphisms (RFLP) and randomly amplified polymorphic DNAs (RAPD). The results showed that the great majority of embryos regenerated from segregants and that different segregants had different genetic constitutions.

Synthesis and toxicity to mammalian cells of the carrot dihydroisocoumarins.

The dihydroisocoumarins (+-)-6-methoxy-8-hydroxy-3-methyl-3,4- dihydroisocoumarin (1), (+-)-6,8-dihydroxy-3-methyl-3,4-dihydroisocoumarin (2), and (+-)-6,8-dimethoxy-3-methyl-3,4-dihydroisocoumarin (3) were synthesized with high yields via metalation of o-methylbenzamides. The toxicity of these compounds and that of (-)-1 extracted from carrot cells were tested, in vitro, on Chinese hamster cells. The toxicity was determined according to the presence or absence of a hydroxyl group in the peri position of the lactonic carbonyl group and according to the stereochemistry of the dihydroisocoumarin.

A carrot cell variant temperature sensitive for somatic embryogenesis reveals a defect in the glycosylation of extracellular proteins.

The temperature-sensitive carrot cell variant ts11c, arrested in somatic embryogenesis after the globular stage, was characterized. The sensitivity to a shift from 24 degrees C (permissive temperature) to 32 degrees C (non-permissive temperature) is greatest at the globular stage of embryogenesis, while cells proliferating in unorganized fashion and plantlets are not affected. Embryogenesis in ts11c is also arrested at the permissive temperature by replacement of conditioned culture medium with fresh medium. The timing of sensitivity of ts11c to medium replacement coincides with the sensitivity to temperature shift. Both sensitivities are recessive in somatic hybrids between ts11c and wild-type cells. Extracellular glycoproteins synthesized by ts11c at the non-permissive temperature contain much less fucose than those synthesized by the wild type. The glycoproteins synthesized by the variant under non-permissive conditions do not accumulate at the periphery of the embryo, as their wild-type counterparts do, but instead show a diffuse distribution throughout the embryo. The defect in ts11c can be fully complemented by the addition of extracellular wild-type proteins. A revertant of ts11c was isolated that simultaneously reacquired temperature insensitivity and normal glycosylation ability. Collectively, these observations indicate that ts11c is not able to perform proper glycosylation at the non-permissive temperature and suggest that the activity of certain extracellular proteins, essential for the transition of globular to heart stage somatic embryos, depends on the correct modification of their oligosaccharide side-chains.

5-azacytidine-induced tumorous transformation and DNA hypomethylation in Nicotiana tissue cultures.

The phenomenon of habituation is considered in plant tissue cultures to be a real process of chemical tumorogenesis; the cultures acquire the capacity of autonomous growth in a hormone-free medium under the influence of a variety of chemical and physical agents. Treatments with 5-azacytidine (AzaC) of in vitro cultured cells of the Nicotiana glauca x N. langsdorffii nontumorous hybrid (NNT) during the culture cycle led to the induction of a habituated phenotype. The repetitive DNA sequences showed a significant lower level of endogenous methylation in the treated cells in comparison with the normal ones. It is worth noting that it was impossible until now to habituate this strain by conventional methods and that the treatments were effective only in the first 5 days of subculturing; various evidence (cytological and biochemical) pointed out a phenomenon of DNA amplification, occurring in the same period. Moreover, analysis of DNA from control and treated cells shows the induction of variations in the endogenous methylation pattern by AzaC in a critical period of cell culture. These results suggest that demethylation can act as a switch from hormone-dependent to autonomous proliferation by activation of genes coding for or regulating the synthesis of growth factors.

DNA methylation of embryogenic carrot cell cultures and its variations as caused by mutation, differentiation, hormones and hypomethylating drugs.

The level of auxin - both natural and synthetic - in the medium has a strong effect on the level of 5-methyl-cytosine in the DNA of carrot cells in culture. This level may vary from approximately 15% to 70% of total cytosine without apparent effects on growth rate and cell morphology. No effect was seen with cytokinin. During somatic embryogenesis, in the absence of hormones, variations were seen in the level of methylation according to a characteristic pattern. If hypomethylation is induced with drugs such as azacytidine, ethionine or ethoxy-carbonyl-pyrimidine, embryogenesis is immediately blocked. A mutant was isolated which is resistant to the action of hypomethylating drugs. It shows variations in the methylation pattern and variations in indole-acetic acid metabolism. In addition its regeneration is often associated with the production of tumors.

Role of permanent dicentric systems in carrot somatic embryogenesis.

A permanent dicentric chromosome system was studied on carrot cultures and regenerated somatic embryos at different stages of development. The large chromosomal variability of the cultures and the presence of the breakage-fusion bridge cycle did not interfere with the initial developmental process up to the seedling stage but subsequent growth proceeded only if healing of the broken ends or dicentric loss had occurred. The behaviour of the dicentric chromosome in culture and during somatic embryogenesis is discussed in relation to chromosomal variability, abnormal development and the somaclonal variation that such mechanisms may generate in regenerated plants.

Stimulation of carrot somatic embryogenesis by proline and serine.

Serine and proline, when added in a wide range of concentrations to Daucus carota cultures during pre-growth in the presence of 2,4-D(2,4-dichlorophenoxyacetic acid), extended in time and quantity the mitotic divisions and stimulated significantly the number of embryos regenerated when the hormone subsequently was omitted from the medium. A specific effect of these amino acids on regeneration is suggested, since proline (as hydroxyproline) and serine are the two major constituents of the cell wall glycoprotein, extensin, which thus may have a morphoregulatory function. The significant role of the conditions during pregrowth of the cultures in the presence of hormone is underlined by the effect of hydroxyproline and D-proline which also stimulate embryogenesis, but alter markedly the development of the embryos.

[Chromosomal variability of 2 species of Nicotiana in fluid culture]. / Variabilità cromosomica di due specie di Nicotiana in coltu ra liquida.

The chromosomal number in relation with the age of suspension culture of nicotiana glauca (NG) and the non tumorous N. glauca x N. langsdorffii hybrid has been studied. In both species, an higher variability of chromosome number is present in younger culture. Aged cultures show a stabilization, for NG, around the hexaploid number (3n = 72) and for NNT, which is an amphidiploid specie, on the tetraploid level (4n = 84). Both species show, with the age of culture, a decrease in plant regeneration capacity, which is not due to chromosomal variability, since young cultures are much more variable than older one.

Genetic effects of griseofulvin on plant cell cultures.

Griseofulvin induces metaphase arrest and polyploidization in plant cells in culture; after which a process of chromosome segregation follows. During this process spontaneous or induced recessive mutations become expressed through the formation of homozygotes or monosomics. This finding can be of use whenever the isolation of recessive mutations is needed and haploid culture is difficult.