Impact of the first Streptomyces genome sequence on the discovery and production of bioactive substances.

An important addition to the field of bacterial genomics is the recent publication of the complete genome sequence of Streptomyces coelicolor. This strain has been for some decades the model organism for streptomycetes and other filamentous actinomycetes, Gram-positive bacteria highly valuable for their ability to produce thousands of bioactive metabolites, many of which have found important applications in medicine and agriculture. We discuss here the impacts that the S. coelicolor genome sequence is likely to have on the production of bioactive metabolites by current industrial strains, on the possible development of future superhost(s) for the production of valuable drugs, and on the search for new bioactive substances from microbial sources.

[Acute pancreatitis: resultos of a diagnostic-therapeutic protocol in 80 consecutive cases]. / Pancreatiti acute: risultati di un protocollo diagnostico-terapeutico in 80 casi consecutivi.

The treatment of acute pancreatitis cannot be standardized in the absence of a prompt diagnosis and of an accurate severity and prognostic score. This study, based on 80 consecutively observed patients, compared the aetiological, clinical, diagnostic (laboratory and imaging) and prognostic data used to select the most appropriate therapy for each patient. The results confirm that the Ranson score shows a satisfactory prognostic relationship between the number of positive parameters and the severity of the disease. Ultrasound, which is useful for defining the aetiologic factors and in the follow-up of peripancreatic effusions, has proved to be limited as a means of imaging abnormalities of the pancreatic parenchyma. CT scans are confirmed as being the only method of accurately demonstrating the presence of necrosis and of evaluating its effective extent. ERCP was performed as soon as possible in the presence of biliary stasis or of suspect ultrasonographic signs. Surgical treatment proved necessary only in 7.5% of cases, on each occasion to drain infected necrotic foci. Promptness of the surgical indication plays an important role in the outcome of necrosectomy and drainage performed with the closed technique. Mortality was limited to 1.25% in our series. A correct diagnostic approach together with prompt treatment can reduce the mortality rate of this disease to a minimum.

Biosynthesis of the thiazolylpeptide antibiotic GE2270.

The biosynthesis of the antibiotic GE2270 in the producing microorganism Planobispora rosea was investigated by adding labelled amino acid precursors. Efficient incorporation of glycine and serine was observed, leading to specific enrichments of selected positions of the thiazole, oxazoline and pyridine rings. Furthermore, efficient enrichment of the C-, N- and O-methyl groups was detected. These results indicate that GE2270 is made through a biosynthetic route similar to that determined for other thiazolylpeptides. At the same time, the result point to an efficient route for the conversion of glycine into serine and methyl equivalents in Planobispora rosea.

[Vascular trauma of the upper extremities]. / I traumi vascolari degli arti superiori.

The aim of this study was to evaluate short- and long-term results of the treatment of upper extremities vascular trauma considering aetiology of the lesions, percentage of limb salvage and residual functional disability. The Authors retrospectively evaluated 17 patients accounting for 21 vascular lesions of the upper extremities (16 arterial and 5 venous injuries). Age, sex, modality of trauma, site of the vascular lesions and of the associated injuries, diagnostic procedures at the admission, ischemic time, arterial and venous repair performed were analyzed. The over all peri-operative mortality was 5.8%. Of the 16 arterial injuries long-term reconstruction viability was obtained in 15 patients (93.7%). In all cases limb salvage was obtained. In 3 patients invalidating functional defects due to associated injuries of the major brachial plexus were observed. The Authors believe that associated nervous lesions are the main factor determining invalidating residual disability. In this series ischemic time, technique of vascular repair performed, associated skeletal injuries didn't influence the functional outcome of the reconstruction.

[Hilar localization of splenic cyst does not always exclude the possibility of resective treatment]. / La localizzazione ilare di una cisti splenica non sempre esclude la possibilità del trattamento resettivo.

