Search

Background: The purpose of this study is to analyse the impact that occupational health and safety policies have on employees’ work and organisational commitment and, in turn, on their intention to leave the company. Methods: For this study, we designed a questionnaire with a five-level Likert scale and distributed it among professionals from different companies in the IT consultancy sector in Spain. The data collected from 458 completed questionnaires were analysed using the partial least squares structural equation modelling (PLS-SEM) technique using the SmartPLS software. Results: From the analysis of the data, it was concluded that there is indeed a positive relationship between occupational health and safety policies and employees’ work and organisational commitment, as well as a negative relationship between these policies and the intention to leave the company. Similarly, there is a negative relationship between employees’ work and organisational commitment and their turnover intention. Conclusions: Although, due to the activity of professionals in the sector, occupational health and safety has not been an area of priority, it was concluded from this study that an improvement in these areas would have a beneficial effect on the commitment of workers to the company, thus helping to reduce the high levels of turnover in the sector. Future lines of research, as well as their practical application and the limitations of the study, are indicated at the end of the paper.

The evaluation of the work performance of health professionals has focused the interest of scientific research in recent decades as a basis for improving the quality of health services. The global COVID-19 pandemic has pushed countries’ health systems to the limit and had previously unknown consequences on the job performance of health professionals. In this context, what are the determinants of performance? There are numerous studies that link job performance with other variables that directly affect it, such as leadership, job satisfaction, organizational commitment, and work environment. However, there are no studies that jointly relate all these variables, and even less in the field of health. The main objective of this work is to analyse how these variables are configured together to generate a good level of performance of health professionals during the times of COVID-19. To do this, a fuzzy set qualitative comparative analysis (fsQCA) is carried out, an appropriate method that will allow finding the joint causal effects of key variables in human resources to ensure a good level of job performance in health organizations. The study reveals that leadership and commitment are the two key drivers of performance. The data confirm that the “recipe” to achieve a good level of performance consists of the combination of leadership, commitment, and a good work environment. Additionally, in the case of less satisfied workers, linking leadership and commitment is a sufficient condition.

The occurrence of toxic metals and metalloids associated with mine tailings is a serious public health concern for communities living in mining areas. This work explores the relationship between metal occurrence (e.g., spatial distribution in street dusts), human health indicators (e.g., metals in urine samples, lifestyle and self-reported diseases) and socioeconomic status (SES) using Chanaral city (in northern Chile) as study site, where a copper mine tailing was disposed in the periurban area. This study model may shed light on the development of environmental and health surveillance plans on arid cities where legacy mining is a sustainability challenge. High concentrations of metals were found in street dust, with arsenic and copper concentrations of 24 ± 13 and 607 ± 911 mg/kg, respectively. The arsenic concentration in street dust correlated with distance to the mine tailing (r = − 0.32, p-value = 0.009), suggesting that arsenic is dispersed from this source toward the city. Despite these high environmental concentrations, urinary levels of metals were low, while 90% of the population had concentrations of inorganic arsenic and its metabolites in urine below 33.2 µg/L, copper was detected in few urine samples (

Emerging contaminants are recently uncovered in natural currents duo to human and industrial activities. Most of them have no legal standard and can probably cause damaging effects in aquatic life at relevant concentrations. The conventional wastewater treatment plant (WWTP) is not always effective for the removal of these huge classes of pollutants and so further water treatments are necessary. In this work, we applied the powdered activated carbon adsorption process to eliminate the selected emerging pollutants, Theobromine and two antibiotics commonly used in Spain: Amoxicillin (AMX) and Penicillin G (Peni G). Different experimental conditions such as pH and temperature, initial concentration, the amount of biomass, and contact time were studied to obtain the best removal conditions. The different contaminants were detected by ultra-performance liquid chromatography and mass spectrometry (UPLC-MS). The whole results obtained confirm the effectiveness of the process of adsorption for the purification of the liquid effluents charged with antibiotics and stimulant. The results we have obtained indicate that coconut shell activated charcoal allows better retention of this compound after 90 minutes of contact and that the elimination yields evolve with the mass of activated charcoal introduced. Furthermore, it has been shown that the initial concentration of contaminants and the variation in pH considerably influence the binding of theobromine, penicillin, and amoxicillin molecules to the sites of powdered activated charcoal. The overall results showed that the adsorption kinetics of three contaminants on activated carbon is well described by the second-order model, and the adsorption isotherms of the absorbent/absorbate systems studied are described satisfactorily by the Langmuir model.

Although the relevant literature has already demonstrated the impact that absorptive capacity has on companies’ innovation capacity, we have found few studies that analyze the role of learning capability in this relationship. The main objective of this study was to examine the role of organizational learning in this relationship. For this purpose, a quantitative research approach was used. A total of 306 valid questionnaires were obtained from small and medium-sized Spanish companies in different sectors. The collected data were analyzed using the multivariate Partial Least Square (PLS) quantitative structural equation technique. According to the result, absorption capacity turns into innovation mainly when learning capacity is involved in this process. This study provides empirical evidence of this relationship and fills this gap. It can also help organizations understand and clarify what would be the most appropriate way in to manage knowledge to improve their innovation levels.

Background People attending drug treatment centres have a high burden of hepatitis C virus (HCV) and face barriers to diagnosis and treatment. Dried blood spot (DBS) testing has been proposed to simplify diagnosis, but many patients remain untreated. In this retrospective study, we evaluated the reasons for non-retention in care in an intervention using on-site DBS testing and the effect of telemedicine and decentralized care compared to standard of care among people attending drug treatment centres who were lost to follow-up. Methods In a first phase, retention in care, adherence to treatment, and predictive factors in the DBS testing program of patients in drug treatment centres were analyzed and compared to a cohort of patients treated at the hospital outpatient clinic. Subsequently, in a second phase we evaluated in patients lost to follow-up from drug treatment centres the efficacy of one-step testing and telemedicine linked to a decentralized dispensation of HCV treatment or standard of care. Results Among 512 patients attending drug treatment centres, 467 (91.2%) agreed to be tested and 53.4% (237 patients/444 valid tests) tested positive (46 ± 9 years, 87.3% male) for HCV antibodies. After excluding patients negative for RNA or under surveillance, 178 patients were scheduled to meet with a specialist. Overall, 44 patients did not attend and 25 did not complete the pre-treatment evaluation. The only factor associated with retention in care was patient's knowledge of HCV infection. Treated patients attending drug treatment centres (n = 68) compared to the hospital outpatient clinic cohort (n = 135) had lower rates of treatment adherence. Among the patients who attended drug treatment centres that were lost to follow-up (n = 69), the proportion of patients who completed the program was significantly higher among those assisted by telemedicine than by standard of care (62.5% vs. 24.3%, p = 0.002). Conclusion Although there was a high participation rate in a DBS testing program in drug treatment centres, non-retention in care is a challenge. Importantly, telemedicine linked to a decentralized dispensation of HCV treatment re-engages patients and may be effective for HCV microelimination.

