Technological innovation of Continuous Glucose Monitoring (CGM) as a tool for commercial aviation pilots with insulin-treated diabetes and stakeholders/regulators: A new chance to improve the directives?

Civil aviation pilots who develop insulin-treated diabetes and want to renew a Commercial Pilot License (CPL) represent a medical, social and regulatory problem. This depends on justified concerns about hypoglycemia, the most threatening event for people who carry out jobs requiring a high level of concentration and reliability. This negatively affects social and working aspects of pilots' lives, who have a high profile and a high-cost professional qualification. It could be possible now to revise this attitude thanks to the availability of Continuous Glucose Monitoring (CGM) devices. CGM clearly showed to prevent hypoglycemic events in insulin-treated diabetic patients by allowing strict monitoring and trend prediction of glucose levels. By systematizing available data on such devices and present regulations in CPL issuance worldwide, our review can be used as handy tool for a fruitful discussion among the scientific community, national and international civil aviation regulators, stakeholders and pilots, aimed at evaluating the evidence-based opportunity to revise CPL issuance criteria for insulin-treated diabetic pilots. For the above-mentioned reasons, there are, among the regulatory administrations of Civil Aviation around the globe, several different approaches and limitations set for the subjects with insulin-treated diabetes who want to obtain, or renew, a CPL.

A novel thermophilic fusion enzyme for trehalose production.

In recent years a number of hyperthermophilic micro-organisms of Sulfolobales have been found to produce trehalose from starch and dextrins. In our laboratory genes encoding the trehalosyl dextrin forming enzyme (TDFE) and the trehalose forming enzyme (TFE) of S. solfataricus MT4 have been cloned and expressed in E. coli (Rb791). Here we report the construction of a new protein obtained by fusion of TFE and TDFE coding sequences which is able to produce trehalose from dextrins at high temperature by sequential enzymatic steps. We demonstrate that the bifunctional fusion enzyme is able to produce trehalose starting from malto-oligosaccharides at 75 degrees C. Furthermore we partially purified the recombinant fusion protein from bacterial cell free extracts and from insoluble fractions in which the fusion protein was also found as aggregate in inclusion bodies.

Ruminant brain ribonucleases: expression and evolution.

Molecular evolutionary analyses of mammalian ribonucleases have shown that gene duplication events giving rise to three paralogous genes occurred in ruminant ancestors. One of these genes encodes a ribonuclease identified in bovine brain. A peculiar feature of this enzyme and orthologous sequences in other ruminants are C-terminal extensions consisting of 17-27 amino acid residues. Evidence was obtained by Western blot analysis for the presence of brain-type ribonucleases in brain tissue not only of ox, but also of sheep, roe deer and chevrotain (Tragulus javanicus), a member of the earliest diverged taxon of the ruminants. The C-terminal extension of brain-type ribonuclease from giraffe deviates much in sequence from orthologues in other ruminants, due to a change of reading frame. However, the gene encodes a functional enzyme, which could be expressed in heterologous systems. The messenger RNA of bovine brain ribonuclease is not only expressed at a high level in brain tissue but also in lactating mammary gland. The enzyme was isolated and identified from this latter tissue, but was not present in bovine milk, although pancreatic ribonucleases A and B could be isolated from both sources. This suggests different ways of secretion of the two enzyme types, possibly related to structural differences. The sequence of the brain-type RNase from chevrotain suggests that the C-terminal extensions of ruminant brain-type ribonucleases originate from deletions in the ancestral DNA (including a region with stop codons), followed by insertion of a 5-8-fold repeated hexanucleotide sequence, coding for a proline-rich polypeptide.

State transitions, cyclic and linear electron transport and photophosphorylation in Chlamydomonas reinhardtii.