Non parasitic cysts of the spleen require surgical treatment because of their progressive growth and in order to prevent the potential severe complications associated with such cysts. Since it is now well known that total splenectomy, especially in young patients, has potential for short- and long-term complications, much emphasis has been placed on splenic salvage, suggesting partial splenectomy as procedure of choice for splenic cysts. However various Authors suggest that many but not all splenic cysts can be treated with partial splenectomy. In particular cystic mass arising from the anterior aspect of the hilum near to vascular peduncle contraindicate partial resection requiring splenectomy. In a case observed TC scan demonstrated a very large epidermoid cyst penetrating hilar parenchyma just above splenic vessels insertion. Preoperative imaging suggested splenectomy as the only possible procedure to remove the cyst. At operation the exposure of the splenic artery extended proximally along the pancreatic tail showed an arterial branch running with satellite vein in the splenopancreatic ligament for inferior segment of the spleen. As we found this branch it was possible to resect cyst preserving a large inferior parenchymal segment normally perfused and functioning at postoperative scintigraphic controls. In conclusion not all hilar cysts must be considered an absolute indication to splenectomy. An accurate and extensive exposure of splenic artery and vein can demonstrate vascular anatomical variations permitting resection also for large cysts located near the splenic hilum.

[Splenectomy in immune thrombocytopenia and other hematological diseases]. / La splenectomia nelle trombocitopenie immuni ed in altre malattie ematologiche.

The Authors report a retrospective study of 74 splenectomies performed for hematologic diseases. The role of splenectomy has changed over recent years with increased indications for immune thrombocytopenic purpura (ITP). The aim of this study was to assess indications to surgery in relation to clinical presentation with typical hemorrhagic features or severe thrombocytopenia only; interval between onset of symptoms and splenectomy; failure of medical management and complications from steroid administration; scintigraphic studies predictive of response to splenectomy and preoperative treatment in patients with severe thrombocytopenia were also studied. The Authors reported response rates to splenectomy of 84% without mortality and only 11% of postoperative complications. These results encouraged to surgery for treatment of those patients with severe thrombocytopenia, who fail to obtain remission or develop serious complications after medical therapy. The splenectomy cured severe thrombocytopenia also in some patients with acquired immunodeficiency (HIV+). Moreover the Authors discuss the indications in patients with chronic lymphatic leukaemia and lymphoma diseases. In selected patients the splenectomy has the potential to relieve symptoms due to splenomegaly, correct cytopenias, specify hystological diagnosis and modify the disease course of malignant lymphomas. In fact splenomegaly sometimes complicated the course of malignant lymphomas because of hematologic abnormalities that are inconsistent with active chemotherapy.

Solitary metachronous splenic metastases: an evaluation of surgical treatment.

Splenic metastases occurring after primary tumor removal and apparently solitary have been documented only recently in Literature. They are, most of the times, clinically asymptomatic and their presence is casually determined by ultrasonographic follow-up in subjects otherwise in good conditions. The belief that splenic metastases occur only in disseminated cancer is today no longer accepted. Some Authors consider solitary splenic metachronous metastases eligible for surgical treatment as well as pulmonary or hepatic metastases. In the case presented, surgery was required due to abscess formation of a splenic metastasis, which was not responding to chemotherapy. Our experience, like others reported in Literature, verified a long-term post-operative survival in spite of limited disease-free time. Surgical treatment by splenectomy can be indicated in selected patients, considering that chemotherapy has been proved to be ineffective in the treatment of splenic metastases.

Isolation and structure determination of a novel complex of the teicoplanin family.

A new teicoplanin-like antibiotic was discovered when using Actinoplanes teichomyceticus strain 3/W, the fermentation medium containing Alburex, and the fermentation time 275 hours. The new product was separated from teicoplanin complex by polyamide resin chromatography and purified by Amberlite XAD-7 and affinity resin chromatographies. The structure was established on the basis of the physico-chemical characteristics of the complex and of its aglycone. The new structure is that of teicoplanin with a carbonyl group substituting for the CHNH2 group of amino acid 1. We hypothesize that the novel complex is a transformation product of teicoplanin due to a simple transamination reaction, as supported by its structure and by the concomitant decrease in teicoplanin concentration during its production.

