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Nonsense mutations are responsible for around 10% of cases of genetic diseases, including cystic fibrosis. 2,6-diaminopurine (DAP) has recently been shown to promote efficient readthrough of UGA premature stop codons. In this study, we show that DAP can correct a nonsense mutation in the Cftrgene in vivoin a new CF mouse model, in utero, and through breastfeeding, thanks, notably, to adequate pharmacokinetic properties. DAP turns out to be very stable in plasma and is distributed throughout the body. The ability of DAP to correct various endogenous UGA nonsense mutations in the CFTRgene and to restore its function in mice, in organoids derived from murine or patient cells, and in cells from patients with cystic fibrosis reveals the potential of such readthrough-stimulating molecules in developing a therapeutic approach. The fact that correction by DAP of certain nonsense mutations reaches a clinically relevant level, as judged from previous studies, makes the use of this compound all the more attractive.

Bacteria under stress produce ribosomally synthesized and post-translationally modified peptides (RiPPs) to target closely related species, such as the lasso peptide microcin J25 (MccJ25). These peptides are also toxic to the producing organisms that utilize dedicated ABC transporters to achieve self-immunity. MccJ25 is exported by the Escherichia coliABC transporter McjD through a complex mechanism of recognition that has remained elusive. Here, we used biomolecular NMR to study this interaction and identified a region of the toxic peptide that is crucial to its recognition by the ABC transporter. Our study provides evidence that McjD is highly specific to MccJ25 and not to other RiPPs or antibiotics, unlike multidrug ABC transporters. Additionally, we show that MccJ25 is not exported by another natural product ABC transporter. Therefore, we propose that specific interactions between natural product ABC transporters and their substrate provides them with their high degree of specificity. Taken together, these findings suggest that ABC transporters might have acquired structural elements in their binding cavity to recognize and allow promiscuous export of a larger variety of compounds.

Lasso peptides are bacterial ribosomally synthesized and post-translationally modified peptides. They have sparked increasing interest in peptide-based drug development because of their compact, interlocked structure, which offers superior stability and protein-binding capacity. Disulfide bond-containing lasso peptides are rare and exhibit highly sought-after activities. In an effort to expand the repertoire of such molecules, we heterologously expressed, in Streptomyces coelicolor, the gene cluster encoding sviceucin, a type I lasso peptide with two disulfide bridges originating from Streptomyces sviceus, which allowed it to be fully characterized. Sviceucin and its reduced forms were characterized by mass spectrometry and peptidase digestion. The three-dimensional structure of sviceucin was determined using NMR. Sviceucin displayed antimicrobial activity selectively against Gram-positive bacteria and inhibition of fsrquorum sensing in Enterococcus faecalis. This study adds sviceucin to the type I lasso peptide family as a new representative. Moreover, new clusters encoding disulfide-bond containing lasso peptides from Actinobacteria were identified by genome mining. Genetic and functional analyses revealed that the formation of disulfide bonds in sviceucin does not require a pathway-encoded thiol–disulfide oxidoreductase. Most importantly, we demonstrated the functional exchangeability of the sviceucin and microcin J25 (a non-disulfide-bridged lasso peptide) macrolactam synthetases in vitro, highlighting the potential of hybrid lasso synthetases in lasso peptide engineering.

ABSTRACTIn this paper we provide the first biochemical evidence of the existence of a family of structure-related antimicrobial peptides, the siderophore-microcins, in the Enterobacteriaceaefamily. We isolated and characterized two novel siderophore-microcins, MccM and MccH47, previously characterized through genetic studies. MccM and MccH47 were expressed from several Escherichia colistrains containing the microcin gene clusters. The spectra of their bactericidal activities were found to be restricted to some species of the Enterobacteriaceae. MccM and MccH47 were unable to inhibit the growth of strains carrying mutations in the fepA, cir, and fiugenes, which showed the requirement of the iron-catecholate receptors for their recognition. The MccM and MccH47 peptide moieties contain 77 and 60 residues, respectively, and are derived from the microcin precursors McmA and MchB, respectively. In addition, both peptides carried a C-terminal posttranslational modification containing a salmochelin-like siderophore moiety also found in MccE492 (X. Thomas et al., J. Biol. Chem., 279:28233-28242, 2004). Interestingly, when MccM was isolated from E. coliNissle 1917, which lacks the two genes necessary for modification biosynthesis, it was devoid of posttranslational modification. Those two genes could be complemented by their homologues from the MccH47 gene cluster, thereby showing their functional interchangeability between at least two members of the siderophore-microcin family. Finally, from the sequence analysis of the MccE492 gene cluster, we hypothesized the existence of an additional member of the siderophore-microcin family. Therefore, we propose that the siderophore-microcin family contains five representatives.