The relationship between state transitions and the kinetic properties of the electron transfer chain has been studied in Chlamydomonas reinhardtii. The same turnover rate of cytochrome f was found in state 1 and 2. However, while DBMIB was inhibitory in both states, DCMU was effective only in state 1. These observations suggest that linear electron transport was active only in state 1, while a cyclic pathway around photosystem (PS) I operated in state 2. The reversible shift from linear to cyclic electron transport was modulated by changes of PSII antenna size, which inactivated the linear pathway, and by oxygen, which inhibited the cyclic one. Attainment of state 2, under anaerobiosis in the dark, was associated with the decline of the ATP/ADP ratio in the cells and the dark reduction of the intersystem carriers. Upon illumination of the cells, the ATP/ADP ratio increased in a few seconds to the aerobic level. Then, several minutes later, the F(m) returned to the state 1 level, and O(2) evolution was reactivated. This suggests that ATP, though required for photosynthesis, is not the rate-limiting factor in the reactivation of photosynthetic O(2) evolution, which is rather controlled by the redox state of the electron carriers.

Interleukin-6 repression is associated with a distinctive chromatin structure of the gene.

Expression of the interleukin-6 (IL-6) gene is usually tightly controlled and may be induced in specific tissues only after treatment with appropriate stimuli. The molecular mechanisms responsible for IL-6 gene repression in specific tissues or cell lines remain poorly defined. In order to address this question we have studied two human breast carcinoma cell lines, MDA-MB-231, in which the IL-6 gene is expressed, and MCF-7, in which it is not. The promoter region of the IL-6 gene was analysed in both cell lines with reference to two different parameters: (i) DNase I hypersensitivity; (ii) the in vivo pattern of DNA-protein interactions. We show herein that the mechanism responsible for silencing IL-6 gene expression in MCF-7 cells most probably involves a modification of chromatin structure, as suggested by a decreased sensitivity of the IL-6 promoter to DNase I relative to the IL-6-expressing cell line MDA-MB-231. Moreover, we show that a 'closed' nucleosomal structure in MCF-7 cells does not inhibit the binding of nuclear proteins to IL-6 gene regulatory sequences in vivo. We suggest, therefore, that, in non-expressing cells, local chromatin remodelling at the proximal promoter is inhibited by negative regulators, as suggested by two specific hallmarks of nuclear factor binding that are not observed in expressing cells: an additional in vivo footprint spanning positions -135/-119 and an additional DNase I hypersensitive site far upstream, around position -1400. Furthermore, a specific factor binding in vitro to the -140/-116 region of the IL-6 promoter is found in MCF-7 cells.

Interaction of the nuclear protein CBF1 with the kappaB site of the IL-6 gene promoter.

The nuclear protein CBF1 has been shown to function as an intermediate to target transcription factors,such as the activated Notch receptor,to specific DNA sites. In this paper,we show that CBF1 from cell lines of different origin is able to bind to the[kappa]B site of the IL-6 promoter. By transfection analyses performed in HeLa cells,we demonstrate that overexpressed CBF1 acts as a negative regulator of IL-6 gene transcription and is unable to elicit Notch-dependent activation of this gene. Analyses of protein-DNA interactions indicate that the topology of the complex formed by CBF1 and the target DNA is subtly affected by sequencessurrounding the recognition site. Furthermore,we show that CBF1 induces DNA bending. This finding suggests that CBF1 may influence IL-6 gene transcription by determining a specific conformation of the promoter region.

Repression of the IL-6 gene is associated with hypermethylation.

The expression of the IL-6 gene is usually tightly controlled and may be induced in specific tissues after treatment with appropriate stimuli. Although much is known about the inducible expression of the IL-6 gene, the molecular mechanisms responsible for its repression in specific tissues or cell types remain poorly defined. To address this question we have studied two human breast carcinoma cell lines, MDA-MB-231, in which the IL-6 gene is expressed, and, MCF-7, in which the IL-6 message is undetectable by Northern blot assay even in the presence of inducers. The expression of the IL-6 message was estimated after treatment with 5-aza-2'deoxycytidine and the methylation state of the IL-6 gene was analyzed. We show herein that treatment of MCF-7 cells with an agent which reduces DNA methylation correlates with IL-6 gene hypomethylation and increases the level of its expression.