Objective To evaluate the change in compliance to thromboprophylaxis guidelines before and after the implementation of a multifaceted patient safety program. Design Longitudinal before and after study. Setting Teaching hospital, Hospital Universitario San Ignacio, Bogotá (Colombia). Participants Adult nonsurgical hospitalized patients. Intervention A multifaceted program for the prevention of venous thromboembolic (VTE) disease among adult nonsurgical hospitalized patients. The strategies of the program included (i) update and communication of thromboprophylaxis guidelines, (ii) the implementation of risk-assessment tools in electronic medical records, (iii) nursing staff activities and (iv) education to health personnel and patients for maintenance of the program. Main Outcome Measure Appropriate use of thromboprophylaxis. Results 221 and 236 patients were evaluated in the pre- and postimplementation periods, respectively. Global appropriate thromboprophylaxis prescription went from 74.66 to 82.6% (P = 0.064). Adequate thromboprophylaxis in high-risk patients did not increase significantly (77.70 vs 80.62%, P = 0.528), but a significant reduction in inappropriate thromboprophylaxis formulation in low-risk patients was found, decreasing from 20.55 to 5.26% (P = 0.005). Conclusions Implementing a quality improvement multifaceted program improves the formulation of adequate thromboprophylaxis. Reducing the inappropriate prescription of VTE prophylaxis in patients at low risk of thrombosis can lead to a reduction in bleeding complications and a better use of economic and human resources.

Trichothecenes are sesquiterpenoid toxins produced by multiple fungi, including plant pathogens, entomopathogens, and saprotrophs. Most of these fungi have the acyltransferase-encoding gene tri18. Even though its function has not been determined, tri18 is predicted to be involved in trichothecene biosynthesis because of its pattern of expression and its location near other trichothecene biosynthetic genes. Here, molecular genetic, precursor feeding, and analytical chemistry experiments indicate that in the saprotroph Trichoderma arundinaceum the tri18-encoded acyltransferase (TRI18) and a previously characterized acyltransferase (TRI3) are required for conversion of the trichothecene biosynthetic intermediate trichodermol to harzianum A, an antifungal trichothecene analog with an octa-2,4,6-trienedioyl acyl group. On the basis of the results, we propose that TRI3 catalyzes trichothecene 4- O-acetylation, and subsequently, TRI18 catalyzes replacement of the resulting acetyl group with octa-2,4,6-trienedioyl to form harzianum A. Thus, the findings provide evidence for a previously unrecognized two-step acylation process during trichothecene biosynthesis in T. arundinaceum and possibly other fungi.

High-speed milling has often been applied in injection mold manufacturing processes, where surface roughness is a significant criterion in product quality demands. It is equally applicable to automotive or industrial engineering and to toy manufacturing, where plastic parts with a high-quality surface finish have been processed using the injection molding technique. High-speed milling involves a number of process parameters that may affect the 3D surface topography formation. Literature analysis reveals that dynamical behavior is a significant factor in the end milling process on surface roughness parameters. To improve the accuracy of predicted surface topography models, it is important to include the dynamical behavior of milling factor. This paper describes the surface prediction model of combined end-milling geometrical and dynamical interaction models. The natural frequency of machine assembly and forced vibrations during the cutting process were measured during the flat-end milling process. Unevenly distributed cutting marks were revealed by surface 3D topography images and microscopy images of the machined samples. A mathematical model to predict surface topography was developed, including dynamical behavior and cutting geometries. Machine accuracy also has to be addressed. 3D surface topography parameters from the experimental sample provided the results for the mathematical prediction model. This model offers a software tool for manufacturers to improve the quality of machined part surfaces, taking into account the behavioral properties of their machining equipment. Relevant conclusions about the manufacturing equipment accuracy have been drawn. Vibrations in the milling system affect the cutting process and contribute to the surface topography prediction model. Local cutting tool vibrations do not have any influence on surface parameter mean values.

Trichoderma harzianum is a filamentous fungus well adapted to different ecological niches. Owing to its ability to antagonize a wide range of plant pathogens, it is used as a biological control agent in agriculture. Selected strains of T. harzianum are also able to increase the tolerance of plants to biotic and abiotic stresses. However, little is known about the regulatory elements of the T. harzianum transcriptional machinery and their role in the biocontrol by this species. We had previously reported the involvement of the transcription factor THCTF1 in the T. harzianum production of the secondary metabolite 6-pentyl-pyrone, an important volatile compound related to interspecies cross-talk. Here, we performed a subtractive hybridization to explore the genes regulated by THCTF1, allowing us to identify a multiprotein bridging factor 1 (mbf1) homolog. The gene from T. harzianum T34 was isolated and characterized, and the generated Thmbf1 overexpressing transformants were used to investigate the role of this gene in the biocontrol abilities of the fungus against two plant pathogens. The transformants showed a reduced antifungal activity against Fusarium oxysporum f. sp. lycopersici race 2 (FO) and Botrytis cinerea (BC) in confrontation assays on discontinuous medium, indicating that the Thmbf1 gene could affect T. harzianum production of volatile organic compounds (VOC) with antifungal activity. Moreover, cellophane and dialysis membrane assays indicated that Thmbf1 overexpression affected the production of low molecular weight secreted compounds with antifungal activity against FO. Intriguingly, no correlation in the expression profiles, either in rich or minimal medium, was observed between Thmbf1 and the master regulator gene cross-pathway control (cpc1). Greenhouse assays allowed us to evaluate the biocontrol potential of T. harzianum strains against BC and FO on susceptible tomato plants. The wild type strain T34 significantly reduced the necrotic leaf lesions caused by BC while plants treated with the Thmbf1-overexpressing transformants exhibited an increased susceptibility to this pathogen. The percentages of Fusarium wilt disease incidence and values of aboveground dry weight showed that T34 did not have biocontrol activity against FO, at least in the 'Moneymaker' tomato variety, and that Thmbf1 overexpression increased the incidence of this disease. Our results show that the Thmbf1 overexpression in T34 negatively affects its biocontrol mechanisms.

The objective of this research was to identify and characterize the encoded peptides present in nut storage proteins ofCarya illinoinensis. It was found, through in silico prediction, proteomic analysis, and MS spectrometry, that bioactive peptides were mainly found in albumin and glutelin fractions. Glutelin was the major fraction with ~53% of the nut storage proteins containing at least 21 peptides with different putative biological activities, including antihypertensives, antioxidants, immunomodulators, protease inhibitors, and inhibitors of cell cycle progression in cancer cells. Data showed that using 50 μg/mL tryptic digests of enriched peptides obtained from nut glutelins is able to induce up to 19% of apoptosis in both HeLa and CasKi cervical cancer cells. To our knowledge, this is the first report that shows the potential value of the nut-encoded peptides to be considered as adjuvants in cancer therapies.