ABSTRACTMicrocin E492-producing bacteria secrete both unmodified and posttranslationally modified microcins. The modification consists of a C-glucosylated linear trimer of N-(2,3-dihydroxybenzoyl)-l-serine, a catecholate siderophore related to salmochelins and enterobactin. We show here that repression of enterobactin biosynthesis inhibits the acquisition of microcin E492 posttranslational modification, as monitored by high-performance liquid chromatography and mass spectrometry. Furthermore, exogenous enterobactin restored the production of posttranslationally modified microcin in a bacterial strain deficient in enterobactin synthesis. We thus concluded that enterobactin serves as a precursor for the synthesis of the posttranslationally modified microcin and that the unmodified microcin is an incompletely processed form of mature microcin E492. Gene disruption experiments showed that MceC and MceD, two enzymes encoded by the mceABCDEFGHIJgene cluster, are involved in the synthesis of the microcin E492 posttranslational modification, as followed by mass spectrometry. Genes homologous to iroBand iroD, required for the conversion (linearization and C-glycosylation) of enterobactin into salmochelins, efficiently complemented mceCand mceD, respectively. Based on our results, a model is proposed for the biosynthesis of the mature siderophore-carrying peptide.

Auto-antibodies (aAbs) to thyroid peroxidase (TPO) interact with a restricted immunodominant region (IDR) divided into two overlapping regions A and B. Among the five major regions structuring the IDR/B, regions 210–225, 353–363, 549–563, 713–720 and 766–775, region 353–363 constitutes an important anchor point for the binding of TPO-specific aAbs in sera from Hashimoto's and Graves' patients. We combined site-directed mutagenesis and expression of TPO mutants in stably transfected CHO cells to precisely define the critical residues in that region. By using flow cytometry and ELISA, we identified four amino acid residues, H353, D358, S359 and R361, that contribute to the interaction between human TPO and anti-TPO aAbs. This identification of these contributing amino acid residues in the IDR allowed us to more precisely depict contours of the IDR.

Microcin E492 (MccE492) is an antibacterial peptide naturally secreted by Klebsiella pneumoniaeRYC492. Initially described as an 84-residue unmodified peptide, it was also recently isolated in a posttranslationally modified form, MccE492m. The production of MccE492m is dependent on the synthesis of enterobactin and the mceABCDEFGHIJgene cluster. The posttranslational modification was characterized as a trimer of N-(2,3-dihydroxybenzoyl)-l-serine (DHBS) linked to the Ser84-carboxylate viaa β-d-glucose moiety. MccE492m was shown to bind ferric ions through the trimer of DHBS. This is the first example of a novel type of antibacterial peptide termed siderophore-peptide. Recognition of MccE492m, but also of the unmodified MccE492, was shown to be mediated by the catecholate siderophore receptors FepA, Cir and Fiu at the outer membrane of E. coli. The siderophore-type modification was shown to be responsible for a significant enhancement of the microcin antibacterial activity. Therefore, we propose that MccE492 and MccE492m use iron-siderophore receptors for uptake into the target bacteria and that improvement of MccE492 antimicrobial activity upon modification results from an increase in the microcin/receptor affinity.

Several studies have shown the efficacy and effectiveness of the combined endoscopic–laparoscopic “rendezvous” technique for treatment of gallbladder and bile duct stones without complications, particularly pancreatitis. The so-called rendezvous technique consists of laparoscopic cholecystectomy standards with intraoperative cholangiography followed by endoscopic sphincterotomy (EST). The sphincterotome is driven across the papilla through a guidewire inserted by the transcystic route. This method allows easier and faster cannulation, thus avoiding papillary edema and pancreatic trauma. The aim of this study was to evaluate whether this method is effective in eliminating ductal stones and to verify whether the risk of postprocedure pancreatitis is diminished.From January 2002 to September 2004, we enrolled 256 patients with cholecystocholedolithiasis detected by transabdominal ultrasound and magnetic resonance cholangiopancreatography. One hundred and twenty of these had one or more patient-related risk factors for post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis, so they were randomized into two groups of 60 patients. In group A, the patients were treated in a single step with videolaparoscopic cholecystectomy, intraoperative cholangiography, and EST during the surgical procedure with the rendezvous technique. In group B, preoperative ERCP and EST were performed by using a traditional method of bile duct cannulation.No cases of post-ERCP pancreatitis were observed in group A, whereas six cases of acute post-ERCP pancreatitis occurred in group B (five mild and one moderate) (p= 0.0274). No procedure-related mortality was recorded.In cholecysthocholedocholithiasis, the combined laparoscopic–endoscopic approach prevents post-ERCP pancreatitis in cases with patient-related risk factors for this complication.

Background: Although laparoscopic transabdominal preperitoneal polypropylene (TAPP) hernia repair is now a recognized procedure for groin hernia repair in patients undergoing elective surgery, there is a scarcity of data on TAPP repair in emergency situations such as those involving strangulated hernias. Methods: Unless contraindicated, the authors’ department considers laparoscopic TAPP repair the procedure of choice for all strangulated hernias. A prospective database of 1,532 consecutive hernia repairs performed between May 1998 and April 2004 was reviewed. Results: A total of 28 irreducible strangulated hernias were observed and repaired through laparoscopic TAPP. There were three conversions (10.7%): one because of extensive adhesions and two because of bowel distention. Resection became necessary in 9 (36%) of the 25 cases involving effective TAPP repair of strangulated hernias. The mean operation time, with and without resection, was 103 and 55 min, respectively. The rate of postoperative morbidity was 4% because of one inguinal hematoma case. During a mean follow-up period of 340 days, there were no recurrences after TAPP repair. Conclusions: For selected patients, the TAPP approach appears to be a good therapeutic option for strangulated hernias.