The differential pattern of tissue-specific expression of ruminant pancreatic type ribonucleases may help to understand the evolutionary history of their genes.

Molecular evolutionary analyses of mammalian ribonucleases have shown that gene duplication events giving three paralogous genes occurred in ruminant ancestors. The enzymes of the bovine species encoded by these genes, isolated from pancreas, brain and seminal vesicles, present similar enzymological properties but distinct structural features. In other ruminant species, genomic sequences orthologous to the bovine genes of pancreas and brain ribonucleases encode active enzymes. In mammalian species other than ruminant artiodactyls, only one gene encoding ribonuclease of the pancreatic type is generally present. In this work, we describe a differential pattern of transcriptional expression of the pancreas and brain ribonuclease genes in the ox species and report transcription of the human ribonuclease gene in brain as well as in pancreas and in mammary gland. We also report the molecular cloning of the gene encoding the bovine seminal ribonuclease in which the structural organization already described for the two paralogous genes is conserved. The seminal RNAase is exclusively expressed in seminal vesicles of Bos taurus, whereas in other ruminant species, the orthologous sequence is a pseudogene. Previous studies from a number of research groups demonstrated that, unlike other mammalian ribonucleases, the seminal enzyme is a covalent dimer, and its unique quaternary structure correlates with special biological activities. The major determinant of dimer formation, i.e. the presence of two adjacent cysteine residues, is absent in the pseudogenes. We advance the hypothesis that the differentiation of distinct expression patterns could represent an important evolutionary determinant for the genes encoding pancreas and brain ribonucleases in ruminants, whereas the differentiation of a quaternary structure endowed with new biological functions could be the main determinant for the evolutionary success of the seminal gene in the bovine species.

Secretory ribonuclease genes and pseudogenes in true ruminants.

Mammalian pancreatic ribonucleases (RNase) form a family of extensively studied homologous proteins. Phylogenetic analyses, based on the primary structures of these enzymes, indicated that the presence of three homologous enzymes (pancreatic, seminal and brain ribonucleases) in the bovine species is due to gene duplication events, which occurred during the evolution of ancestral ruminants. In this paper the sequences are reported of the coding regions of the orthologues of the three bovine secretory ribonucleases in hog deer and roe deer, two deer species belonging to two different subfamilies of the family Cervidae. The sequences of the 3' untranslated regions of the three different secretory RNase genes of these two deer species and giraffe are also presented. Comparison of these and previously determined sequences of ruminant ribonucleases showed that the brain-type enzymes of giraffe and these deer species exhibit variations in their C-terminal extensions. The seminal-type genes of giraffe, hog deer and roe deer show all the features of pseudogenes. Phylogenetic analyses, based on the complete coding regions and parts of the 3' untranslated regions of the three different secretory ribonuclease genes of ox, sheep, giraffe and the two deer species, show that pancreatic, seminal- and brain-type RNases form three separate groups.

Molecular mechanisms regulating induction of interleukin-6 gene transcription by interferon-gamma.

The multifunctional cytokine interleukin-6 (IL-6) plays a central role in host defence mechanisms and hematopoiesis. Furthermore, dysregulation of IL-6 gene expression is associated with the pathogenesis of various immunologically related diseases such as myeloma, systemic lupus erythematosus, rheumatoid arthritis, psoriasis and Kaposi's sarcoma. The regulation of IL-6 gene expression occurs mainly at transcriptional level, although mechanisms of post-transcriptional regulation have also been described. In the present study we demonstrate that in HeLa cells, induction of IL-6 by interferon-gamma (IFN-gamma) is transcriptionally controlled, as shown by run on assays and analysis of the IL-6 mRNA stability. Gel-retardation experiments using antibodies specific for factors of the IRF family identified four protein-DNA complexes, which bind to the interferon regulatory factor (IRF) binding site at position -267 to -254, in nuclear extracts from IFN-gamma treated cells. Furthermore, transient transfection analyses of the 5'-flanking region of IL-6 gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene demonstrated that the -267 to -254 IRF site is necessary for IL-6 induction by IFN-gamma. However, transfection experiments in which IRF-1 and I kappa B alpha were overexpressed show that full-scale transcriptional activation of the IL-6 promoter directing CAT expression requires the co-operation between IRF-1 and NF-kappa B at a low constitutive level.