Resumen: Introducción: El objetivo del presente trabajo es demostrar si la aplicación del índice biespectral (BIS®) en la monitorización de la anestesia general en respiración espontánea en endoscopias digestivas altas (EDA) diagnósticas en el paciente pediátrico es útil para: a) disminuir la dosis de fármaco necesaria; b) disminuir el tiempo del despertar, y c) mejorar la seguridad del paciente. Pacientes y método: estudio prospectivo cuasi experimental de casos y controles en el ámbito de una unidad de cuidados intensivos pediátricos y neonatales de segundo nivel. Pacientes: niños entre 12 meses y 13 años. Caso: paciente ASA I que precisa EDA diagnóstica; sujetos elegibles 36, participantes 30. Población control: serie histórica de pacientes que precisaron EDA (años 2008-2010): 50 pacientes. Intervenciones: realización de EDA, aplicando protocolo de anestesia, monitorización de constantes vitales, nivel de sedación (escala de Ramsay) y nivel BIS. Variables de interés: dosis total de propofol (mg/kg), tiempo de inducción, tiempo de EDA y tiempo de despertar (min); índice BIS al inicio de la EDA (BISi) y durante la EDA; efectos adversos. Resultados: Sin diferencias significativas entre casos (B) y controles (C) respecto a sexo, edad y peso. Sin diferencias significativas en: dosis total de propofol (B 4,9 ± 1,4 mg/kg; C 5,2 ± 1,6 mg/kg, p = 0,492), Tiempo de despertar (B 12,2 ± 4,6 min; C 12,8 ± 4,4 min, p = 0,402), tiempo de procedimiento (B 9,5 ± 4,8 min; C 11,3 ± 6,5 min, p = 0,335) y tiempo de inducción (B 11,1 ± 2,6 min; C 10,1 ± 4,2 min, p = 0,059). BISi 55,4 ± 6,9. Sin diferencias significativas en efectos adversos (2 casos de desaturación leve en el grupo control). Conclusiones: La monitorización anestésica con índice biespectral en endoscopias digestivas altas en respiración espontánea en la población pediátrica es factible, pero no parece disminuir ni la dosis de fármaco necesaria ni el tiempo de despertar. Tampoco disminuye la incidencia de efectos adversos de forma significativa. Abstract: Introduction: The objective of this investigation is to determine whether bispectral index (BIS®) monitoring during intravenous anaesthesia with spontaneous breathing for upper gastrointestinal endoscopy (UGE) in a pediatric population is useful for: a) decreasing the amount of drug, b) decreasing the time for awakening, and c) improving patient safety. Patients and method: A quasi-experimental case-control prospective study was conducted in the setting of a second level hospital pediatric intensive care unit. Patients: Children aged 1-13 years. Case: ASA I patient who needed a diagnostic UGE; eligible, 36, participants, 30. Control: historical cohort of patients who needed UGE (years 2008-2010): 50 patients. Intervention: UGE performed with anaesthetic protocol, vital signs monitoring, sedation level (Ramsay scale) and BIS monitoring. Variables of interest: propofol total dose (mg/kg), induction time, time in performing the UGE, awakening time (min); initial BIS (iBIS), and BIS during the UGE; adverse effects. Results: There were no significant differences in sex, age or weight between case (B) and control (C) population. No significant differences in total propofol doses: (B 4.9 ± 1.4 mg/kg; C 5.2 ± 1.6 mg/kg, P=.492), awakening time (B 12.2 ± 4.6 min; C 12.8 ± 4.4 min, P=.402), time for execution of UGE (B 9.5 ± 4.8 min; C 11.3 ± 6.5 min, P=.335) and induction time (B 11.1 ± 2.6 min; C 10.1 ± 4.2 min, P=.059), iBIS 55.4 ± 6.9. There were no significant differences in adverse effects: 2 patients suffered from mild desaturation in the control group. Conclusions: BIS monitoring for diagnostic UGE in spontaneous breathing in a pediatric population is feasible, but does not appear to decrease awakening time or the amount of propofol needed. Furthermore, there was no statistically significant decrease in the number of adverse effects.

El carcinoma adenoide quístico del conducto auditivo externo es un tumor extremadamente raro, con alta probabilidad de invasión perineural y de metástasis. Los objetivos de este trabajo son presentar un caso típico de una mujer de 63 años de edad, quien consultó por hipoacusia, plenitud aural y otalgia; así como describir la intervención quirúrgica realizada para su resección y su evolución postoperatoria.

ObjectivesTo outline the methods used to build a discrete-event simulation model for use in decision-making in the context of waiting list management strategies for cataract surgery by comparing a waiting list prioritization system with the routinely used first-in, first-out (FIFO) discipline.MethodsThe setting was the Spanish health system. The model reproduced the process of cataract, from incidence of need of surgery (meeting indication criteria), through demand, inclusion on a waiting list, and surgery. “Nonexpressed Need” represented the population that, even with need, would not be included on a waiting list. Parameters were estimated from administrative data and research databases. The impact of introducing a prioritization system on the waiting list compared with the FIFO system was assessed. For all patients entering the waiting list, the main outcome variable was waiting time weighted by priority score. A sensitivity analysis with different scenarios of mean waiting time was used to compare the two alternatives.ResultsThe prioritization system shortened waiting time (weighted by priority score) by 1.55 months (95% CI: 1.47 to 1.62) compared with the FIFO system. This difference was statistically significant for all scenarios (which were defined from a waiting time of 4 months to 24 months under the FIFO system). A tendency to greater time savings in scenarios with longer waiting times was observed.ConclusionsDiscrete-event simulation is useful in decision-making when assessing health services. Introducing a waiting list prioritization system produced greater benefit than allocating surgery by waiting time only. Use of the simulation model would allow the impact of proposed policies to reduce waiting lists or assign resources more efficiently to be tested.

In the present article, we describe the cloning and characterization of the Trichoderma harzianum hmgR gene encoding a hydroxymethylglutaryl CoA reductase (HMGR), a key enzyme in the biosynthesis of terpene compounds. In T. harzianum , partial silencing of the hmgR gene gave rise to transformants with a higher level of sensitivity to lovastatin, a competitive inhibitor of the HMGR enzyme. In addition, these hmgR -silenced transformants produced lower levels of ergosterol than the wild-type strain in a minimal medium containing lovastatin. The silenced transformants showed a decrease in hmgR gene expression (up to a 8.4-fold, after 72 h of incubation), together with an increase in the expression of erg7 (up to a 15.8-fold, after 72 h of incubation), a gene involved in the biosynthesis of triterpenes. Finally, hmgR -silenced transformants showed a reduction in their antifungal activity against the plant-pathogen fungi Rhizoctonia solani and Fusarium oxysporum .

