Alterations in pro- and anti-inflammatory mediators are involved in microvascular dysfunction in postmenopausal women with type 2 diabetes mellitus

Evidence has shown that women with type 2 diabetes mellitus (T2DM) have a greater risk of cardiovascular complications compared with men, but this sex difference is not clearly understood. This study assessed the microvascular function and circulatory biomarkers in postmenopausal women (PMW) with T2DM compared with diabetic men and their non-diabetic counterparts. Sixty participants were divided into nondiabetic PMW, PMW with T2DM, non-diabetic men, and diabetic men. Microvascular function was assessed using non-invasive equipment (EndoPAT®) and reported as reactive hyperemia index (RHI). Anthropometric and cardiovascular parameters were also measured. Two-way ANOVA was performed using sex (women or men) and T2DM (non-diabetic and diabetic) as the two factors. RHI impairment (1.97±0.14) was detected in diabetic PMW compared with women without T2DM (2.5±0.13) accompanied by lower adiponectin levels (T2DM: 9.3±1.2 and CTL: 13.8±1.8 ug/mL, P<0.05). An increase in the Nε-carboxymethyllysine (CML), nitrate/nitrite, and C-reactive protein (CRP) levels were observed in diabetic PMW compared to the other groups. Although a poor glycemia control was seen in diabetic men, neither RHI nor circulatory biomarkers were affected by T2DM. Multiple linear regression stratified by sex and T2DM identified some variables with RHI only in PMW with T2DM: HbA1c (P=0.003), body mass index (P=0.029), CML (P=0.032), and CRP (P=0.006). Diabetic PMW were more susceptible to the deleterious effects of hyperglycemia than men, showing microvascular dysfunction with high levels of pro-inflammatory mediators (CML and CRP) and a lower adiponectin concentration.

Role of IGF2 in the Study of Development and Evolution of Prostate Cancer.

Prostate Cancer (PC) is commonly known as one of the most frequent tumors among males. A significant problem of this tumor is that in early stages most of the cases course as indolent forms, so an active surveillance will anticipate the appearance of aggressive stages. One of the main strategies in medical and biomedical research is to find non-invasive biomarkers for improving monitoring and performing a more precise follow-up of diseases like PC. Here we report the relevant role of IGF2 and miR-93-5p as non-invasive biomarker for PC. This event could improve current medical strategies in PC.

Evidence for a New Compact Symmetric Fission Mode in Light Thorium Isotopes.

Taking benefit of the R3B/SOFIA setup to measure the mass and the nuclear charge of both fission fragments in coincidence with the total prompt-neutron multiplicity, the scission configurations are inferred along the thorium chain, from the asymmetric fission in the heavier isotopes to the symmetric fission in the neutron-deficient thorium. Against all expectations, the symmetric scission in the light thorium isotopes shows a compact configuration, which is in total contrast to what is known in the fission of the heavier thorium isotopes and heavier actinides. This new main symmetric scission mode is characterized by a significant drop in deformation energy of the fission fragments of about 19 MeV, compared to the well-known symmetric scission in the uranium-plutonium region.

Endothelial dysfunction is an early indicator of sepsis and neutrophil degranulation of septic shock in surgical patients.

