The activity of the CCAAT-box binding factor NF-Y is modulated through the regulated expression of its A subunit during monocyte to macrophage differentiation: regulation of tissue-specific genes through a ubiquitous transcription factor.

In this study, we analyzed the regulation of NF-Y expression during human monocyte to macrophage maturation. NF-Y is a ubiquitous and evolutionarily conserved transcription factor that binds specifically to the CCAAT motif present in the 5' promoter region of a wide variety of genes. We show here that in circulating monocytes, NF-Y binding activity is not detected on the CCAAT motif present in the promoters of genes such as major histocompatibility complex (MHC) class II, gp91-phox, mig, and fibronectin, whereas during macrophage differentiation, a progressive increase in NF-Y binding activity is observed on these promoters. Analysis of NF-Y subunit expression indicates that the absence of NF-Y activity in circulating monocytes is caused by a lack of the A subunit. Furthermore, addition of the recombinant NF-YA subunit restores NF-Y binding. We show that the lack of NF-YA protein is due to posttranscriptional regulation and not to a specific proteolytic activity. In fact, NF-YA mRNA is present at the same level at all days of monocyte cultivation, whereas the protein is absent in freshly isolated monocytes but is progressively synthesized during the maturation process. We thus conclude that the NF-YA subunit plays a relevant role in activating transcription of genes highly expressed in mature monocytes. In line with this conclusion, we show that the cut/CDP protein, a transcriptional repressor that inhibits gpc91-phox gene expression by preventing NF-Y binding to the CAAT box, is absent in monocytes.

Transcriptional regulation of the ferritin heavy-chain gene: the activity of the CCAAT binding factor NF-Y is modulated in heme-treated Friend leukemia cells and during monocyte-to-macrophage differentiation.

The ferritin H-chain gene promoter regulation was analyzed in heme-treated Friend leukemia cells (FLCs) and during monocyte-to-macrophage differentiation. In the majority of cell lines studied, the regulation of ferritin expression was exerted mostly at the translational level. However, in differentiating erythroid cells, which must incorporate high levels of iron to sustain hemoglobin synthesis, and in macrophages, which are involved in iron storage, transcriptional regulation seemed to be a relevant mechanism. We show here that the minimum region of the ferritin H-gene promoter that is able to confer transcriptional regulation by heme in FLCs to a reporter gene is 77 nucleotides upstream of the TATA box. This cis element binds a protein complex referred to as HRF (heme-responsive factor), which is greatly enhanced both in heme-treated FLCs and during monocyte-to-macrophage differentiation. The CCAAT element present in reverse orientation in this promoter region of the ferritin H-chain gene is necessary for binding and for gene activity, since a single point mutation is able to abolish the binding of HRF and the transcriptional activity in transfected cells. By competition experiments and supershift assays, we identified the induced HRF as containing at least the ubiquitous transcription factor NF-Y. NF-Y is formed by three subunits, A, B, and C, all of which are necessary for DNA binding. Cotransfection with a transdominant negative mutant of the NF-YA subunit abolishes the transcriptional activation by heme, indicating that NF-Y plays an essential role in this activation. We have also observed a differential expression of the NF-YA subunit in heme-treated and control FLCs and during monocyte-to-macrophage differentiation.

Regulation of expression of ferritin H-chain and transferrin receptor by protoporphyrin IX.

The effect of protoporphyrin IX (hemin without iron) on the expression of transferrin receptor and ferritin was investigated in Friend leukemia cells. Cells treated with protoporphyrin IX exhibit enhanced transferrin-receptor expression and markedly reduced ferritin synthesis. Stimulation of transferrin-receptor expression is observed at both the mRNA and protein level. The effect on ferritin synthesis is mediated by translational inhibition of the mRNA, which, in contrast, is transcriptionally stimulated by protoporphyrin IX treatment. The regulation of transferrin receptor and ferritin in response to iron perturbations has been studied extensively and is mediated by the binding of iron-regulatory proteins (IRP) to the iron-responsive elements (IRE) present in the 3' and 5' untranslated regions of the transferrin-receptor and ferritin mRNA, respectively. To elucidate the molecular mechanisms underlying the effects of protoporphyrin IX on ferritin and transferrin-receptor expression, the role of the IRE sequence was investigated both in vivo by transfection experiments, with a construct containing the coding region for the chloramphenicol acetyltransferase (CAT) reporter gene under the translational control of the ferritin IRE, and in vitro by RNA band-shift assays. Whereas, examination of IRP binding to the IRE by in vitro assays suggests an apparent inactivation of IRP by protoporphyrin IX treatment, CAT assays indicate that protoporphyrin IX is able to induce in vivo a translational inhibition similar to that obtained by treatment with the iron chelator Desferal. This observation raises the possibility of different effects on the IRP activity exerted by porphyrin treatment in intact tissue-culture cells and in vitro. We conclude that translation of ferritin mRNA and degradation of transferrin-receptor mRNA are inhibited in intact tissue-culture cells by protoporphyrin IX through a mechanism similar to that exerted by iron chelation, thus involving depletion of the intracellular iron pool. These results can improve the understanding of the regulation of ferritin gene expression in some pathological conditions associated with disturbed heme synthesis.