The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB–ExbB–ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA− strain and by using isogenic strains mutated in genes encoding the protein complex respectively. In addition, MccJ25 was shown to block phage T5 infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as MccJ25 inhibited phage T5 DNA ejection triggered by purified FhuA. Direct interaction of MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry. MccJ25 bound to FhuA with a 2:1 stoichiometry and a Kd of 1.2 μM. Taken together, our results demonstrate that FhuA is the receptor for MccJ25 and that the ligand–receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that MccJ25 binding involves external loops of FhuA. Unlike native MccJ25, a thermolysin-cleaved MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5 infection of E. coli. Therefore the Val11–Pro16 β-hairpin region of MccJ25, which is disrupted upon cleavage by thermolysin, is required for microcin recognition.

Microcin J25 (MccJ25) is a cyclic antibacterial peptide secreted by a fecal isolate of Escherichia coli. It exerts highly potent activity on Salmonella and Escherichia species. The microcin is recognized at the outer membrane of sensitive strains by the FhuA multifunctional protein, which belongs to the iron / siderophore receptor family, and inhibits bacterial transcription through binding to the RNA-polymerase β' subunit. The mcjABCD genetic system carried by the wild type 50-kb pTUC100 plasmid contains four genes involved in MccJ25 production and immunity. MccJ25 results from the proteolytic cleavage of a 58-residue precursor at a specific Lys-Gly bond. The resulting mature peptide consists of 21 unmodified amino acids, mostly hydrophobic and includes a single dehydration. The initially described macrocyclic structure of MccJ25, which mostly relied on manual Edman sequencing of the thermolysin-cleaved form (t-MccJ25), involved a head-to-tail cyclisation of the 21-residue precursor. This structure did not prove to be consistent with recent ITMS CID experiments conducted either on the native microcin or on peptides resulting from acidic or enzymatic cleavages, which are in favour of an 8-residue ring followed by a 13-residue tail. Cyclisation thus occurs between the N-terminus (Gly1) and the Glu8 side chain carboxyl group. The solution three-dimensional structure shows threading of the tail into the ring, thus forming a highly stable lasso type structure. Such a structure was described previously for enzyme inhibitors from Actinobacteria and is consistent with the ability of MccJ25 to inhibit RNA polymerase. The lasso structure is discussed in terms of phylogenetical and biotechnological perspectives.

ABSTRACTMicrocin C51 (MccC51) is an antimicrobial nucleotide-heptapeptide produced by a natural Escherichia colistrain. A 5.7-kb fragment of the pC51 plasmid carrying the genes involved in MccC51 production, secretion, and self-immunity was sequenced, and the genes were characterized. The sequence of the MccC51 gene cluster is highly similar to that of the MccC7 gene. Recombinant plasmids carrying different combinations of the mccgenes involved in the MccC51 production or immunity were constructed to characterize their functional roles. The mccA, mccB, mccD, and mccEgenes are involved in MccC51 production, while the mccCand mccEgenes are responsible for immunity to MccC51. The mccgene cluster is flanked by 44-bp direct repeats. Amino acid sequence comparisons allowed us to propose functions for each Mcc polypeptide in MccC51 biosynthesis. Plasmid pUHN containing the cloned mccA, mccB, mccC, and mccEgenes, but lacking mccD, directed the synthesis of MccC51p, a substance chemically related to MccC51. MccC51p exhibited weak antibiotic activity against E. coliand was toxic to the producing cells. The immunity to exogenous MccC51 determined by the mccCand mccEgenes did not overcome the toxic action of MccC51p on the producing cells. The G+C content of the MccC51 operon, markedly lower than that of the E. coligenome, and the presence of direct repeats suggest the possibility of horizontal transfer of this gene cluster.

En 2002, le Prix Nobel de Chimie a été décerné à trois grands scientifiques, John B. Fenn et Koichi Tanaka pour la spectrométrie de masse et Kurt Wüthrich pour la résonance magnétique nucléaire. Il récompense ainsi les idées innovantes qui ont conduit à développer, à l’interface chimie-biologie, des méthodologies spectroscopiques majeures initialement introduites en chimie analytique. La découverte de nouvelles méthodes douces d’ionisation en spectrométrie de masse, et la conception d’une stratégie d’attributions séquentielles des signaux de résonance magnétique nucléaire de biopolymères, ont permis l’application de ces deux méthodologies puissantes et complémentaires à la détermination de structures de macromolécules du vivant, protéines, acides nucléiques (ADN, ARN) et polysaccharides, etc. Par l’élucidation de leurs édifices (structures planes et tridimensionnelles) et des mécanismes moléculaires régissant leurs interactions, ces travaux fournissent des clés précieuses pour la compréhension des mécanismes moléculaires du vivant.