BACKGROUND: Stratification of the severity of infection is currently based on the Sequential Organ Failure Assessment (SOFA) score, which is difficult to calculate outside the ICU. Biomarkers could help to stratify the severity of infection in surgical patients. METHODS: Levels of ten biomarkers indicating endothelial dysfunction, 22 indicating emergency granulopoiesis, and six denoting neutrophil degranulation were compared in three groups of patients in the first 12 h after diagnosis at three Spanish hospitals. RESULTS: There were 100 patients with infection, 95 with sepsis and 57 with septic shock. Seven biomarkers indicating endothelial dysfunction (mid-regional proadrenomedullin (MR-ProADM), syndecan 1, thrombomodulin, angiopoietin 2, endothelial cell-specific molecule 1, vascular cell adhesion molecule 1 and E-selectin) had stronger associations with sepsis than infection alone. MR-ProADM had the highest odds ratio (OR) in multivariable analysis (OR 11·53, 95 per cent c.i. 4·15 to 32·08; P = 0·006) and the best area under the curve (AUC) for detecting sepsis (0·86, 95 per cent c.i. 0·80 to 0·91; P < 0·001). In a comparison of sepsis with septic shock, two biomarkers of neutrophil degranulation, proteinase 3 (OR 8·09, 1·34 to 48·91; P = 0·028) and lipocalin 2 (OR 6·62, 2·47 to 17·77; P = 0·002), had the strongest association with septic shock, but lipocalin 2 exhibited the highest AUC (0·81, 0·73 to 0·90; P < 0·001). CONCLUSION: MR-ProADM and lipocalin 2 could be alternatives to the SOFA score in the detection of sepsis and septic shock respectively in surgical patients with infection.

ANTECEDENTES: La estratificación de la gravedad de una infección se basa actualmente en la puntuación SOFA (Sequential Organ Failure Assessment), que es difícil de calcular fuera de la unidad de cuidados intensivos. Los biomarcadores podrían ayudar a estratificar la gravedad de la infección en pacientes quirúrgicos. MÉTODOS: Se compararon las concentraciones de 10 biomarcadores que denotan disfunción endotelial, 22 que indican granulopoyesis de emergencia y 6 que expresan la degranulación de neutrófilos en tres grupos de pacientes de tres hospitales españoles (100 con infección, 95 con sepsis y 57 con shock séptico) en las primeras doce horas después del diagnóstico. RESULTADOS: Siete biomarcadores que expresan disfunción endotelial (proadrenomedulina, sindecan-1, trombomodulina, angiopoyetina-2, endocan-1, molécula de adhesión endotelial 1 y E-selectina) mostraron una fuerte asociación con la sepsis en comparación con la infección aislada. La proadrenomedulina presentó el valor más alto de la razón de oportunidades (odds ratio, OR) en el análisis multivariable (OR 11,53, i.c. del 95% 4,15-32,08, P = 0,006) y la mejor área bajo la curva para detectar sepsis (AUC 0,86, i.c. del 95% 0,80-0,91, P < 0,001). En la comparación entre sepsis y shock séptico, los biomarcadores que mostraron la asociación más estrecha con el shock séptico fueron dos biomarcadores de degranulación de neutrófilos (proteinasa-3 y lipocalina-2) (OR 8,09, i.c. del 9% 1,34-48,91, P = 0,028; OR 6.62, i.c. del 95% 2,47-17,77, P = 0,002), pero la lipocalina-2 presentó la mejor AUC (0,81, i.c. del 95% 0,73-0,90, P < 0,001). CONCLUSIÓN: la proadrenomedulina y la lipocalina-2 podrían representar alternativas a la puntuación SOFA para detectar sepsis y shock séptico en pacientes quirúrgicos con infección.

Role of the neutrophil/lymphocyte ratio in patients with metastatic castration-resistant prostate cancer treated first-line with abiraterone. / Papel del índice neutrófilo/linfocito en pacientes con cáncer de próstata resistente a castración metastásicos tratados en primera línea con abiraterona.

INTRODUCTION: In patients with prostate cancer, high NLR seems to be associated with worse survival. Abiraterone acetate (AA) is a new generation hormonal treatment that has shown to increase PFS and OS in mCRPC. MATERIAL AND METHODS: Retrospective analysis of patients treated with AA in our center (December 2012-September 2018). We analyzed the association of the NLR (< or ≥ 3) before and after 6 months of treatment with PSA response, PFS, OS, and hormone sensitivity prior to AA (< or> 12 months). RESULTS: We have treated 56 patients with a median age of 82 (62-94), of which 22 (39%) had NLR ≥ 3 before treatment. There is a statistically significant association between the NLR prior to treatment<3 and PSA response, OR=9,444, P=.001, and there was no association with the NLR at 6 months of treatment. Statistically significant differences were found between the groups of NLR 3 prior to treatment with abiraterone in PFS with 15 months of median vs. 9 and P=.008, and in OS with 20 months vs. 9 with P=.014. With respect to the determination of NLR at 6 months, there are no differences in the survival curves between both groups. There are significant differences between the NLR prior to treatment according to the length of hormone sensitivity (P=.026). CONCLUSIONS: Our results suggest that NLR could provide relevant information and could act as an early and accessible prognostic marker in patients with mCRPC in first line treatment with Abiraterone.