Determination of the acyl moieties of the antibiotic complex A40926 and their relation with the membrane lipids of the producer strain.

Structures of the fatty acid residues characterizing the various components of A40926 were determined by gas chromatography/mass spectrometry on the methyl esters obtained by methanolysis of the complex. The results confirm the residues previously assigned to Factor A (n-undecanoic acid) and B (10-methyl-undecanoic acid) and establish the residues of Factor A1 (9-methyl-decanoic acid), B1 (n-dodecanoic acid), RS1 (8-methyl-nonanoic acid), RS2 (n-decanoic acid), and RS3 (n-tridecanoic acid). As the Actinomadura species contain in their mycelia large quantities of C15-C17 fatty acid residues as membrane phospholipids, these mycelia were saponified and the fatty acids obtained were analyzed as above. There is a close correlation between the fatty acid content of A40926 complex and that of the longer homologues in the producer mycelia.

Microbial de-mannosylation and mannosylation of teicoplanin derivatives.

The single components of the teicoplanin complex, glycopeptide antibiotics active against Gram-positive bacteria, can be converted in the corresponding de-mannosyl derivatives by cultures of Nocardia orientalis NRRL 2450 or Streptomyces candidus NRRL 3218. Conversely, teicoplanin aglycone and other teicoplanin de-mannosyl derivatives can be converted in the corresponding teicoplanin mannosyl derivatives by cultures of Actinoplanes teichomyceticus ATCC 31121. The biological transformation yields are approximately 40% for de-mannosylation and 90% for mannosylation. The processes allow for the preparation of gram quantities of the de-mannosyl derivatives of teicoplanin and of teicoplanin mannosyl derivatives. De-mannosyl teicoplanin and teicoplanin mannosyl-pseudoaglycone were not amenable to preparation by either acidic or basic chemical hydrolysis.

Time course of changes in pancreatic enzymes, isoenzymes and, isoforms in serum after endoscopic retrograde cholangiopancreatography.

Kinetics of the catalytic activities of total amylase (AMY; EC 3.2.1.1), pancreatic (P)-AMY isoenzyme, P2 and P3 isoforms, and pancreatic lipase (LPS; EC 3.1.1.3), and of the mass concentration of LPS in serum were studied in 10 patients who underwent endoscopic retrograde cholangiopancreatography (ERCP) and showed a distinct pancreatic injury. The temporal characteristics of enzyme changes described were (a) the maximal rate (Ka) at which enzymes are released into blood, (b) the time lag from ERCP until maximum concentration value, (c) the peak value of each serum enzyme, and (d) the rate (Kd) at which each enzyme is cleared from serum. LPS activity and mass concentrations increased and decreased faster than AMY and isoamylases, and the time of the LPS peak tended to be earlier than that of the other enzymes, but not significantly. The average peak increase of LPS values was higher than that of total AMY, P-AMY, and P2 isoform (P less than 0.001). The P-AMY time-activity curve was a composite of curves attributable to its isoforms; the isoforms increased and peaked sequentially, with P3 returning to normal more slowly than did P2. LPS mass and activity concentrations showed excellent parallelism, with no important differences. At 50 h after ERCP, only LPS values still exceeded the upper reference limit, returning to normal 70 h after the examination.

Factors affecting the normal and branched-chain acyl moieties of teicoplanin components produced by Actinoplanes teichomyceticus.