Trichoderma species are commonly used as biocontrol agents of different plant-pathogenic fungi. Terpene compounds are involved in the biocontrol process due to their antifungal properties (e.g., ergokonins and viridins) but additionally their structural function in the cell membranes (ergosterol) is essential. We report here the characterization of the T. harzianum erg1 gene, encoding a squalene epoxidase, a key enzyme in the biosynthesis of triterpene derivatives such as ergosterol. In T. harzianum the partial silencing of the erg1 gene gave rise to transformants with a higher level of sensitivity to terbinafine, an antifungal compound that acts specifically over the squalene epoxidase activity. In addition, these silenced transformants produced lower levels of ergosterol than the wild type strain. Finally, the silencing of the erg1 gene resulted in an increase in the expression level of the erg7 gene that encodes the oxidosqualene lanosterol-cyclase, another enzyme of the terpene biosynthesis pathway.

Targeted gene disruption efficiency in Acremonium chrysogenum was increased 10-fold by applying the double-marker enrichment technique to this filamentous fungus. Disruption of the mecB gene by the double-marker technique was achieved in 5% of the transformants screened. Mutants T6 and T24, obtained by gene replacement, showed an inactive mecB gene by Southern blot analysis and no cystathionine-γ-lyase activity. These mutants exhibited lower cephalosporin production than that of the control strain, A. chrysogenum C10, in MDFA medium supplemented with methionine. However, there was no difference in cephalosporin production between parental strain A. chrysogenum C10 and the mutants T6 and T24 in Shen's defined fermentation medium (MDFA) without methionine. These results indicate that the supply of cysteine through the transsulfuration pathway is required for high-level cephalosporin biosynthesis but not for low-level production of this antibiotic in methionine-unsupplemented medium. Therefore, cysteine for cephalosporin biosynthesis in A. chrysogenum derives from the autotrophic (SH 2 ) and the reverse transsulfuration pathways. Levels of methionine induction of the cephalosporin biosynthesis gene pcbC were identical in the parental strain and the mecB mutants, indicating that the induction effect is not mediated by cystathionine-γ-lyase.

Penicillins and cephalosporins are synthesized by a series of enzymatic reactions that form the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and convert this tripeptide into the final penicillin or cephalosporin molecules. One of the enzymes, isopenicillin N synthase has been crystallyzed and its active center identified. The three genes pcbAB, pcbC and penDE involved in penicillin biosynthesis are clustered in Penicillium chrysogenum, Aspergillus nidulans and Penicillium nalgiovense. Carbon catabolite regulation of penicillin biosynthesis is exerted by glucose and other easily utilizable carbon sources but not by lactose. The glucose effect is enhanced by high phosphate concentrations. Glucose represses the biosynthesis of penicillin by preventing the formation of the penicillin biosynthesis enzymes. Transcription of the pcbAB, pcbC and penDE genes of P. chrysogenum is strongly repressed by glucose and the repression is not reversed by alkaline pHs. Carbon catabolite repression of penicillin biosynthesis in A. nidulans is not mediated by CreA and the same appears to be true in P. chrysogenum. The first two genes of the penicillin pathway (pcbAB and pcbC) are expressed from a bidirectional promoter region. Analysis of different DNA fragments of this bidirectional promoter region revealed two important DNA sequences (boxes A and B) for expression and glucose catabolite regulation of the pcbAB gene. Using protein extracts from mycelia grown under carbon catabolite repressing or derepressing conditions DNA-binding proteins that interact with the bidirectional promoter region were purified to near homogeneity.

La diálisis peritoneal aguda (DPA) continúa siendo una medida útil en el paciente pediátrico crítico. En el shock séptico el fracaso renal agudo puede precisar medidas invasivas de depuración y aunque la hemofiltración es muy efectiva, en ciertas Unidades de Cuidados Intensivos Pediátricas no se dispone aún de ella. La DPA pediátrica se suele iniciar con pases horarios, permanencias cortas y volumen por pase de unos 10 ml/kg. Mostramos la evolución de la DPA de dos pacientes críticos con fallo renal en los que la monitorización de los cocientes dializado/plasma de urea y creatinina y del cociente de concentración de glucosa del dializado (efluente)/concentración de glucosa de la solución de diálisis infundida fue útil para la prescripción de la DPA.

Several strains of Penicillium chrysogenum with different productions of penicillin were characterized at the molecular level in order to establish the basis of the increased penicillin production rates. The cluster of penicillin biosynthetic genes was located in an amplified genomic region of 57.9 kb in a high-producing strain (E1) and 106.5 kb in two strains (AS-P-78 and P2) producing moderately high levels of penicillin. This region was shown to be present in multiple tandemly repeated copies with a different copy number depending on the strain. The sequence TTTACA appeared at the junction points between repeats and at the borders of the amplified region in strains AS-P-78 and P2, while its reverse complementary TGTAAA was found in strain E1. The tandem reiteration and deletion appear to arise by site-specific recombination induced by mutagenic treatments. Finally, the relationship between glucose repression and pH regulation was studied in strain AS-P-78.

The organization of the genes of the penicillin cluster has been studied in three different mutants of P. chrysogenum impaired in penicillin biosynthesis. The three blocked mutants (derived from the parental strain P. chrysogenum Bb-1) lacked the genes pcbAB, pcbC and penDE of the penicillin biosynthetic pathway and were unable to form isopenicillin N synthase and isopenicillin N acyltransferase. All strains were identified as P. chrysogenum derivatives by fingerprinting analysis with (GTG)n as a probe. The borders of the deleted region were cloned and sequenced, showing the same junction point in the three mutants. The deleted DNA region was found to be identical to that described in P. chrysogenum npe10. The frequent deletion of the pen gene cluster at this point may indicate that this cluster is located in an unstable genetic region, flanked by hot spots of recombination, that is easily lost by mutagen-induced recombination.

