In Penicillium chrysogenum the beta-lactam biosynthetic pathway is compartmentalized. This fact forces the occurrence of transport processes of penicillin-intermediate molecules across cell ...membranes. Many aspects around this molecular traffic remain obscure but are supposed to involve transmembrane transporter proteins. In the present work, an in-depth study has been developed on a Major Facilitator-type secondary transporter from P. chrysogenum named as PenM. The reduction of penM expression level reached by penM targeted silencing, leads to a decrease in benzylpenicillin production in silenced transformants, especially in SilM-35. On the contrary, the penM overexpression from a high efficiency promoter increases the benzylpenicillin production and the expression of the biosynthetic genes. Moreover, when the silenced strain SilM-35 is cultured under penicillin production conditions with 6-aminopenicillanic acid supplementation, an increase in the benzylpenicillin production proportional to the 6-aminopenicillanic acid availability is observed. By this phenomenon, it can be concluded that due to the penM silencing the benzylpenicillin transport remains intact but the peroxisomal isopenicillin N import results affected. As a culminating result, obtained by the expression of the fluorescent recombinant PenM-DsRed protein, it was determined that PenM is naturally located in P. chrysogenum peroxisomes. In summary, our experimental results suggest that PenM is involved in penicillin production most likely through the translocation of isopenicillin N from the cytosol to the peroxisomal lumen across P. chrysogenum peroxisomal membrane.
•The penM gene encodes a membrane protein in Penicillium chrysogenum.•penM gene silencing and oveerexpresion affects the benzylpenicillin production.•6-APA feeding increases the benzylpenicillin production in SilM35 (silenced strain).•Localization studies showed that PenM protein is located in the peroxisomal membrane.•PenM could be implicated in the import of IPN to the peroxisome from the cytosol.
The
Acremonium chrysogenum cephalosporin biosynthetic genes are divided in two different clusters. The central step of the biosynthetic pathway (epimerization of isopenicillin N to penicillin N) ...occurs in peroxisomes. We found in the “early” cephalosporin cluster a new ORF encoding a regulatory protein (CefR), containing a nuclear targeting signal and a “Fungal_trans” domain. Targeted inactivation of
cefR delays expression of the
cefEF gene, increases penicillin N secretion and decreases cephalosporin production. Overexpression of the
cefR gene decreased (up to 60%) penicillin N secretion, saving precursors and resulting in increased cephalosporin C production. Northern blot analysis revealed that the CefR protein acts as a repressor of the exporter
cefT and exerts a small stimulatory effect over the expression level of
cefEF that explains the increased cephalosporin yields observed in transformants overexpressing
cefR. In summary, we describe for the first time a modulator of beta-lactam intermediate transporters in
A. chrysogenum.
► A regulatory gene cefR modulates cephalosporin biosynthesis. ► Inactivation of CefR increases PenN secretion and decreases CPC. ► CefR controls expression of cefT and cefEF. ► In summary, CefR saves intermediates for CPC production.
This study reports mortality and morbidity rates observed with the Omniscience valve prosthesis. Complication rates were calculated from a prospective, long-term follow up study of patients with the ...Omniscience valve prosthesis in the aortic, mitral, or both aortic and mitral positions.
The Omniscience heart valve was implanted into 200 consecutive patients (42.5% aortic valve replacement (AVR); 51.5% mitral valve replacement (MVR); 6% AVR/MVR), with an average age of 57 +/- 12 years, at the author's institution. Patients were checked regularly at a clinic which specialized in prosthetic valve follow up. Only one patient was lost to follow up during this 13-year period (99.5% completeness of follow up).
The total follow up was 1475 patient-years (pt-yr); mean follow up 8.0 +/- 2.9 years. Survival probability at 12 years, including freedom from both early and late mortalities, was 67.1 +/- 6.7% for AVR and 62.8 +/- 6.0% for MVR patients. There were no structural valve failures. Twelve-year freedom from thromboembolism was 85.5 +/- 5.0% and 85.2 +/- 6.5% for AVR and MVR, respectively. For thromboembolism + anticoagulation-related hemorrhage, the 12-year freedom rates were 79.4 +/- 5.3% for AVR and 77.3 +/- 6.7% for MVR patients.