Teicoplanin, a glycopeptide antibiotic produced by Actinoplanes teichomyceticus, comprises five main components, denoted T-A2-1 to T-A2-5, differing in the structure of their acyl side chain, which is linear in T-A2-1 and T-A2-3 and branched in the other components. Production of T-A2-1, characterized by a linear C10:1 acyl moiety, is entirely dependent on the presence of linoleate in the fermentation medium. Addition to the medium of oleic acid esters at 2 g l-1 increases the yields of T-A2-3, characterized by a linear C10:0 acyl chain, about threefold. The antibiotic linear side chains thus appear to originate from C18 unsaturated acid by beta-oxidation degradation. The percentage of T-A2-2, T-A2-4 and T-A2-5, bearing the iso-C10:0, anteiso-C11:0 and iso-C11:0 acyl moieties, respectively, is strongly influenced by the presence in the medium of the amino acids known to be precursors of branched-chain fatty acids. Thus, valine increases the production of T-A2-2 whereas isoleucine or leucine increase the relative yields of T-A2-4 or T-A2-5, respectively. Analysis of the total cell lipids upon addition of the same amino acid shows corresponding increases in the proportion of the iso-C16:0, iso-C15:0 or anteiso-C17:0. A mutant A. teichomyceticus strain, which produces a novel teicoplanin with a linear C9:0 chain, differs from the wild strain in the presence of the linear C17:1 acid in its lipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and structure determination of two new analogs of teicoplanin, a glycopeptide antibiotic.

Teicoplanin is an antibiotic produced by fermentation of Actinoplanes teichomyceticus as a complex formed by five closely related glycopeptides characterized by different fatty acid chains of ten and eleven carbon atoms. In addition, minor quantities of related substances are present. Two of them, named RS-1 and RS-2, were shown to be teicoplanins having as fatty acid chains 10-methylundecanoic acid and n-dodecanoic acid, respectively. Other two related substances, named RS-3 and RS-4, have now been isolated and purified starting from fermentation broths of a mutant of the same microorganism producing them in substantial amounts. This was achieved by semipreparative reversed-phase liquid chromatography carried out on high-pressure scale. The structures were assigned on the basis of 1H NMR spectra and homonuclear COSY 2D experiments and fast atom bombardment MS spectrometry, in comparison with the large mass of data till now accumulated on teicoplanin. RS-3 and RS-4 are teicoplanins having as fatty acid chains 6-methyloctanoic acid and n-nonanoic acid, respectively.

Cisplatin, high dose folinic acid and 5-fluorouracil in squamous cell carcinoma of the esophagus. A pilot study.

We treated 20 patients with squamous cell carcinoma of the esophagus with the following regimen: cisplatin = 100 mg/m2 i.v. day 1, HDFA = 200 mg/m2 i.v. day 1 to 5, 5-fluorouracil = 370 mg/m2 i.v. day 1 to 5 repeated every 28 days. Among the 17 evaluable patients, 4 (23%) had a partial remission, 6 had stable disease while 7 progressed. The median survival for all patients was 6+ months. Grade III or IV toxicity included 2 patients with grade III leukopenia, 1 with grade III thrombocytopenia, 1 patient with grade IV and 3 patients with grade III oral mucositis, 3 patients with grade III diarrhea. The regimen did not seem particularly active in the treatment of squamous cell carcinoma of the esophagus and had manageable but substantial toxicity.

On the mode of action of a new contragestational agent (DL 111-IT).

Non-hormonal compounds belonging to 3,5-diphenyl-1H,1,2,4 triazoles and chemically related classes (triazoles in short) are known to be endowed with high contragestational activity in rodents, dogs and primates. The data herein reported for one of the leaders of this family of compounds (DL 111-IT) along with those previously reported for some analogues, demonstrate that triazoles cause pregnancy arrest by a direct action on conception product. This action is expressed through a progressive slowing down of the conceptus development with a consequent onset of a state of anoxia, pregnancy arrest, degeneration of placentae and adnexae and their absorption or expulsion. The selective time of gestation during which they elicit the antifertility effect (early post-implantation period) and the histological examinations revealed that the target of their action are the ectoplacental and decidual cells. Biochemical studies on conceptus product demonstrate that, although they do not bind to progesterone receptors or inhibit ornithine decarboxylase activity, triazoles induce an early and marked increase in the number of cytosol progesterone receptors accompanied by a steep decrease in the ornithine decarboxylase activity in the product of conception. These findings are discussed in relation to the possibility that triazoles may trigger pregnancy arrest by interfering with the chain of events by which progesterone regulates the mitotic activity of decidual and trophoblastic cells.