The genes pcbAB, pcbC and penDE encoding enzymes involved in the biosynthesis of penicillin have been cloned from Penicillium chrysogenum and Aspergillus nidulans. They are clustered in chromosome I (10.4 Mb) of P. chrysogenum, but they are located in chromosome II of Penicillium notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. Expression studies have shown that each gene is expressed as a single transcript from separate promoters. Enzyme regulation studies and gene expression analysis have provided useful information to understand the control of gene expression leading to overexpression of the genes involved in penicillin biosynthesis. Cephalosporin genes have been studied in Cephalosporium acremonium and also in cephalosporin-producing bacteria. In C. acremonium the genes involved in cephalosporin biosynthesis are separated in at least two clusters. Cluster I (pcbAB-pcbC) encodes the first two enzymes of the cephalosporin pathway which are very similar to those involved in penicillin biosynthesis. Cluster II (cefEF-cefG), encodes the last three enzymatic activities of the cephalosporin pathway. It is unknown, at this time, if the cefD gene encoding isopenicillin epimerase is linked to any of the two clusters. In cephamycin producing bacteria the genes encoding the entire biosynthetic pathway are located in a single cluster extending for about 30 kb in Nocardia lactamdurans, and in Streptomyces clavuligerus. The cephamycin clusters of N. lactamdurans and S. clavuligerus include a gene lat which encodes lysine-6-aminotransferase an enzyme involved in formation of the precursor alpha-aminoadipic acid. The N. lactamdurans cephamycin cluster includes, in addition, a beta-lactamase (bla) gene, a penicillin binding protein (pbp), and a transmembrane protein gene (cmcT) that is probably involved in secretion of the cephamycin. Little is known however about the mechanism of control of gene expression in the different beta-lactam producers. The availability of most of the structural genes provides a good basis for further studies on gene expression. This knowledge should lead in the next decade to a rational design of strain improvement procedures. The origin and evolution of beta-lactam genes is intriguing since their nucleotide sequences are extremely conserved despite their restricted distribution in the microbial world.

No DNA sequence homologous to the penDE gene of Penicillium chrysogenum was found in the genome of three different strains of Cephalosporium acremonium. The pcbC-penDE gene cluster of P. chrysogenum complemented the isopenicillin N synthase deficiency of C. acremonium mutant N2 and resulted in the production of penicillin, in addition to cephalosporin, in cultures supplemented with phenylacetic acid. The penicillin formed was identified as benzylpenicillin by HPLC and NMR studies. The penDE gene of P. chrysogenum is expressed in C. acremonium forming a transcript of 1.15 kb. The transcript is processed and translated in C. acremonium resulting in the formation of acyl CoA: isopenicillin N acyl transferase. When the penDE gene was introduced into a cephalosporin producing strain, the total titre of beta-lactam antibiotics comprised distinct proportions of penicillin and cephalosporin in different transformants. Analysis of the hybridization patterns of the DNA of C. acremonium transformed with the pcbC or penDE genes indicated that integration occurs by non-homologous recombination.

BACKGROUND: Celiac disease (CD) is a common disorder in children and adults. However, limited data are available when comparing differences between both populations. AIMS: To prospectively evaluate and compare the clinical and histological features present at diagnosis in a cohort of celiac children and adults. METHODS: Consecutive new cases diagnosed between 2000 and 2006 were prospectively included (66 children and 54 adults). The clinical spectrum was categorized in two groups: (a) typical (malabsorption, chronic diarrhea, or failure to thrive) and (b) oligosymptomatic (abdominal pain, anemia, hypertransaminasemia, or screening in risk groups or in relatives). The histological results were divided into mild (i.e., Marsh I, II, and IIIA) and severe (i.e., Marsh IIIB, IIIC). In all cases, the human antitissue transglutaminase IgA antibodies (TTGA) were determined. RESULTS: Overall, a female/male ratio (2.6:1) was observed. This ratio was significantly higher in adults (5.7:1) than in children (1.6:1) (P = 0.009). Typical symptoms were present in 62.5% children versus 31% adults (P = 0.01). The average time to diagnosis after the appearance of symptoms was 7.6 months for children and 90 months for adults (P < 0.001). TTGA levels were higher in children and correlated with age (P < 0.001) and with the degree of villous atrophy (P < 0.001). Histological analysis revealed a marked atrophy in 86% children versus 52% adults (P < 0.001). The degree of villous atrophy was inversely correlated with age (P < 0.001). Classic symptoms were also associated with more severe villous atrophy. CONCLUSIONS: At initial diagnosis, CD shows age-related differences, which consist of more evident clinical and histological features in children. Furthermore, IgA TTGA levels correlate both with the degree of villous atrophy and with the patient's age.

Background In Spain, there are substantial variations in the utilization of health resources among regions. Because the need for surgery differs in patients with appropriate surgical indication, introducing a prioritization system might be beneficial. Our objective was to assess geographical variations in the impact of applying a prioritization system in patients on the waiting list for cataract surgery in different regions of Spain by using a discrete-event simulation model. Methods A discrete-event simulation model to evaluate demand and waiting time for cataract surgery was constructed. The model was reproduced and validated in five regions of Spain and was fed administrative data (population census, surgery rates, waiting list information) and data from research studies (incidence of cataract). The benefit of introducing a prioritization system was contrasted with the usual first-in, first-out (FIFO) discipline. The prioritization system included clinical, functional and social criteria. Priority scores ranged between 0 and 100, with greater values indicating higher priority. The measure of results was the waiting time weighted by the priority score of each patient who had passed through the waiting list. Benefit was calculated as the difference in time weighted by priority score between operating according to waiting time or to priority. Results The mean waiting time for patients undergoing surgery according to the FIFO discipline varied from 1.97 months (95% CI 1.85; 2.09) in the Basque Country to 10.02 months (95% CI 9.91; 10.12) in the Canary Islands. When the prioritization system was applied, the mean waiting time was reduced to a minimum of 0.73 months weighted by priority score (95% CI 0.68; 0.78) in the Basque Country and a maximum of 5.63 months (95% CI 5.57; 5.69) in the Canary Islands. The waiting time weighted by priority score saved by the prioritization system varied from 1.12 months (95% CI 1.07; 1.16) in Andalusia to 2.73 months (95% CI 2.67; 2.80) in Aragon. Conclusion The prioritization system reduced the impact of the variations found among the regions studied, thus improving equity. Prioritization allocates the available resources within each region more efficiently and reduces the waiting time of patients with greater need. Prioritization was more beneficial than allocating surgery by waiting time alone.

La utilidad del óxido nítrico inhalado en el tratamiento del síndrome de distrés respiratorio agudo (SDRA) con hipertensión pulmonar (HPP) es controvertida y hay pocos estudios en la literatura que evalúen sus efectos durante una administración prolongada. Analizamos el caso de un paciente de 45 días de vida con tetralogía de Fallot no intervenida, que desarrolló SDRA e HPP de causa no cardíaca, que precisó ventilación mecánica prolongada (30 días) y administración de óxido nítrico inhalado (ONI). Antes del ONI el índice de oxigenación era 19 y a los pocos minutos de su aplicación descendió a 15. El paciente desarrolló dependencia del ONI, tolerando un destete gradual, pudiendo interrumpir la administración a los 27 días de su inicio. No observamos efectos tóxicos durante la administración del ONI. Utility of inhaled nitric oxide in the treatment of acute respiratory distress syndrome (ARDS) with pulmonary hypertension (PHT) is controversial and there are few studies in the literature that evaluate its effects during prolonged administration. We analyze the case of a 45 day old male patient with un-operated Fallot's tetralogy who developed ARDS and PHT with non-cardiac cause, that required prolonged mechanical ventilation (30 days) and administration of inhaled nitric oxide (INO). Before the INO, the oxygenation index was 19, and at a few minutes of its application it decreased to 15. The patient developed a dependence on INO, tolerating its gradual weaning. Administration could be interrupted at 27 days on onset. We did not observe toxic effects during the administration of INO.