Clinical factors influencing mortality risk in hospital-acquired sepsis.

BACKGROUND: Identification of factors that confer an increased risk of mortality in hospital-acquired sepsis (HAS) is necessary to help prevent, and improve the outcome of, this condition. AIM: To evaluate the clinical characteristics and factors associated with mortality in patients with HAS. METHODS: Retrospective study of patients with HAS in a major Spanish Hospital from 2011 to 2015. Data from adults receiving any of the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes associated with sepsis were collected. Those fulfilling the SEPSIS-2 definition with no evidence of infection during the first 48 h following hospitalization were included (N = 196). Multivariate analysis was employed to identify the risk factors of mortality. FINDINGS: HAS patients were found to have many of the risk factors associated with cardiovascular disease (male sex, ageing, antecedent of cardiac disease, arterial hypertension, dyslipidaemia, smoking habit) and cancer. Vascular disease or chronic kidney disease were associated with 28-day mortality. Time from hospital admission to sepsis diagnosis, and the presence of organ failure were risk factors for 28-day and hospital mortality. Experiencing more than one episode of sepsis increased the risk of hospital mortality. 'Sepsis code' for the early identification of sepsis was protective against hospital mortality. CONCLUSION: This study identifies several major factors associated with mortality in patients suffering from HAS. Implementation of surveillance programmes for the early identification and treatment of sepsis translate into a clear benefit.

Hippo Pathway: An Emerging Regulator of Craniofacial and Dental Development.

The evolutionarily conserved Hippo signaling pathway is a vital regulator of organ size that fine-tunes cell proliferation, apoptosis, and differentiation. A number of important studies have revealed critical roles of Hippo signaling and its effectors Yap (Yes-associated protein) and Taz (transcriptional coactivator with PDZ binding motif) in tissue development, homeostasis, and regeneration, as well as in tumorigenesis. In addition, recent studies have shown evidence of crosstalk between the Hippo pathway and other key signaling pathways, such as Wnt signaling, that not only regulates developmental processes but also contributes to disease pathogenesis. In this review, we summarize the major discoveries in the field of Hippo signaling and what has been learned about its regulation and crosstalk with other signaling pathways, with a particular focus on recent findings involving the Hippo-Yap pathway in craniofacial and tooth development. New and exciting studies of the Hippo pathway are anticipated that will significantly improve our understanding of the molecular mechanisms of human craniofacial and tooth development and disease and will ultimately lead to the development of new therapies.

Full Spectrum of Postnatal Tooth Phenotypes in a Novel Irf6 Cleft Lip Model.

Clefting of the lip, with or without palatal involvement (CLP), is associated with a higher incidence of developmental tooth abnormalities, including hypodontia and supernumerary teeth, aberrant crown and root morphologies, and enamel defects, although the underlying mechanistic link is poorly understood. As most CLP genes are expressed throughout the oral epithelium, the authors hypothesized that the expression of CLP genes may persist in the dental epithelium and thus, in addition to their earlier role in labiopalatine development, may play an important functional role in subsequent tooth patterning and amelogenesis. To address this, the authors generated a unique conditional knockout model involving the major CLP gene, Irf6, that overcomes the previously reported perinatal lethality to enable assessment of any posteruption dental phenotypes. A dental epithelium-specific Irf6 conditional knockout (Irf6-cKO) mouse was generated via a Pitx2-Cre driver line. Dental development was analyzed by microcomputed tomography, scanning electron microscopy, histology, immunohistochemistry, and quantitative polymerase chain reaction. Irf6-cKO mice displayed variable hypodontia, occasional supernumerary incisors and molars, as well as crown and root patterning anomalies, including peg-shaped first molars and taurodontic and C-shaped mandibular second molars. Enamel density was reduced in preeruption Irf6-cKO mice, and some shearing of enamel rods was noted in posteruption incisors. There was also rapid attrition of Irf6-cKO molars following eruption. Histologically, Irf6-cKO ameloblasts exhibited disturbances in adhesion and polarity, and delayed enamel formation was confirmed immunohistochemically. Altered structure of Hertwig's epithelial root sheath was also observed. These data support a role for IRF6 in tooth number, crown and root morphology and amelogenesis that is likely due to a functional role of Irf6 in organization and polarity of epithelial cell types. This data reinforce the notion that various isolated tooth defects could be considered part of the CLP spectrum in relatives of an affected individual.