The fungus Trichoderma virensis a ubiquitous soil saprophyte that has been applied as a biological control agent to protect plants from fungal pathogens. One mechanism of biocontrol is mycoparasitism, and T. virensproduces antifungal compounds to assist in killing its fungal targets. Peptide synthetases produce a wide variety of peptide secondary metabolites in bacteria and fungi. Many of these are known to possess antibiotic activities. Peptaibols form a class of antibiotics known for their high α-aminoisobutyric acid content and their synthesis as a mixture of isoforms ranging from 7 to 20 amino acids in length. Here we report preliminary characterization of a 62.8-kb continuous open reading frame encoding a peptaibol synthetase from T. virens. The predicted protein structure consists of 18 peptide synthetase modules with additional modifying domains at the N- and C-termini. T. virenswas shown to produce a mixture of peptaibols, with the largest peptides being 18 residues. Mutation of the gene eliminated production of all peptaibol isoforms. Identification of the gene responsible for peptaibol production will facilitate studies of the structure and function of peptaibol antibiotics and their contribution to biocontrol activity.

Aggregation of the human amyloid β-peptide (Aβ) into insoluble plaques is a key event in Alzheimer's disease. Zinc sharply accelerates the Aβ aggregation in vitro, and the Aβ region 6–28 was suggested to be the obligatory zinc binding site. However, time-dependent aggregation of the zinc-bound Aβ species investigated so far prevented their structural analysis. By using CD spectroscopy, we have shown here for the first time that (i) the protected synthetic peptide spanning the fragment 1–16 of Aβ binds specifically zinc with 1:1 and 1:2 stoichiometry under physiologically relevant conditions; (ii) the peptide–zinc complex is soluble and stable for several months; (iii) zinc binding causes a conformational change of the peptide towards a more structured state. These findings suggest the region 1–16 to be the minimal autonomous zinc binding domain of Aβ.

Longibrachins are members of the class of natural Aib-containing peptides designated as peptaibols. Six longibrachins, LGA I−IV and LGB II and III, were purified from a Trichoderma longibrachiatum strain by a procedure employing several chromatography steps including reversed-phase HPLC. The amino acid sequence determination was based on a combination of liquid secondary ion mass spectrometry (LSIMS) and two-dimensional ¹H and ¹³C NMR spectroscopy. Longibrachins are 20-residue peptaibols with a C-terminal phenylalaninol and either neutral (LGA; Gln¹⁸) or acidic (LGB; Glu¹⁸) character. Longibrachins LGB II and III have novel sequences. Both longibrachins LGA and LGB show significant bactericidal activity against mycoplasmas (Acholeplasma, Mycoplasma, and Spiroplasma), with minimal inhibitory concentrations in the range 1.56−12.5 μM (3−25 μg/mL), and also perturb the permeability of membrane bilayers. Longibrachin LGA IV is the most potent of the presently known 18−20-residue peptaibols. The antimicrobial and membrane-perturbing properties of longibrachins, which are described here for the first time, were shown to be correlated.

Pseudokonins KL III and KL VI are two natural ten‐residue peptides, which both contain the (Xaa‐Yaa‐Aib‐Pro) motif and exhibit an unusual C‐terminus. They have been isolated from the fungus Trichoderma pseudokoningii by intensive reversed‐phase HPLC, beside peptaibols classically C‐ended by a β‐amino alcohol. The amino acid sequences and the chemical structures of the C‐ends have been determined by the combined use of positive ion LSI‐MS and two‐dimensional homo‐ and heteronuclear NMR, including COSY, TOCSY, ROESY, 13C heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC). Instead of one of the amino alcohols usually found as C‐terminal residue in peptaibols, pseudokonins KL III and KL VI are characterized by ‐Pro‐NH2and cyclo‐(Aib‐𝓁‐Proal) (Proal, prolinal), respectively. Such backbone modifications are described here for the first time for peptaibol antibiotics. The unusual cyclo‐(Aib‐𝓁‐Proal) C‐terminus is probably the result of an intramolecular cyclization of the two last Aib and Pro residues of a ten‐amino acid precursor, via a Proal intermediate. A secondary structure stabilized by –CO…H–N‐hydrogen bonds of the 1←4 type has been deduced for both peptides from ROESY data, 3JNHCαHcouplings and amide proton temperature coefficient values. The (Xaa‐Yaa‐Aib‐Pro) β‐bend ribbon spiral, which has been described for the first time in the case of a 14‐residue peptaibol containing three repetitive (Xaa‐Yaa‐Aib‐Pro) motifs (Ségalas G et al. Biopolymers 1999; 50: 71–85) appears to be maintained in the two shortened modified peptides. The β‐bend ribbon structure thus appears to be initiated by a single (Xaa‐Yaa‐Aib‐Pro) motif and unaffected by the C‐terminal modifications. However, the membrane and antibiotic properties of pseudokonins KL III and KL VI, point to the unfavourable effect of both shortening and cyclization of the peptide chain. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.