Methanol extracts from 24 Trichoderma isolates, selected as biocontrol agents and representating different species and genotypes from three of the four taxonomic sections of this genus (T. sect. Trichoderma, T. sect. Pachybasium and T. sect. Longibrachiatum) were screened for antibacterial, anti-yeast and antifungal activities against a panel of seven bacteria, seven yeasts and six filamentous fungi previously used in similar studies. Two different growth media were tested (potato dextrose broth and CYS80), and all isolates included in the antimicrobial tests showed at least one inhibitory activity against one of the target microorganisms in one of the two culture media. No statistically significant differences were detected in the number of active strains between the two culture media, but the highest number of inhibitory strains against bacteria and fungi were found in strains from Trichoderma sect. Pachybasium, whereas strains from T. sect. Longibrachiatum showed the highest anti-yeast values. In all cases, a correlation was found between the strains that were active against yeasts and fungi. However, some degree of variability was detected for strains within the same taxonomic section. In general terms, strains from T. asperellum (mainly in CYS80 medium), and T. longibrachiatum gave the best non-enzymatic antimicrobial profiles.

The isopenicillin N acyltransferases (IATs) of Aspergillus nidulans and Penicillium chrysogenum differed in their ability to maintain the 40-kDa proacyltransferase alphabeta heterodimer in an undissociated form. The native A. nidulans IAT exhibited a molecular mass of 40 kDa by gel filtration. The P. chrysogenum IAT showed a molecular mass of 29 kDa by gel filtration (corresponding to the beta subunit of the enzyme) but the undissociated 40-kDa heterodimer was never observed even in crude extracts. Heterologous expression experiments showed that the chromatographic behaviour of IAT was determined by the source of the penDE gene used in the expression experiments and not by the host itself. When the penDE gene of A. nidulans was expressed in P. chrysogenum npe6 and npe8 or in Acremonium chrysogenum, the IAT formed had a molecular mass of 40 kDa. On the other hand, when the penDE gene originating from P. chrysogenum was expressed in A. chrysogenum, the active IAT had a molecular mass of 29 kDa. The intronless form of the penDE gene cloned from an A. nidulans cDNA library and overexpressed in Escherichia coli formed the enzymatically active 40-kDa proIAT, which was not self-processed as shown by immunoblotting with antibodies to IAT. This 40-kDa protein remained unprocessed even when treated with A. nidulans crude extract. In contrast, the P. chrysogenum penDE intronless gene cloned from a cDNA library was expressed in E. coli, and the IAT was self-processed efficiently into its alpha (29 kDa) and beta (11 kDa) subunits. It is concluded that P. chrysogenum and A. nidulans differ in their ability to self-process their respective proIAT protein and to maintain the alpha and beta subunits as an undissociated heterodimer, probably because of the amino-acid sequence differences in the proIAT which affect the autocatalytic activity.

Transcriptional analysis of the region downstream of the pcbAB gene (which encodes the alpha-aminoadipyl-cysteinyl-valine synthetase involved in cephalosporin synthesis) of Acremonium chrysogenum revealed the presence of two different transcripts corresponding to two new ORFs. ORF3 encodes a putative D-hydroxyacid dehydrogenase and cefT (for transmembrane protein) encodes a multidrug efflux pump belonging to the Major Facilitator Superfamily (MFS) of membrane proteins. The CefT protein has 12 transmembrane segments (TMS) and contains motifs A, B, C, D2 and G characteristic of the Drug:H(+) antiporter 12-TMS group of the major facilitator superfamily. The CefT protein confers resistance to some toxic organic acids, including isovaleric acid and phenylacetic acid. Targeted inactivation of ORF3 and cefT by gene replacement showed that they are not essential for cephalosporin biosynthesis. However, amplification of the cefT gene results in increments of up to 100% in cephalosporin production in the A. chrysogenum C10 strain. Amplification of a truncated form of the cefT insert did not lead to cephalosporin overproduction. It seems that the CefT protein is involved in cephalosporin export from A. chrysogenum or in transmembrane signal transduction, and that there are redundant systems involved in cephalosporin export.

An important problem during the production of cephalosporin C by Acremonium chrysogenum is the hydrolysis of cephalosporin C to deacetylcephalosporin C, since the latter compound has no commercial value and represents an unwanted side-product. Characterization of the enzymatic process that gives rise to deacetylcephalosporin C will help to avoid the accumulation of this side-product. An extracellular cephalosporin C acetylhydrolase (CPC-AH) from Acremonium chrysogenum C10 was purified to near homogeneity. This enzyme had a molecular mass of 31 kDa, a pI of 4.0, and showed relatively little affinity for cephalosporin C (Km 33.7 mM). We sequenced twenty amino acids at the amino-terminal end; a probe based on this sequence was then used to clone the cephalosporin acetylhydrolase (cahB) gene. cahB encodes a pre-protein of 383 amino acids with a deduced molecular mass of 38,228 Da. The sequenced 20 amino acids of the purified protein corresponded to amino acids 107–127 deduced from the cahB gene, suggesting that mature CPC-AH results from processing of the pre-protein after Gln-106. cahB is located on chromosome VIII of A. chrysogenum C10 and is not linked to the cephalosporin early or late gene clusters. It is expressed as a single 1.4-kb transcript after 72 h of cultivation. Expression declined in batch cultures after 120 h even though CPC-AH activity was observed until 144 h. The CPC-AH protein resembles other wide-spectrum substrate fungal esterases that are functionally related to serine proteases. The cahB gene does not seem to be related to the cephalosporin biosynthesis genes and encodes an esterase active on several substrates in addition to cephalosporin C.