Primary treatment of painful varicocoele through percutaneous retrograde embolization with fibred coils.

The literature on the treatment of painful varicocoele is limited, likely because of the short period since it was recognized as a clinical entity and the limitations posed by the subjectivity of pain. Our aim was to systematically analyse the results of percutaneous embolization as the chosen treatment for this condition. We conducted a retrospective study of patients undergoing percutaneous embolization as primary treatment for painful varicocoele from January 2007 to November 2013. Radiologic and ultrasonographic successes were evaluated according to the existence or absence of venous reflux on venography after embolization and on Echo Doppler control at 3-6 months. Clinical success was assessed by Visual Analog Scale pain questionnaires before surgery and at 3-6 months; in addition, at the time of the study, telephone interviews were conducted to update the clinical situation and development. A total of 154 patients received operations. The median pain before surgery, at 3-6 months and at the time of interview was 7, 1 and 0 points respectively (p < 0.001). The ultrasonographic success rate at 3-6 months was 68.6%. With a median follow-up of 39 months, the success and relapse/clinical persistence rates were 86.9 and 13.1% respectively. By studying the degree of agreement between clinical success and ultrasonographic success, a kappa index = 0.443 was obtained. Patients with success recounted greater pre-operative pain scores than those who relapsed or persisted (7.5 vs. 5.0; p = 0.004). In patients with painful varicocoele, the ultrasonographic recurrence of venous reflux does not imply the recurrence of pain; hence, the proper assessment of success in these patients should include a systematic assessment of their pain and grade of reflux. Percutaneous retrograde embolization as a primary treatment for painful varicocoele is a clinically effective option with a high success rate that can be maintained in the long term, especially in patients with high pre-operative pain.

Sphenoid sinus metastasis as the presenting manifestation of a prostatic adenocarcinoma: case report and overview of the literature.

Although a metastatic presentation of an occult prostatic adenocarcinoma is not uncommon, the majority of these patients present with bone metastasis affecting the axial skeleton. Cranial metastases to the paranasal sinuses are extremely rare. A 56-year-old man presented with loss of vision and numbness of the right side of the face. Computed tomography (CT) scan and cranial magnetic resonance imaging (MRI) revealed a mass invading the sphenoid sinus. The patient underwent surgery to remove the lesion, and the histopathological examination suggested metastasis of an adenocarcinoma, with positive staining to prostatic specific antigen (PSA). However, serum PSA was 4 ng/mL, and the patient did not report any lower urinary tract symptoms or bone pain. Transrectal ultrasound-guided biopsy revealed prostatic adenocarcinomas with a Gleason score of 8 [4 + 4]. The subsequent treatment consisted of radiotherapy and androgen deprivation, followed by first- and second-line chemotherapy (docetaxel and cabazitaxel) when the disease progressed. The patient achieved a good response with the last cycle of cabazitaxel and after a 5-year followup is currently alive. Cranial metastases of prostate adenocarcinoma are rare, and there is currently no standard treatment for these patients. Whenever possible, surgery combined with radiotherapy and hormonotherapy is the recommended option.

MicroRNAs play a critical role in tooth development.