The Omniscience valve demonstrated a low risk of late complications, comparable with that of other modern mechanical valves. Omniscience valve patients also exhibited excellent hematology values and good functional improvement during this long-term prospective follow up. It is concluded that the Omniscience valve has satisfactory long-term clinical performance in both the aortic and mitral positions.
A case of spontaneous hemothorax in a 7-year-old child secondary to erosion of the diaphragm by an exostosis coming from the left sixth rib is reported. This rare case of hemothorax with hereditary ...multiple exostoses is made even rarer by the concomitant perforation of the diaphragm.
The Perma-Flow prosthetic coronary graft is a 5-mm polytetrafluoroethylene tube into which is incorporated a Venturi flow restrictor. An aorto–superior vena caval fistula is created and coronary ...anastomoses are constructed proximal to the resistor in side-to-side fashion, where arterial pressure is maintained. From November 1992 through December 1995, eight investigational centers in North America have implanted this graft in 40 patients with inadequate autologous alternatives.
Patients were selected for inclusion in this study if coronary artery bypass grafting was required and adequate autologous conduit to complete revascularization was not available. Operative data were completed by the implantating surgeon and referred to a central center, the Minneapolis Heart Institute, for correlation. Follow-up was conducted by data coordinators at each institution, and follow-up data were obtained directly from these coordinators for inclusion in the study.
Patient age ranged from 53 to 82 years, and 15 patients were undergoing reoperations (38%). On each Perma-Flow graft one to four coronary side-to-side anastomoses were constructed. In addition, left internal mammary artery (n = 26), greater saphenous vein (8), right internal mammary artery (4), and gastroepiploic artery (4) were used to complete revascularization. Aortic (2) or mitral valve replacement (1) was also carried out. There were seven operative deaths (18%) and two late deaths (4 and 6 months). After 1 to 37 months (mean, 13±9 months) of follow-up, 29 of 31 surviving patients are asymptomatic. Echocardiographic heart size has not increased from the postoperative value, indicating limited volume load has not affected heart size. Protocol catheterization (n = 32) in 28 patients 1 week to 1 year postoperatively revealed 7 of 73 studied coronary anastomoses (9.5%) and two distal extensions and resistors were occluded (7%). In 1 patient during sternal debridement at 1 year, no flow was found in the graft.
The Perma-Flow graft is a useful adjunct to complete revascularization in patients with deficient autologous conduit.
In this work we report the development and validation of a new RNA interference vector (pJL43-RNAi) containing a double-stranded RNA expression cassette for gene silencing in the filamentous fungi ...Penicillium chrysogenum and Acremonium chrysogenum. Classical targeted gene disruption in these fungi is very laborious and inefficient due to the low frequency of homologous recombination. The RNAi vector has been validated by testing the attenuation of two different genes of the b-lactam pathway; pcbC in P. chrysogenum and cefEF in A. chrysogenum. Quantification of mRNA transcript levels and antibiotic production showed knockdown of pcbC and cefEF genes in randomly isolated transformants of P. chrysogenum and A. chrysogenum, respectively. The process is efficient; 15 to 20% of the selected transformants were found to be knockdown mutants showing reduced penicillin or cephalosporin production. This new RNAi vector opens the way for exploring gene function in the genomes of P. chrysogenum and A. chrysogenum.