Advances in perinatal care have made it possible to improve survival of infants with low birth weight. The aim of study was to analyse the clinical impact of respiratory disease in premature infants with low birth weight. Methods: Between January-2011 and November-2012 were included 81 preterm infants with low birth weight and ≤32 weeks gestation. Data are expressed in according to birth weight defined as: extremely low 28 weeks gestation. The incidence of morbidities such as bronchopulmonary dysplasia, pulmonary haemorrhage and pneumothorax was very low: 6.2%, 4.9% and 1.25%, respectively. The mean number of days requiring mechanical ventilation and treatment with caffeine for apnea was higher for ELBW compared to VLBW and LBW (6.7 ± 5.5 vs. 4.1 ± 4.7 vs. 3.38 ± 2.7, p = 0.023 and 38.1 ± 7.3 vs. 26.8 ± 15 vs. 12 ± 7.8, p = 0.01, respectively). The onset of bronchopulmonary dysplasia occurred in 5 preterm with ELBW. In all preterm infants who died required mechanical ventilation vs. who those survived 14 (100%) vs. 32 (47.8%), p = 0.001 and greater need of surfactant 10 (71.4%) vs. 24 (35.8%), p = 0.047. Conclusions: Although the most preterm with low birth weight require respiratory support, the incidence of complications in our series is low.

Background Advances in perinatal care have made it possible to improve survival of infants with low birth weight. The aims of this study were to analyse the clinical impact of morbidities and to identify predictors of in-hospital mortality in preterm infants with low birth weight. Methods Between January-2011 and November-2012 were included 81 preterm infants at our centre with low birth weight or ≤ 32 weeks gestation. Perinatal variables were included in risk factor analysis. Data are expressed as gestational age (GA) Conclusion The survival in preterm infants in addition of GA or birth weight, it depends on the presence of morbidities. The use of risk scores on admission is useful for prediction in-hospital mortality.

Maternal risk factors can cause prematurity. The aim of our study was to analyse the impact of maternal characteristics in preterm low birth weight. Methods A retrospective cohort study of a total of 81 preterm infants at our centre with low birth weight and ≤32 weeks of gestation was performed. We analysed perinatal risk factors. Data are expressed as a function of gestational age (GA) Results The mean maternal age was 30.3 ± 5.8 years. There were no statistically significant differences when comparing maternal age in according to birth weight (29.2 ± 5.7 vs. 31.5 ± 6.6 vs. 30.2 ± 4.6, p = 0.460 respectively) or gestational age (30.4 ± 5 vs. 30.3 ± 6, p = 0.964). The presence of maternal risk factors was very low: 3.7% were smokers, 2.5% had obesity. There were preeclampsia and diabetes gestational in 5 and 3 cases, respectively. Chorioamnionitis was diagnosed in 13.6% of pregnancies and 71.3% received antenatal steroids. The pregnancy was twins in 29.6% of cases. No significant differences were found when perinatal characteristics were examined in terms of birth weight, except for gestational diabetes (0% vs. 0% vs. 13.6% in low birth, p = 0,016). Maternal age did not influence in neonatal mortality (30.2 ± 5 years in preterm infants living vs. 30.7 ± 6 years in those died, p = 0.784. Conclusions In our series, the presence of maternal risk factors was very low. We were not found significant differences in according to birth weight or impact on neonatal mortality.

α-Aminoadipic acid is a well known precursor in the biosynthesis of penicillin. α-Aminoadipic acid is formed in Penicillium chrysogenum as an intermediate in the lysine biosynthetic pathway. The lysine pool has a great influence on penicillin production since lysine is a potent inhibitor of penicillin biosynthesis. The relationship between intracellular α-aminoadipic acid availability and the penicillin production rate of one strain is well established. Metabolic engineering of the lysine pathway has shown that it is possible to channel the metabolic flux of lysine precursors towards penicillin biosynthesis by target inactivation of the α-aminoadipate reductase (the first enzyme of the lysine pathway after the branch point). In addition, lys1 (encoding homocitrate synthase) gene amplification and gene overexpression studies showed that there are more than one rate-limiting step in the direct supply of α-aminoadipic acid for penicillin biosynthesis from the first part of the lysine pathway. In summary, gene disruption of diverging branches of a biosynthetic pathway leads to channelling of the intermediates towards the desired end products whereas amplification of a specific gene encoding a putative bottleneck enzyme in a biosynthetic pathway does not necessarily result in overproducing strains since other bottlenecks may still occur after removal of the first one.

Homocitrate synthase activity (encoded by the lys1 gene) catalyzes the first step of the lysine and penicillin pathway and is highly sensitive to feedback regulation by L-lysine. The transcript levels of the lys1 gene and the homocitrate synthase activity are high during the growth phase and decrease during the antibiotic production phase, except in the high penicillin producer strain AS-P-99 which maintained high levels of homocitrate synthase activity in cultures at 96 h and 120 h. The lys1 gene was overexpressed in Penicillium chrysogenum using additional copies of lys1 with its own promoter or under the control of the pcbC promoter in either autonomously replicating or integrative vectors. Transformants containing 3 to 32 additional copies of the lys1 gene were selected. Some of these transformants, particularly Ti-C4 (integrative) and TAR-L9 (with autonomously replicating plasmids) showed very high levels of lys1 transcript and, in the case of TAR-L9, high levels of homocitrate synthase activity in cultures of 120 h. However, these transformants did not show increased alpha-aminoadipate or lysine pools. A mutant P. chrysogenum L-G- disrupted in the lys2 gene (therefore lacking the lysine branch of the pathway) showed increased alpha-aminoadipate levels and produced higher levels of penicillin than non-disrupted control strains. Overexpression of the lys1 gene in the L-G- mutant resulted in high homocitrate synthase levels but no additional increase of the alpha-aminoadipate pool or penicillin production levels. These results suggest that after amplification of the homocitrate synthase levels there are other limiting steps in the common stem of the lysine and penicillin pathways.

Constructions starting at each of the three in-frame ATG codons of the Acremonium chrysogenum cefG gene (Met1, Met46 and Met60) were expressed in Escherichia coli, obtaining proteins of 49, 44 and 43 kDa, respectively. All three proteins showed deacetylcephalosporin C (DAC) acetyltransferase activity. The native A. chrysogenum DAC acetyltransferase was purified to electrophoretic homogeneity by immunoaffinity chromatography. It showed a molecular mass of 50 kDa by filtration in calibrated Sephadex G-75 SF or Superose 12 (FPLC) columns. The N-terminal end of the pure DAC acetyltransferase was Met-Leu-Pro-Ser-Ala-Gln-Val-Ala-Arg-Leu, which matched perfectly the deduced amino acid sequence starting at Met1. The putative α- and β-subunits of DAC acetyltransferase were also obtained in E. coli but showed no enzymic activity either separately or in combination. Immunoblotting (Western) analysis revealed that the 50 kDa DAC acetyltransferase showed high protein levels in A. chrysogenum cultures at 72 and 96 h and decreased sharply thereafter, but in all cases no detectable processing of the enzyme into subunits was found. Three different A. chrysogenum strains (including the wild-type Brotzu strain and two high-cephalosporin-producing mutants) showed the same unprocessed 50 kDa DAC acetyltransferase. The non-producer mutant ATCC 20371 showed no DAC acetyltransferase protein band but formed a normal transcript of 1.4 kb.