MicroRNAs are known to regulate gene function in many tissues and organs, but their expression and function, if any, in tooth development are elusive. We sought to identify them by microRNA screening analyses and reveal their overall roles by inactivating Dicer1 in the dental epithelium and mesenchyme. Discrete sets of microRNAs are expressed in molars compared with incisors as well as epithelium compared with mesenchyme. Conditional knockout (cKO) of Dicer1 (mature microRNAs) in the dental epithelium of the Pitx2-Cre mouse results in multiple and branched enamel-free incisors and cuspless molars, and change in incisor patterning and in incisor and molar size and shape. Analyses of differentiating dental epithelial markers reveal a defect in ameloblast differentiation. Conversely, the cervical loop (stem cell niche) is expanded in Dicer1 cKO. These results demonstrate that tooth development is tightly controlled by microRNAs and that specific microRNAs regulate tooth epithelial stem cell differentiation.

Proteolytic activity, mycotoxins and andrastin A in Penicillium roqueforti strains isolated from Cabrales, Valdeón and Bejes-Tresviso local varieties of blue-veined cheeses.

High quality local varieties of blue-veined cheese are made in the villages of the valleys of Cabrales, Valdeón and Bejes-Tresviso in North Spain. Penicillium roqueforti strains have been isolated from each of those blue cheeses and compared with the collection strain P. roqueforti CECT 2905 (ATCC 10110) and a strain 'Valdeón-industrial' used for large scale production of Valdeón cheese. Using molecular genetics techniques and 5.8S and 18S rRNAs and the D1-D2 regions of 28S rRNA all strains were identified as authentic P. roqueforti. These strains from local varieties of blue cheese could be distinguished from the Valdeón-industrial strain and the control strain CECT 2905 by the mitochondrial DNA restriction pattern. The industrial strain showed high levels of aspartylprotease AspA, whereas the culture collection strain showed barely detectable levels of this enzyme, as shown by proteolysis tests and by immunodetection with anti-AspA antibodies. The lipolytic activity was similar in the strains isolated from the three types of local blue cheeses. The strains isolated from the local varieties of the blue cheese produced moderate levels of PR toxin, whereas the Valdeón-industrial strains showed a higher content of this mycotoxin. All strains (except the control strain CECT 2905) showed similar levels of roquefortine C. The antitumoral compound andrastin A was produced by all strains at different levels. P. roqueforti CECT 2905 showed high ability to synthesize this compound. Andrastin A was present in all industrial and local varieties of blue cheese. The content of andrastin A was similar to that of other well-known blue cheeses from France and Denmark.

20p12.3 microdeletion predisposes to Wolff-Parkinson-White syndrome with variable neurocognitive deficits.

BACKGROUND: Wolff-Parkinson-White syndrome (WPW) is a bypass re-entrant tachycardia that results from an abnormal connection between the atria and ventricles. Mutations in PRKAG2 have been described in patients with familial WPW syndrome and hypertrophic cardiomyopathy. Based on the role of bone morphogenetic protein (BMP) signalling in the development of annulus fibrosus in mice, it has been proposed that BMP signalling through the type 1a receptor and other downstream components may play a role in pre-excitation. METHODS AND RESULTS: Using the array comparative genomic hybridisation (CGH), we identified five individuals with non-recurrent deletions of 20p12.3. Four of these individuals had WPW syndrome with variable dysmorphisms and neurocognitive delay. With the exception of one maternally inherited deletion, all occurred de novo, and the smallest of these harboured a single gene, BMP2. In two individuals with additional features of Alagille syndrome, deletion of both JAG1 and BMP2 were identified. Deletion of this region has not been described as a copy number variant in the Database of Genomic Variants and has not been identified in 13 321 individuals from other cohort examined by array CGH in our laboratory. CONCLUSIONS: Our findings demonstrate a novel genomic disorder characterised by deletion of BMP2 with variable cognitive deficits and dysmorphic features and show that individuals bearing microdeletions in 20p12.3 often present with WPW syndrome.

Relevance of apolipoprotein E4 for the lipid profile of Brazilian patients with coronary artery disease.