Cells resistant to interferon-beta respond to interferon-gamma via the Stat1-IRF-1 pathway.

The mechanism responsible for the induction of the 2-5A synthetase gene by Interferon-gamma (IFN-gamma) (type II) was studied in Friend leukemia cells. It was previously shown that activation of 2-5A synthetase gene expression by IFN-gamma in the 3Cl8 cell, a clone resistant to IFN-alpha,beta (type I), correlates with the formation of two major complexes, designated Fg and Fc, that bind to the interferon-stimulated responsive element of the gene. Conversely, in a clone resistant to both types of IFNs (3 gamma R8), no induction of DNA-protein complexes or of 2-5A synthetase gene expression was detected. In the present report the Fg complex has been characterized as including the interferon regulatory factor 1 (IRF-1), whereas the Fc factor, present also in control cells, has been characterized as composed of IRF-2. Incubation of cell extracts with antibodies to IRF-1 abolishes the formation of the Fg complex, and antibodies to IRF-2 abolish the formation of the Fc complex. Moreover, in the 3Cl8 cell, IFN-gamma is able to induce in few minutes the formation of a complex between a DNA element identified as the IFN-gamma activation site (GAS), present on the IRF-1 gene promoter, and the STAT1 protein. These findings suggest that in cells resistant to type I IFN, IFN-gamma is able, through the activation of the STAT1 protein, to induce the expression of the IRF-1 factor which in turn seems to be sufficient to transactivate the 2-5A synthetase gene.

Differential regulation of ferritin expression in Friend leukemia cells by iron compounds.

Ferritin is an ubiquitous iron storage protein that plays a key role in determining the intracellular fate of iron. Therefore, ferritin synthesis is highly regulated by the iron status of the cell through post-transcriptional mechanisms that involve a specific high-affinity interaction between an iron-responsive element (IRE) in the 5' untranslated region of ferritin mRNA and a 98 kDa cytoplasmic protein, the iron-regulatory factor (IRF). The mechanisms that regulate the expression of the iron storage protein ferritin were investigated in erythroid (Friend erythroleukemia cell, FLC) and fibroblastic (L929 and B6) cell lines after exposure to various iron compounds. Both hemin (ferric protoporphyrin IX) and iron (as ferric ammonium citrate, FAC) were used as inducers of ferritin synthesis. Administration of hemin increases ferritin synthesis 8-12 fold both in erythroid and in non-erythroid cell lines, whereas FAC is a weak inducer of ferritin in FLC (only 5-fold). These results correlate with ferritin mRNA expression in FLC observed after hemin treatment compared to the effect exerted by FAC administration. This differential effect suggests that heme is the physiological compound able to stimulate ferritin synthesis in erythroid cell lines and that it plays an important physiological role in regulating gene expression in developing erythroid cells.

Carbon dioxide anesthesia in phlebotomine sand flies (Diptera: Psychodidae): CO2 effect upon two laboratory colonies.

Gravid females of 2 sand fly species, Phlebotomus papatasi and P. perniciosus, were exposed to carbon dioxide anesthesia for 5, 10 and 20 minutes. Recovery time, mortality at 0 min and 24 h, percentage of females laying eggs, time to oviposition, and egg productivity for each exposure time were registered. Survival, fecundity and oviposition time in the 2 species were not adversely affected by the short period of CO2 anesthesia routinely used in the sand fly colony maintenance.