The three-dimensional solution structure of harzianin HC IX, a peptaibol antibiotic isolated from the fungus Trichoderma harzianum, was determined using CD, homonuclear, and heteronuclear two-dimensional nmr spectroscopy combined with molecular modeling. This 14-residue peptide, Ac Aib¹ Asn² Leu³ Aib⁴ Pro⁵ Ala⁶ Ile⁷ Aib⁸ Pro⁹ Iva¹⁰ Leu¹¹ Aib¹² Pro¹³ Leuol¹⁴ (Aib, α-aminoisobutyric acid; Iva, isovaline; Leuol, leucinol), is a main representative of a short-sequence peptaibol class characterized by an acetylated N-terminus, a C-terminal amino alcohol, and the presence of three Aib-L-Pro motifs at positions 4–5, 8–9, and 12–13, separated by two dipeptide units. In spite of a lower number of residues, compared to the 18/20-residue peptaibols such as alamethicin, harzianin HC IX exhibits remarkable membrane-perturbing properties. It interacts with phospholipid bilayers, increasing their permeability and forming voltage-gated ion channels through a mechanism slightly differing from that proposed for alamethicin. Sequence-specific ¹H- and ¹³C-nmr assignments and conformational nmr parameters (³JNHCαH coupling constants, quantitative nuclear Overhauser enhancement data, temperature coefficients of amide and carbonyl groups, NH–ND exchange rates) were obtained in methanol solution. Sixty structures were calculated based on 98 interproton distance restraints and 6 Φ dihedral angle restraints, using high temperature restrained molecular dynamics and energy minimization. Thirty-seven out of the sixty generated structures were consistent with the nmr data and were convergent. The peptide backbone consists in a ribbon of overlapping β-turns twisted into a continuous spiral from Asn² to Leuol¹⁴ and forming a 26 Å long helix-like structure. This structure is slightly amphipathic, with the three Aib–Pro motifs aligned on the less hydrophobic face of the spiral where the Asn² side chain is also present, while the more hydrophobic bulky side chains of leucines, isoleucine, isovaline, and leucinol are located on the concave side. The repetitive (Xaa–Yaa–Aib–Pro) tetrapeptide subunit, making up the peptide sequence, is characterized by four sets of (Φ,Ψ) torsional angles, with the following mean values: Φi = −90°, Ψi = −27°; Φi+1 = −98°, Ψi+1 = −17°; Φi+2 = −49°, Ψi+2 = −50°; Φi+3 = −78°, Ψi+3 = +3°. We term this particular structure, specifically occurring in the case of (Xaa–Yaa–Aib–Pro)n sequences, the (Xaa–Yaa–Aib–Pro)-β-bend ribbon spiral. It is stabilized by 4 → 1 intramolecular hydrogen bonds and differs from both the canonical 310-helix made of a succession of type III β-turns and from the β-bend ribbon spiral that has been described in the case of (Aib–Pro)n peptide segments. © 1999 John Wiley & Sons, Inc. Biopoly 50: 71–85, 1999

Fifteen Beagle dogs underwent oesophagogastric anastomosis with a new device which enables a ‘sutureless’ compression anastomosis. The device fragmented and was passed in bits anally without causing obstruction. Immediate bursting pressure, tested in five dogs, was between 175 and 190 mmHg. The anastomoses of the remaining dogs were examined macroscopically and microscopically from day 6 to day 30. Healing was excellent with good muscular apposition and minimal residual inflammation.

Mazzocchi, G., L. K. Malendowicz, P. Rebuffat, C. Tortorella and G. G. Nussdorfer. Arginine-vasopressin stimulates Crh and Acth release by rat adrenal medulla, acting via the V1receptor subtype and a protein kinase C-dependent pathway. Peptides 18(2) 191–195, 1997.—Arginine-vasopressin (AVP) is a hypothalamic hormone that, like CRH, stimulates the pituitary release of ACTH, thereby activating adrenal glucocorticoid secretion. Evidence indicates that rat adrenal medulla contains a CRH-ACTH system duplicating that existing at the hypothalamo-pituitary level and involved in the paracrine stimulation of the cortex secretion. Therefore, we investigated by RIA the effect of AVP on the release of CRH and ACTH immunoreactivities (IR) by rat adrenal medulla in vitro. AVP concentration-dependently enhanced the release of both CRH-IR and ACTH-IR, and the effect was blocked by a selective antagonist of the V1subtype of AVP receptors. The CRH receptor antagonist α-helical-CRH partially reversed AVP-evoked rise in ACTH-IR release, without altering either CRH response or basal secretions of CRH and ACTH. The specific inhibitors of protein kinase C Ro31-8220 and calphostin C abolished both CRH and ACTH responses to AVP. In conclusion, our present findings suggest that AVP stimulates intramedullary the CRH-ACTH system, acting via V1receptors and activating protein kinase C.

We have synthesized by solution-phase methods two analogues of the 11-residue lipopeptaibol antibiotic trichogin GA IV in which the N-terminal n-octanoyl group is replaced either by an N-acetylated 2-amino-2-methyl-l-undecanoic acid or by an N-acetylated α-aminoisobutyric acid. CD, FTIR absorption, and NMR analyses unequivocally show that the main structural features of trichogin GA IV are preserved in these analogues. Since only the peptide containing the lipophilic chain exhibits membrane-modifying properties, these results strongly support the view that moving the long acyl moiety from the N^α-blocking group to the side chain of the N-terminal extra-residue does not affect the conformational properties or the membrane activity of trichogin GA IV. © 1998 European Peptide Society and John Wiley & Sons, Ltd.