Five mutants of Penicillium chrysogenum blocked in penicillin biosynthesis (npe) which are deficient in isopenicillin N-acyltransferase were isolated previously. Three of these mutants, npe6, npe7, and npe8, have been characterized at the molecular level and compared with npe10, a deletion mutant. Transcripts of normal size (1.15 kb) of the penDE genes, which encode isopenicillin N-acyltransferase, and also of the pcbAB (11.5 kb) and pcbC (1.1 kb) genes were observed in all mutants except for the npe10 mutant. Immunoblotting studies using antibodies against isopenicillin N-acyltransferase showed that all mutants (except npe10) formed the 40-kDa (unprocessed) protein and the 29-kDa subunit of the isopenicillin N-acyltransferase. The 11-kDa subunit could not be observed in the immunoblots. The mutant penDE genes of strains npe6, npe7, and npe8 were cloned and sequenced. These three strains showed a mutation in the penDE genes which results in a single amino acid change in each modified isopenicillin N-acyltransferase. The mutation in npe6 resulted in a change of Gly-150 to Val, whereas the mutation in both npe7 and npe8 introduced a change of Glu-258 to Lys. Replacement of the Val-150 and Lys-258 mutations by constructing hybrid isopenicillin N-acyltransferase molecules led to the recovery of the isopenicillin N-acyltransferase activity. The mutations in npe6, npe7, and npe8 do not affect the ability of the 40-kDa isopenicillin N-acyltransferase to be processed into the component subunits.

Methionine stimulated cephalosporin production in cultures of three different strains of Acremonium chrysogenum when added either at inoculation time or at 72 h to cells grown previously in the absence of methionine. When methionine was added at 72 h, the stimulation of cephalosporin biosynthesis was observed only 12 h later and required de novo protein synthesis. Methionine increased the levels of enzymes (isopenicillin N synthase and deacetylcephalosporin C acetyltransferase) expressed from genes (pcbC and cefG, respectively) located in the two clusters of cephalosporin biosynthesis genes in the wild-type A. chrysogenum strain and also in the two improved strains, CW19 and C10. Methionine-supplemented cells showed higher levels of transcripts of the four known genes (pcbAB, pcbC, cefEF and, to a slight extent, cefG) of the cephalosporin biosynthetic pathway than cells grown in the absence of methionine. The levels of the cefG transcript were much lower than those of the pcbAB, pcbC, and cefEF transcripts. The induction by methionine of transcription of the four cephalosporin biosynthesis genes and the known effect of this amino acid on the differentiation of A. chrysogenum indicate that methionine exerts a pleiotropic effect that coordinately regulates cephalosporin biosynthesis and differentiation.

A DNA vector for expressing an oxygen-binding heme protein (Vitreoscilla hemoglobin, or VHb) in filamentous fungi was constructed and introduced into a cephalosporin C-producing strain of Acremonium chrysogenum. Expression of VHb in transformants was demonstrated by Western immunoblot analysis and by increased carbon monoxide binding activity of cell extracts. Several VHb-expressing transformants produced significantly higher yields of cephalosporin C than control strains in batch culture experiments. Using the same vector system, VHb was also expressed in the related fungus Penicillium chrysogenum.

Nine mutants of Penicillium chrysogenum (npe1 to npe8 and npe10) impaired in penicillin biosynthesis were screened after nitrosoguanidine mutation. Mutants npe1, npe4, npe5, npe6, npe7, npe8, and npe10 failed to synthesize significant levels of penicillin, whereas strains npe2 and npe3 synthesized about 20% of the penicillin level produced by the parental strain. Mutants npe5 and npe10 did not show alpha-aminoadipylcysteinyl-valine (ACV) synthetase activity in vitro and did not form ACV in vivo. Immunoblotting analysis of the different mutants using antibodies raised against Aspergillus nidulans ACV-synthetase showed that mutants npe5 and npe10 lacked this multienzyme protein, which in the parental strain had a molecular mass of about 420 kDa, and mutants npe2 and npe3 formed reduced level of this protein. All mutants showed normal levels of isopenicillin N synthase, as shown by Western blot analysis and enzyme assays (except npe10 that lacked this enzyme and npe2 and npe3 that formed reduced levels); npe1, npe4, npe6, npe7, npe8, and npe10 lacked isopenicillin N acyltransferase. Southern hybridizations of total DNA of the parental strain and mutants npe5, npe6, npe8, and npe10 with probes internal to the pcbAB, pcbC, and penDE genes showed that mutants npe5, npe6, and npe8 had the same arrangement of the penicillin gene cluster carrying probably point mutations, but mutant npe10 lacked the three penicillin biosynthetic genes, suggesting that it had suffered a deletion of the entire penicillin cluster. Southern hybridization with a pyrG probe as control and fingerprinting analysis of total DNA of npe10 as compared to several P.chrysogenum strains and other Penicillium and Aspergillus species, confirmed that npe10 is a deletion mutant of P. chrysogenum that had lost the penicillin biosynthetic genes.

The isopenicillin-N acyltransferase of Penicillium chrysogenum catalyzes the conversion of the biosynthetic intermediate isopenicillin N to the hydrophobic penicillins. The isopenicillin-N acyltransferase copurified with the acyl-CoA:6-aminopenicillanic acid (6-APA) acyltransferase activity which transfers an acyl residue from acyl-CoA derivatives (e.g. phenylacetyl-CoA, phenoxyacetyl-CoA) to 6-APA. Other thioesters of phenylacetic acid were also used as substrates. An amino acid sequence similar to that of the active site of thioesterases was found in the isopenicillin-N acyltransferase, suggesting that this site is involved in the transfer of phenylacetyl residues from phenylacetyl thioesters. Purified isopenicillin-N acyltransferase also showed isopenicillin-N amidohydrolase, penicillin transacylase and penicillin amidase activities. The isopenicillin-N amidohydrolase (releasing 6-APA) showed a much lower specific activity than the isopenicillin-N acyltransferase of the same enzyme preparation, suggesting that in the isopenicillin-N acyltransferase reaction the 6-APA is not released and is directly converted into benzylpenicillin. Penicillin transacylase exchanged side chains between two hydrophobic penicillin molecules; or between one penicillin molecule and 6-APA. The penicillin amidase activity is probably the reverse of the biosynthetic acyl-CoA:6-APA acyltransferase. Four P. chrysogenum mutants deficient in acyl-CoA:6-APA acyltransferase lacked the other four related activities. Transformation of these mutants with the penDE gene restored all five enzyme activities.