Studies on mating plug of two sandfly species, Phlebotomus perniciosus and Phlebotomus papatasi (Diptera: Psychodidae).

A study was undertaken to interpret the nature and function of the "plug like formation" observed inside the spermathecae of many phlebotomine sandflies dissected during field surveys. Trials were carried out on two laboratory reared species, Phlebotomus perniciosus and P. papatasi. The results showed that the "plug like formation" is a true mating plug (MP) containing immotile sperms. Our studies showed also that the MP evolves differently according to the physiological state of the mated female. In blood-fed females, it persists 22-25 hrs post mating. Follows a period of 3-4 days in which the spermathecae acquire different form from that of the virgin spermathecae, but sperms are still undistinguishable. Active sperms appear only a few hours before oviposition. After oviposition, however, spermathecae are void as in the virgin female. Thus, during the complete gonotrophic cycle the spermathecae display five different forms: a) normal and void; b) showing MP; c) distended and yellowish, MP absent; d) active sperms present; e) normal and void. In unfed females, MP was observed to last for 6 days post copula. The results show also that MP is interfering with reinsemination.

Electrophoretic studies on two Phlebotomus species (Diptera: Psychodidae) from Italy.

Two sandfly species of the genus Phlebotomus from three different regions of Italy were examined by horizontal starch gel electrophoresis. Eleven loci were used for allozyme analysis. Data showed that there are two diagnostic loci, Idh and Lap-1, (for which different alleles are fixed in the two species) useful for the identification of these species. The degree of polymorphism for both species is low, with only Mpi and Pgm showing a relatively high polymorphism in P. perniciosus and P. perfiliewi respectively. Estimates of genetic variability are discussed.

Marriage trends in the Italo-Greeks of Italy.

The Italo-Greek ethnolinguistic minority, living in thirteen villages of southern Italy, marry largely amongst themselves but there are some intermarriages with native Italians. The majority of marriages are within the villages, but there is some marriage movement from one Italo-Greek village to another. Data on marriage and birthplace of parents and grandparents obtained by questionnaires to families of primary school children (aged 6-13 years) are analysed, to show the trends in breakdown of isolation over the last two generations.

Inbreeding coefficients from isonymy in the Italian-Greek villages.

There are four Italian-Greek communes in one area of Calabria and nine in an area of Apulia. The communes are too small to give good information on marital isonymy for each, but in the Calabrian area the weighted mean inbreeding coefficient and its random and non-random components are F = 0.01442, Fr = 0.00450 and Fn = 0.00997, respectively. The weighted means in the more populous Apulian area are significantly lower, F = 0.00423, Fr = 0.00379, Fn = 0.00045.

Relationships estimated by isonymy among the Italo-Greco villages of southern Italy.

Surnames of parents and grandparents were collected from 1993 children in the primary schools of the thirteen Italo-Greco communes that lie in two areas, four communes in Reggio Calabria in the "toe" of Italy and nine in Lecce in the "heel." The coefficients of relationship by isonymy show almost no relationship between the two areas. The smaller area in Reggio Calabria Province has consistently larger coefficients of relationship between communes than the larger area in Lecce Province. The difference can be ascribed to greater accumulated random isonymy in the smaller area. These populations are not genetic isolates, but each area shows a degree of cohesiveness with respect to surnames that suggests that they are genetically somewhat distinct. Contiguous pairs of communes tend to have higher coefficients of relationship than pairs of communes separated by intervening communes.

[Rupture of the diaphragm in childhood]. / La rottura del diaframma nell'innfanzia.

Two patients had acute rupture of the diaphragm from blunt trauma. One was a nine-year-old girl with an associated fractured pelvis and intraperitoneal injuries. She required emergency repair through a laparotomy-thoracotomy. The second patient, a six-year-old boy, underwent repair of a diaphragmatic laceration, diagnosed after a latent interval. Thoracotomy was used.

[Diverticulosis of Meckel's diverticulum in childhodd. 1 year's experience]. / La diverticolosi di Meckel nell'infanzia. L'esperienza di un anno

12 cases of Meckel's diverticulosis, submitted to operation, are examined. In the 41% of the patients, the ascertained seat of the disease was the diverticulum. The following complications were observed: phlogosis, enterorrhagia due to ulcer and occlusion for invagination. Mortality was of 8.3%.