Contragestational profile of the tumor-inhibiting agent, L-alanosine, in the rat and the hamster.

L-Alanosine [L-2-amino-3(N-hydroxy-N-nitrosamino)propionic acid], a tumor-inhibiting agent, induces pregnancy arrest after single or multiple SC or PO administration to rats and hamsters. Its contragestational effects are dose- and route-dependent, with no important differences in species-sensitivity or administration schedules. L-Alanosine is maximally effective shortly (3-4 days) after implantation. Both placenta and fetus appear to be target tissues. Consistent with previous in vitro findings, adenine but not aspartic acid counteracts the contragestational action of L-alanosine. The 'contragestational test', i.e., the effect on conceptus growth, appears to be an interesting approach for learning more about the antiproliferative activity of an antineoplastic agent.

DL 111, a new non-hormonal antifertility agent: contragestational and kinetic profile in baboons.

It was previously shown that 3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4 triazole (DL 111) given parenterally in single or multiple doses during the early stage of embryonal development terminates pregnancy in the mouse, the hamster, the rat, the rabbit and the dog. In the present work, the studies have been extended to the baboon. In this sub-human primate, single and/or multiple intramuscular injections of the compound terminated pregnancy when given between day 34 and 54 of gestation. The effectiveness of DL 111 was greater when earlier in gestation and the optimal mode of treatment appears to be a multiple one. DL 111 appears to act by a direct action on the conceptus, with consequent suppression of the endocrine function of the placenta, progesterone withdrawal and abortion. In all the baboons that aborted, the menstrual cycles resumed within a reasonable length of time and subsequent cycles were regular. All the animals that did not abort have given birth to normal and health term infants. Fertility appears to be unimpaired and the progeny resulting form these pregnancies did not show any abnormalities. No significant drug-related side-effects or alterations in plasma enzymes or haematological parameters were observed. Pharmacokinetic and activity relationships strongly suggest that sustained exposure of the conceptus to the drug action is indispensable for optimizing the pregnancy-terminating effect.

A new non-hormonal pregnancy-terminating agent.

DL 111-IT, 3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4 triazole, a compound belonging to a new class of non-hormonal antifertility agents, when given subcutaneously, intramuscularly, intravaginally or orally terminates pregnancy in the rat, the mouse, the hamster and the dog. Time-course and dose-activity studies indicate that its effectiveness is dependent on dose, vehicle, route and time of pregnancy. DL 111-IT has no pre-implantation activity. The most effective time for treatment is the early post-implantation period. The compound has an antifertility effect through a slow and continuing action that results in the degeneration and subsequent resorption or expulsion of conceptuses. As a result, there must be sustained availability of active principle to arrest the pregnancy. Administered parenterally in a proper vehicle (oily) and with a suitable schedule of treatment (x 2-5 days), it demonstrates a very high pregnancy terminating activity (ED50: 0.04-0.7 mg/kg/day). Multiple intravaginal and oral administrations are also effective but the daily doses required are 10-20 and 40-100 times higher than the parenteral ones. Studies of the mechanism of action indicate that the site of action is the utero-placental complex. In fact, in pregnant rats, mice, hamsters and dogs, both plasma progesterone levels and the ineffectiveness of progesterone therapy rule out luteolysis as a basis for the activity. Moreover in hypophysectomized, ovariectomized animals whose pregnancies were maintained with proper hormonal treatments, DL 111-IT terminates pregnancy and adrenalectomy does not prevent its effect, which suggests that pituitary, ovaries and adrenals are not required for the antifertility action.

Rifamycin R, a novel metabolite from a mutant of Nocardia mediterranea.

Rifamycin R is a novel ansamycin produced by a mutant of Nocardia mediterranea; both physical and chemical data indicate that it is 30-demethyl-30-hydroxymethyl rifamycin S.