Apolipoprotein E (apoE - e2, e3, e4 alleles) plays a role in the regulation of lipid metabolism, with the e4 considered to be a risk factor for coronary artery disease (CAD). We aimed to evaluate the apoE polymorphisms in Brazilians with CAD and their influence on the lipid profile and other risk factors (hypertension, diabetes mellitus, smoking). Two hundred individuals were examined: 100 patients with atherosclerosis confirmed by coronary angiography and 100 controls. Blood samples were drawn to determine apoE polymorphisms and lipid profile. As expected, the e3 allele was prevalent in the CAD (0.87) and non-CAD groups (0.81; P = 0.099), followed by the e4 allele (0.09 and 0.14, respectively; P = 0.158). The e3/3 (76 and 78%) and e3/4 (16 and 23%) were the most common genotypes for patients and controls, respectively. The lipid profile was altered in patients compared to controls (P < 0.05), independently of the e4 allele. However, in the controls this allele was prevalent in individuals with elevated LDL-cholesterol levels only (odds ratio = 2.531; 95% CI = 1.028-6.232). The frequency of risk factors was higher in the CAD group (P < 0.05), but their association with the lipid profile was not demonstrable in e4 carriers. In conclusion, the e4 allele is not associated with CAD or lipid profile in patients with atherosclerosis. However, its frequency in the non-CAD group is associated with increased levels of LDL-cholesterol, suggesting an independent effect of the e4 allele on lipid profile when the low frequency of other risk factors in this group is taken into account.

Relevance of apolipoprotein E4 for the lipid profile of Brazilian patients with coronary artery disease

Apolipoprotein E (apoE - e2, e3, e4 alleles) plays a role in the regulation of lipid metabolism, with the e4 considered to be a risk factor for coronary artery disease (CAD). We aimed to evaluate the apoE polymorphisms in Brazilians with CAD and their influence on the lipid profile and other risk factors (hypertension, diabetes mellitus, smoking). Two hundred individuals were examined: 100 patients with atherosclerosis confirmed by coronary angiography and 100 controls. Blood samples were drawn to determine apoE polymorphisms and lipid profile. As expected, the e3 allele was prevalent in the CAD (0.87) and non-CAD groups (0.81; P = 0.099), followed by the e4 allele (0.09 and 0.14, respectively; P = 0.158). The e3/3 (76 and 78 percent) and e3/4 (16 and 23 percent) were the most common genotypes for patients and controls, respectively. The lipid profile was altered in patients compared to controls (P < 0.05), independently of the e4 allele. However, in the controls this allele was prevalent in individuals with elevated LDL-cholesterol levels only (odds ratio = 2.531; 95 percent CI = 1.028-6.232). The frequency of risk factors was higher in the CAD group (P < 0.05), but their association with the lipid profile was not demonstrable in e4 carriers. In conclusion, the e4 allele is not associated with CAD or lipid profile in patients with atherosclerosis. However, its frequency in the non-CAD group is associated with increased levels of LDL-cholesterol, suggesting an independent effect of the e4 allele on lipid profile when the low frequency of other risk factors in this group is taken into account.

Lysine is catabolized to 2-aminoadipic acid in Penicillium chrysogenum by an omega-aminotransferase and to saccharopine by a lysine 2-ketoglutarate reductase. Characterization of the omega-aminotransferase.