To assess the safety of reusing single-use intraaortic balloon devices (IABs), 112 used devices were investigated in terms of physical integrity, gas leakage inspection, mechanical performance, ...surface chemistry and morphology, and physical stability. These IABs were all used clinically only once, and the duration of the IABs in vivo ranged from 6 to 312 h. Macroscopic examination of the balloons and the outer catheters revealed no obvious change in either shape or color. No discernible abrasions or cracks were observed on the balloons. However, 61% of the balloons were creased, and 40% of the central lumens and 21% of the sheaths showed visible bending flaws. Moreover, 65% of the balloons and 38% of the central lumens were contaminated by visible residual organic debris. The physical integrity of each device was verified in a specially designed leakage-fatigue tester for 72 h. Ninety-seven percent of the devices passed the leakage inspection. Stress-strain testing, differential scanning calorimetry, attenuated total reflection-Fourier transform infrared, and scanning electron microscopy analyses clearly indicated that there were no significant differences in the mechanical properties, bulk material morphology, surface chemistry, and external surface morphology between the used balloons and virgin controls. Although some surface modifications occurred on the internal side of the balloons, the external surfaces of most balloons suffered no trauma. Most of the used IABs examined in this study maintained physical and mechanical properties similar to those of the virgin devices. The chemistry of the balloon material was stable after short-term in vivo use. However, it does not seem possible to establish a rigorous protocol of cleaning, sterilization, and inspection to guarantee a safer reuse of these devices. The presence of residual organic debris that cannot be eliminated results in an imperative preclusion not to reuse the IABs.
Hydrophilic, microporous polyurethane vascular prostheses displayed good mechanical characteristics and the behaviour in vitro was excellent. The in-vivo results were, however, disappointing. To ...obtain a better understanding of the phenomena involved in the acute and early thrombosis, we implanted the polyurethane graft in the canine left femoral artery and an autologous femoral vein in the right for 4 and 24 h. At 4 h, one polyurethane graft was totally occluded and the other two were close to complete occlusion; at 24 h, none was patent. On the other hand, all autologous veins were patent. The thrombotic matrix incorporating both platelets and fibrinogen, quantified by labelling, was anchored along the anastomotic lines. This study confirms that polyurethane occlusion is initiated by hyperplastic reaction, but does not explain why.
The Penicillium chrysogenum oat1 gene, which encodes a class III omega-aminotransferase, was cloned and characterized. This enzyme converts lysine into 2-aminoadipic semialdehyde, and plays an ...important role in the biosynthesis of 2-aminoadipic acid, a precursor of penicillin and other beta-lactam antibiotics. The enzyme is related to ornithine-5-aminotransferases and to the lysine-6-aminotransferases encoded by the lat genes found in bacterial cephamycin gene clusters. Expression of oat1 is induced by lysine, ornithine and arginine, and repressed by ammonium ions. AreA-binding GATA and GATT sequences involved in regulation by ammonium, and an 8-bp direct repeat associated with arginine induction in Emericella (Aspergillus nidulans and Saccharomyces cerevisiae, were found in the oat1 promoter region. Deletion of the oat1 gene resulted in the loss of omega-aminotransferase activity. The null mutants were unable to grow on ornithine or arginine as sole nitrogen sources and showed reduced growth on lysine. Complementation of the null mutant with the oat1 gene restored normal levels of omega-aminotransferase activity and the ability to grow on ornithine, arginine and lysine. The role of the oat1 gene in the biosynthesis of 2-aminoadipic acid is discussed.
This study was undertaken to compare the chemically processed internal mammary artery (BIMA) and the autologous femoral vein as arterial grafts. The BIMA prosthesis was implanted as a left femoral ...artery bypass and the femoral vein as a right femoral artery bypass graft in 27 dogs. In groups of three dogs the grafts were implanted for predetermined durations: 4, 24 and 48 hours (short term), 1, 2 and 4 weeks (medium term) and 3, 6 and 9 months (long term). All autologous veins were patent when the dogs were killed. The patency rates of the BIMA grafts were 100% in the short-term group, 67% in the medium-term group and 29% in the long-term group. The deposition of labelled fibrinogen and platelets on flow surfaces, the structural preservation of the wall of the BIMA prosthesis and accumulation of thrombi during the period of implantation were studied.