DNA uptake can be facilitated by addition of physiological amounts of 11β-hydroxy glucocorticosteroids (such as cortisol) during transfection. In the presence of cortisol, but not of the inactive 11-keto glucocorticoid cortisone, twice as many cells uptake and express the reporter gene. The effect is specific and dose-dependent; the amounts of glucocorticosteroids needed to enhance transfection efficiency are in the nanomolar range, which corresponds to the dissociation constant of glucocorticoids for the glucocorticoid receptor in vitro. This effect can be abolished by an excess of the glucocorticoid antagonist RU486. We infer that the activated cytoplasmic glucocorticoid receptors enhance nuclear translocation of the incoming transfected DNA.

We have synthesized by solution‐phase methods two analogues of the 11‐residue lipopeptaibol antibiotic trichogin GA IV in which the N‐terminal n‐octanoyl group is replaced either by an N‐acetylated 2‐amino‐2‐methyl‐l‐undecanoic acid or by an N‐acetylated α‐aminoisobutyric acid. CD, FTIR absorption, and NMR analyses unequivocally show that the main structural features of trichogin GA IV are preserved in these analogues. Since only the peptide containing the lipophilic chain exhibits membrane‐modifying properties, these results strongly support the view that moving the long acyl moiety from the Nα‐blocking group to the side chain of the N‐terminal extra‐residue does not affect the conformational properties or the membrane activity of trichogin GA IV. © 1998 European Peptide Society and John Wiley & Sons, Ltd.

Videothoracoscopy, routinely performed as the initial step of an operation, opens interesting opportunities for both the operative staging and treatment of lung cancer. Videosurgical maneuvers ensure thorough exploration of the cavity, thus avoiding unnecessary exploratory thoracotomies, confirming resectability of the lesion by open or, in selected cases, by a direct video-assisted approach. We report our experience of 155 patients submitted to videothoracoscopic operative staging between October 1991 and January 1994. Videothoracoscopic operative staging showed unresectability in 13 patients (8.3%) due to preoperatively unexpected (10 patients) or suspected conditions (3 patients). The remaining 142 patients were divided by staging of the lesion and general conditions into three groups. Group A consisted of 13 elderly patients with small peripheral tumor who could not tolerate lobectomy and who underwent thoracoscopic wedge resection. Group B consisted of 63 patients with peripheral clinical T1 N0 or T2 N0 tumor. Fifty-two lobectomies and 4 pneumonectomies were carried out thoracoscopically. Seven conversions to thoracotomy were necessary due to technical problems. The postoperative course was uneventful in 51, 5 had prolonged air leakage, and a bronchial fistula developed in 1 because of positive-pressure postoperative ventilation. Group C consisted of 66 patients with stage II or IIIa neoplasm. Thoracotomy after thoracoscopy proved unresectability in 4, whereas 62 were submitted to a radical pulmonary resection. In the literature the incidence of exploratory thoracotomies for conditions missed by preoperative staging still remains high. After adoption of videothoracoscopic operative staging we reported a 2.6% exploratory thoracotomy rate. This is sufficient to justify routine performing of videothoracoscopic operative staging as the first step of operation for lung cancer. Furthermore, videothoracoscopic operative staging permits confirmation of resectability of the lesion and, in selected patients, even direct video-excision. In our experience videothoracoscopic treatment proved a safe and concrete opportunity.

To establish the effectiveness of adjuvant chemotherapy in patients with colon cancer after radical surgery, from 1980 to December 1983, 263 patients were randomized in a multicentric study to no further treatment (131 patients) or to a combination of fluorouracil (5-FU) (400 mg/m2i.v., days 1–5) and lomustine (CCNU) (100 mg/m2per os on day 5) every 6 weeks for 9 cycles (132 patients). The two groups were well balanced for age, sex, histology, tumor and nodal extent. Chemotherapy was not given to 30 of the 132 randomized patients, and of 98 treated patients only 38 completed the entire protocol. Analysis, as intention to treat, at 54 months did not show any significant difference between the two treatment groups in terms of relapse-free survival (surgery alone, 74.5%; surgery+adjuvant chemotherapy, 70.9%; p = 0.91). In contrast, a significant difference was observed in overall survival (surgery alone, 78.8%; surgery+adjuvant chemotherapy, 60.8%; p = 0.04). The sites of relapse were identical in the two treatment arms. In conclusion, from this study it appears that adjuvant chemotherapy with 5-FU and CCNU seems to have no efficacy in the cure rate of colon cancer patients.

In the hamster, the weight of the adrenal glands increases during the course of gestation, with the highest value at day 5. In comparison to non-pregnant control animals, there were no changes in the volume of the zona glomerulosa (ZG) and zona fasciculata (ZF), while the volume of the zona reticularis (ZR) increased notably. The average volume of ZG-cells rose at day 5 of pregnancy and thereafter gradually decreased to that of control hamsters. A marked drop in the volume of ZF-cells was seen at days 5 and 10 of pregnancy, whereas at day 15 the cells were larger than in controls. At day 5 of pregnancy, a conspicuous increase in the cell volume was found in ZR, followed by lower values at day 10 and again higher than in control hamsters at day 15. The total number of parenchymal cells in hamster adrenal cortex increased at day 10 of gestation, then underwent a marked decrease, reaching the control value at the final day of pregnancy; this drop was mainly due to a reduction in the number of ZF-cells. The changes in the cell volume were paralleled by rather proportional changes in the volume of the mitochondrial compartment and in the quantity of smooth endoplasmic reticulum. The volume of the lipid-droplet compartment significantly rose in the course of gestation in both ZF- and ZR-cells. The cortisol output by adrenal homogenates gradually decreased during pregnancy.