The biosynthesis and catabolism of lysine in Penicillium chrysogenum is of great interest because these pathways provide 2-aminoadipic acid, a precursor of the tripeptide delta-L-2-aminoadipyl-L-cysteinyl-D-valine that is an intermediate in penicillin biosynthesis. In vivo conversion of labelled L-lysine into two different intermediates was demonstrated by HPLC analysis of the intracellular amino acid pool. L-lysine is catabolized to 2-aminoadipic acid by an omega-aminotransferase and to saccharopine by a lysine-2-ketoglutarate reductase. In lysine-containing medium both activities were expressed at high levels, but the omega-aminotransferase activity, in particular, decreased sharply when ammonium was used as the nitrogen source. The omega-aminotransferase was partially purified, and found to accept L-lysine, L-ornithine and, to a lesser extent, N-acetyl-L-lysine as amino-group donors. 2-Ketoglutarate, 2-ketoadipate and, to a lesser extent, pyruvate served as amino group acceptors. This pattern suggests that this enzyme, previously designated as a lysine-6-aminotransferase, is actually an omega-aminotransferase. When 2-ketoadipate is used as substrate, the reaction product is 2-aminoadipic acid, which contributes to the pool of this intermediate available for penicillin biosynthesis. The N-terminal end of the purified 45-kDa omega-aminotransferase was sequenced and was found to be similar to the corresponding segment of the OAT1 protein of Emericella (Aspergillus) nidulans. This information was used to clone the gene encoding this enzyme.

Agrobacterium tumefaciens-mediated transformation of the antitumor clavaric acid-producing basidiomycete Hypholoma sublateritium.

The basidiomycete Hypholoma sublateritium produces clavaric acid, an antitumor isoprenoid compound. Arthrospores of this fungus were transformed by Agrobacterium tumefaciens-mediated conjugation. Five plasmids carrying different regulatory sequences to drive expression of the hph (hygromycin phosphotransferase) gene were tested. The promoter used was critically important in order to express heterologous genes in H. sublateritium. Constructions carrying the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter (P gpd) showed a good transformation efficiency, whereas constructions with the gpd promoter from ascomycetes were ineffective. Transformant clones showed a random integration pattern of plasmid DNA. Most transformants showed a single integrated copy of the transforming plasmid, but about 1.5% showed double or multiple integrations. All the analyzed transformants were mitotically stable and maintained the integrated exogenous DNA in the absence of antibiotic. The green fluorescent protein gene was expressed from the A. bisporus gpd promoter, as shown by RT-PCR studies, but no significant fluorescence was observed. Transformation of H. sublateritium opens the way for the genetic manipulation of clavaric acid biosynthesis in this fungus.

Stem cells and repair of the heart.

Stem-cell therapy provides the prospect of an exciting and powerful treatment to repair the heart. Although research has been undertaken in animals to analyse the safety and efficacy of this new approach, results have been inconclusive. The mechanism by which stem cells could improve cardiac function remains unclear. We describe the background to the concept of natural repair and the work that has been done to establish the role of stem cells in cardiac repair. Controversies have arisen in interpretation of experimental data. The important issues surrounding the application of stem-cell therapy to man are discussed critically. We discuss the future of this pioneering work in the setting of growing concerns about clinical studies in man without understanding the biological mechanisms involved, with the difficulties in funding this type of research.

Expression of a synthetic copy of the bovine chymosin gene in Aspergillus awamori from constitutive and pH-regulated promoters and secretion using two different pre-pro sequences.

A copy of the bovine chymosin gene (chy) with a codon usage optimized for its expression in Aspergillus awamori was constructed starting from synthetic oligonucleotides. To study the ability of this filamentous fungus to secrete bovine prochymosin, two plasmids were constructed in which the transcriptional, translational, and secretory control regions of the A. nidulans gpdA gene and pepB genes were coupled to either preprochymosin or prochymosin genes. Secretion of a protein enzymatically and immunologically indistinguishable from bovine chymosin was achieved in A. awamori transformants with each of these constructions. In all cases, the primary translation product (40.5 kDa) was self-processed to a mature chymosin polypeptide having a molecular weight of 35.6 kDa. Immunological assays indicated that most of the chymosin was secreted to the extracellular medium. Hybridization analysis of genomic DNA from chymosin transformants showed chromosomal integration of prochymosin sequences and, in some transformants, multiple copies of the expression cassettes were observed. Expression from the gpdA promoter was constitutive, whereas expression from the pepB promoter was strongly influenced by pH. A very high expression from the pepB promoter was observed during the growth phase. The A. awamori pepB gene terminator was more favorable for chymosin production than the S. cerevisiae CYC1 terminator.