Protein synthesis inhibitors cycloheximide and anisomycin induce interleukin-6 gene expression and activate transcription factor NF-kappaB.

In two human cell lines, MDA-MB-231 and HeLa, the inducible expression of the interleukin-6 (IL-6) gene by two protein synthesis inhibitors, cycloheximide and anisomycin, was compared with the induction by the most potent physiological inducer of IL-6 described to date, interleukin-1beta (IL-1beta). In cycloheximide or anisomycin treated cells, the accumulation of the IL-6 message and the activation of transcription factors required for IL-6 gene expression occurs at an extent similar to that obtained with IL-1beta. Furthermore, IL-6 mRNA accumulation stimulated by cycloheximide or anisomycin is almost completely inhibited in the presence of actinomycin D, indicating that this effect occurs mainly through the activation of the transcriptional machinery. These data indicate that transcriptional induction of the IL-6 gene by inhibitors of protein synthesis is triggered by the same nuclear signals as other inducers.

Nuclear factor kappa B (NF-kappa B), nuclear factor interleukin-6 (NFIL-6 or C/EBP beta) and nuclear factor interleukin-6 beta (NFIL6-beta or C/EBP delta) are not sufficient to activate the endogenous interleukin-6 gene in the human breast carcinoma cell line MCF-7. Comparative analysis with MDA-MB-231 cells, an interleukin-6-expressing human breast carcinoma cell line.

A comparative study of the molecular mechanism of interleukin-6 (IL-6) gene induction on two breast-carcinoma-derived cell lines has been performed. MDA-MB-231 cells produce constitutive detectable levels of both secreted IL-6 and mRNA which, as expected, are dramatically enhanced following induction by either IL-1 beta or tumor necrosis factor-alpha (TNF-alpha). The levels of both secreted IL-6 and IL-6 mRNA are significantly higher in response to IL-1 beta in spite of the fact that stimulation by TNF-alpha alone enhances the half life of IL-6 mRNA. The protein synthesis inhibitor cycloheximide is also a fairly strong inducer of IL-6 in these cells. In contrast, MCF-7 cells fail to produce detectable IL-6 protein or mRNA, even after stimulation with proper inducers. Analysis of transcription factors NF-kappa B, NFIL6 and NFIL6 beta, which have been described to be sufficient to activate the IL-6 gene in other cell systems, shows a similar pattern of expression in both MCF-7 and MDA-MB-231 cells. Furthermore, transfection of a recombinant plasmid carrying the IL-6 promoter linked to a luciferase reporter gene shows that both cell lines are able to drive IL-1 beta or TNF-alpha activation of this construction in a very similar manner. Finally, when MCF-7 cells were treated with IL-1 beta or TNF-alpha in the presence of cycloheximide, transcription of IL-6 mRNA from the endogenous IL-6 gene was observed. These data suggest that a mechanism of IL-6 gene repression is active in MCF-7 cells.

Molecular evolution of genes encoding ribonucleases in ruminant species.

Phylogenetic analysis, based on the primary structures of mammalian pancreatic-type ribonucleases, indicated that gene duplication events, which occurred during the evolution of ancestral ruminants, gave rise to the three paralogous enzymes present in the bovine species. Herein we report data that demonstrate the existence of the orthologues of the bovine pancreatic, seminal, and cerebral ribonucleases coding sequences in the genomes of giraffe and sheep. The "seminal" sequence is a pseudogene in both species. We also report an analysis of the transcriptional expression of ribonuclease genes in sheep tissues. The data presented support a model for positive selection acting on the molecular evolution of ruminant ribonuclease genes.