The bolus administration of prolactin (PRL) to adult rats did not cause any apparent change in the basal and luteinizing hormone (LH)-stimulated blood levels of testosterone (as estimated by radioimmune assay). Prolonged PRL infusion did not affect either basal testosterone plasma concentration or the morphology of Leydig cells (as evaluated by electron microscopy and stereology). Conversely, prolonged PRL treatment notably increased the gonadotrophic effects of chronic LH administration; this mainly consisted of a rise in the blood concentration of testosterone and a conspicuous hypertrophy of Leydig cells. The LH-induced increase in the volume of Leydig cells was the result of an increase in the volumes of all the organelles involved in steroid synthesis (i.e., smooth endoplasmic reticulum, peroxisomes and mitochondria). However, the trophic effects of PRL infusion exclusively concerned smooth endoplasmic reticulum and peroxisomes. In the light of these findings, the hypothesis is advanced that the mechanism underlying the gonadotrophic action of PRL involves an enhancement of the endogenous cholesterol synthesis, which could provide an abundance of precursors for testosterone synthesis, the post-cholesterol steps of which, in turn, would be exclusively controlled by LH.

Transgenic rats for the murine Ren-2 gene display high blood pressure, low circulating levels of angiotensin II, and high renin content in the adrenal glands. Moreover, transgenic rats possess and increased aldosterone secretion (maximal from 6 to 18 weeks of age), paralleling the development of hypertension. To investigate further the cytophysiology of the adrenal glands of this strain of rats, we performed a combined morphometric and functional study of the zona glomerulosa of 10-week-old female transgenic rats. Morphometry did not reveal notable differences between zona glomerulosa cells of transgenic and age- and sex-matched Sprague-Dawley rats, with the exception of a marked accumulation of lipid droplets, in which cholesterol and cholesterol esters are stored. The volume of the lipid-droplet compartment underwent a significant decrease when transgenic rats were previously injected with angiotensin II or ACTH. Dispersed zona glomerulosa cells of transgenic rats showed a significantly higher basal aldosterone secretion, but their response to angiotensin II and ACTH was similar to that of Sprague-Dawley animals. Angiotensin II-receptor number and affinity were not dissimilar in zona glomerulosa cells of transgenic and Sprague-Dawley rats. These data suggest that the sustained stimulation of the adrenal renin-angiotensin system in transgenic animals causes an increase in the accumulation in zona glomerulosa cells of cholesterol available for steroidogenesis, as indicated by the expanded volume of the lipid-droplet compartment and the elevated basal steroidogenesis. However, the basal hyperfunction of the zona glomerulosa in transgenic animals does not appear to be coupled with an enhanced responsivity to its main secretagogues, at least in terms of aldosterone secretion.

Abstract: Epidemiological studies of sudden infant death syndrome (SIDS) reveal a progressive increase in the incidence of cases, suggesting that the in utero environment and postneonatal care were less than optimal. Subtle differences in symptoms could indicate that, since birth, the SIDS infants are different from control infants, some having an autonomic dysfunction of the airway controls. These observations could contribute to a better understanding of some of the causes of sudden death in infants and could eventually lead to new therapeutic approaches.

Abstract. The effects of short and prolonged treatments with prolactin on the morphology and hormone secretion of the rat adrenal zona glomerulosa were investigated by coupled morphometric and radioimmunological techniques. Short-term prolactin administration did not alter plasma aldosterone concentration or zona glomerulosa morphology. Conversely, chronic prolactin treatment caused both a notable hypertrophy of zona glomerulosa cells and a significant rise in the blood level of aldosterone. The possibility is discussed that prolactin may be involved in the control of the growth and steroidogenic capacity of the rat adrenal zona glomerulosa.

We studied sleeping infants in order to investigate whether a temporal relation exists between acid reflux extending to the proximal portion of the oesophagus and cardiorespiratory events. One hundred infants with occasional regurgitations were studied: 50 infants admitted after an apparently life-threatening event (ALTE) that occurred during sleep and that remained unexplained despite medical investigation, and 50 asymptomatic infants (non-ALTE). The infants had a median age of 8 weeks (range 4–26 weeks); 54 were boys. In each child a pH probe was placed in the proximal portion of the thoracic oesophagus, under radiological control. Polygraphic monitoring of state of alertness, cardiorespiratory activity, and proximal oesophageal pH changes was conducted continuously during 1 night. The data were analysed blind. In 80 infants a total of 186 decreases in oesophageal pH below 4 units were seen; 37% occurred during wakefulness, and 40% during rapid eye movement (REM) sleep. A total of 7029 central and 61 obstructive apnoeas were monitored, mainly during REM sleep. Within 5 min before, and 5 min after the drops in pH, there was no difference in the number, or the duration of bradycardia, central, mixed, or obstructive apnoea. The infants with an ALTE could not be differentiated from the non-ALTE infants for any of the variables studied. It is concluded that spontaneous acid refluxes extending to the proximal portion of the oesophagus during sleep are usually not temporally related with the development of apnoeas or bradycardias.

Summary: Two methods of management of the perineal wound after rectal excision for neoplastic disease were compared in a prospective randomized clinical trial in 65 patients. Twenty-eight patients (Group A) were treated by partial closure of the perineal wound and cigarette drainage of the pelvic space, according to the method proposed by Gabriel. Thirty-seven patients (Group B) were treated by complete suture of the perineal wound and suction drainage of the pelvic space according to the method proposed by Altemeier. Immediate and late results were better in patients in Group B. They achieved healingper primam in 62.85 per cent of cases and had significantly shorter hospital stays than patients in Group A. Three months postoperatively, 91.42 per cent of patients in Group B and 59.25 per cent of those in Group A had complete healing of the perineal wounds, with a statistically significant difference (χ2 with Yates correction =7.27, P<0.01). The development in 10 patients of Group B of perineal abscesses or hematomas necessitated reopening of the perineal wounds: however, the postoperative courses were similar to those of patients in Group A. It is concluded that healing by primary intention with use of the method described herein is the treatment of choice for managing the perineal wounds in patients undergoing rectal excision for neoplastic disease.

Trichorzianin TA VII, Ac⁰ U¹ A² A³ U⁴ J⁵ Q⁶ U⁷ U⁸ U⁹ S¹⁰ L¹¹ U¹² P¹³ V¹⁴ U¹⁵ I¹⁶ Q¹⁷ Q¹⁸ Fol¹⁹, is a nonadecapeptide member of the peptaibol antibiotics biosynthesized by Trichoderma soil fungi, which is characterized by a high proportion of the α,α-dialkylated amino acids, α-aminoisobutyric acid (Aib, U) and isovaline (Iva, J), an acetylated N-terminus and a C-terminal phenylalaninol (Pheol, Fol). The main interest in such peptides stems from their ability to interact with phospholipid bilayers and form voltage-dependent transmembrane channels in planar lipid bilayers. In order to provide insights into the lipid-peptide interaction promoting the voltage gating, the conformational study of TA VII in the presence of perdeuterated sodium dodecyl sulfate (SDS-d25) micelles has been carried out. ¹H sequential assignments have been performed with the use of two-dimensional homo- and -heteronuclear nmr techniques including double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser effect spectroscopy, ¹H-¹³C heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. Conformational parameters, such as ³JNHCαH coupling constants, temperature coefficients of amide protons (Δδ/ΔTNH) and quantitative nuclear Overhauser enhancement data, lead to detailed structural information. Ninety-eight three-dimensional structures consistent with the nmr data were generated from 231 interproton distances and six Φ dihedral angle restraints, using restrained molecular dynamics and energy minimization calculations. The average rms deviation between the 98 refined structures and the energy-minimized average structure is 0.59 Å for the backbone atoms. The structure of trichorzianin TA VII associated with SDS micelles, as determined by these methods, is characterized by two right-handed helical segments involving residues 1–8 and 11–19, linked by a β-turn that leads to an angle about 90°–100° between the two helix axes; residues 18 and 19 at the end of the C-terminal helix exhibit multiple conformations. © 1998 John Wiley & Sons, Inc. Biopoly 46: 75–88, 1998

During sleep, infants with obstructive sleep apneas are characterised by snoring, laborious breathing, and profuse sweating. During wakefulness, they may have breath-holding spells, and during feeding, difficult breathing and swallowing coordination. Abnormal weight, difficult growth, and recurring ear infections may also develop. During sleep apneas, cinefiuoroscopy shows approximation of tongue and hypopharyngeal tissues, with an obliteration of the air space. The obstructed breaths occur mainly in REM, and light NREM sleep, associated with total short sleep time, and frequent arousals. Preterm infants, and term neonates are more prone to obstructive apneas than older healthy infants. Apneas are more frequently seen in boys and in case of excess in body weight. Obstructive apneas are frequently associated with upper airway anatomic abnormalities: malformations, soft tissue infiltration, and neurologic lesions impairing muscle contractions. Alterations of the autonomic nervous control may induce airways obstructions. Contributing factors include mucopolysaccharide storage disease, hypothyroidism, or Down’s syndrome. Superimposed factors may occur, such as nasal obstruction, secretions in the airways, or tissue edema. Pressure- and chemo-sensitive reflexes may also favor obstruction. Environmental factors also contribute to the development of sleep apneas: body position, neck flexion, sleep deprivation, or the effects of sedative drugs.

The effects of a chronic treatment with angiotensin II (up to 15 consecutive days) on the mitochondria of the rat zona glomerulosa cells were investigated by electron microscopic and stereological methods. Angiotensin induced a significant increase in the volume of the mitochondrial compartment. Up to the 3rd day of treatment this was due only to the hypertrophy of the organelles, and from the 3rd to the 15th day exclusively to mitochondrial proliferation. The hypothesis that angiotensin controls the growth and proliferation of rat zona glomerulosa mitochondria is discussed.

The ultrastructural changes associated with the compensatory hypertrophy of the zona fasciculata cells on monoadrenalectomized rats were investigated by stereological techniques. It was found that these subcellular changes display a different pattern from those underlying the ACTH-induced adrenocortical cell growth in the intact rats. This result supports the view that compensatory adrenal hypertrophy does not involve activation of the hypothalamo-hypophyseal-adrenal axis.