An efficient system for active bovine pancreatic ribonuclease expression in Escherichia coli.

Bovine pancreatic ribonuclease (RNase A) is a member of a homologous group of extensively studied proteins. It is a small, basic protein, containing 124 amino acid residues and four stabilizing disulfide bridges. Ribonuclease A catalyzes the hydrolysis of the phosphodiester bonds in ribonucleic acids. Since this degradation of RNA interferes with normal cell functions, the signal peptide of alkaline phosphatase (phoA, Escherichia coli) was cloned onto the gene coding for RNase A, directing the protein to the periplasm. Several expression systems have been evaluated which use T7, trc, or PR promoters to transcribe the RNase A gene. Also, variation in host strains was tested to optimize the protein yield. It was found that the PR system gave better expression than the two other systems. E. coli strain BL21 was shown to be the strain in which export to the periplasm was most effective and recombinant RNase A could be isolated from the periplasmic fraction of these cells. The system provides a stable yield of active recombinant bovine pancreatic RNase of about 45-50 mg/liter of cell culture.

Sequences related to the ox pancreatic ribonuclease coding region in the genomic DNA of mammalian species.

Mammalian pancreatic ribonucleases form a family of homologous proteins that has been extensively investigated. The primary structures of these enzymes were used to derive phylogenetic trees. These analyses indicate that the presence of three strictly homologous enzymes in the bovine species (the pancreatic, seminal, and cerebral ribonucleases) is due to gene duplication events which occurred during the evolution of ancestral ruminants. In this paper we present evidence that confirms this finding and that suggests an overall structural conservation of the putative ribonuclease genes in ruminant species. We could also demonstrate that the sequences related to ox ribonuclease coding regions present in genomic DNA of the giraffe species are the orthologues of the bovine genes encoding the three ribonucleases mentioned above.

Protein-DNA interactions in the 5'-flanking region of the bovine pancreatic ribonuclease gene.

In the 5'-flanking region of the bovine pancreatic ribonuclease gene a sequence has been identified which specifically binds one or more factors present in nuclear protein extracts prepared from bovine pancreas. The binding site, as delineated by footprinting analysis, is located in a region extending from positions -113 to -146 relative to the transcription initiation site of the ribonuclease gene. This region contains consensus sequences for known control transcriptional elements. The observed pattern of protein-DNA interactions is likely to be pancreas-specific as it could not be detected with nuclear extracts prepared from HeLa or bovine aorta endothelium cells.

A pancreatic-like ribonuclease is synthesized in rat brain.

The distribution and cell localization of a pancreatic-like ribonuclease (RNAase) in the rat brain has been studied by RNA blot analysis and in situ hybridization using as a probe the cDNA coding for the rat pancreas RNAase, and by immunocytochemistry using an antiserum raised against the rat pancreas RNAase. RNA blot analysis and in situ hybridization experiments have shown that the RNAase mRNA is present in all the cerebral areas investigated and that neurons appeared to be actively expressing RNAase mRNA while glial cells were devoid of hybridization signals. In agreement with these results the immunocytochemical analysis has shown that neurons are specifically immunostained. These experiments demonstrate that a pancreatic-like ribonuclease is synthesized in the neurons of the rat brain.

Molecular cloning of the gene encoding the bovine brain ribonuclease and its expression in different regions of the brain.

In this paper we report the molecular cloning of the gene encoding the bovine brain ribonuclease. The nucleotide sequence determined in this work shows a high degree of identity to the homologous gene encoding the bovine pancreatic ribonuclease. Processing of the primary transcripts of these genes also follows a similar pathway, splicing of the unique intron in the 5' untranslated region occurs at corresponding positions. Expression of the bovine brain ribonuclease gene can be detected both at the transcriptional and translational levels in all the regions of the brain examined.