Regulation of trehalase activity by multi-site phosphorylation and 14-3-3 interaction.

Protein phosphorylation enables a rapid adjustment of cellular activities to diverse intracellular and environmental stimuli. Many phosphoproteins are targeted on more than one site, which allows the integration of multiple signals and the implementation of complex responses. However, the hierarchy and interplay between multiple phospho-sites are often unknown. Here, we study multi-site phosphorylation using the yeast trehalase Nth1 and its activator, the 14-3-3 protein Bmh1, as a model. Nth1 is known to be phosphorylated by the metabolic kinase PKA on four serine residues and by the cell cycle kinase CDK on one residue. However, how these five phospho-sites adjust Nth1 activity remains unclear. Using a novel reporter construct, we investigated the contribution of the individual sites for the regulation of the trehalase and its 14-3-3 interactor. In contrast to the constitutively phosphorylated S20 and S83, the weaker sites S21 and S60 are only phosphorylated by increased PKA activity. For binding Bmh1, S83 functions as the high-affinity "gatekeeper" site, but successful binding of the Bmh1 dimer and thus Nth1 activation requires S60 as a secondary site. Under nutrient-poor conditions with low PKA activity, S60 is not efficiently phosphorylated and the cell cycle dependent phosphorylation of S66 by Cdk1 contributes to Nth1 activity, likely by providing an alternative Bmh1 binding site. Additionally, the PKA sites S20 and S21 modulate the dephosphorylation of Nth1 on downstream Bmh1 sites. In summary, our results expand our molecular understanding of Nth1 regulation and provide a new aspect of the interaction of 14-3-3 proteins with their targets.

Effects of different amounts and type of diet during weaning-to-estrus interval on reproductive performance of primiparous and multiparous sows.

During weaning-to-estrus interval (WEI), the sows are usually fed with high feed level to improve the reproductive performance. However, the WEI has been reduced over the years which may reduce the impact of feed level on performance in the modern genetic lines. The aim of this study was to evaluate the effect of two feeding levels (moderate feeding level (MFL): 2.7 kg/day and high feeding level (HFL): 4.3 kg/day) and two diet types (gestation: 13.67 MJ/kg of metabolizable energy (ME) and 0.62% of standard ileal digestible lysine (SID Lys) and lactation: 14.34 MJ ME/kg and 1.20% of SID Lys) offered during the WEI on reproductive performance. In total, 19.0% of sows were excluded from the analysis due to feed intake below 75% (9.6% and 28.5% in MFL and HFL groups, respectively), remaining 254 primiparous and 806 multiparous sows. Follicular size and change in BW were measured in subsamples of 180 and 227 females, respectively. Data were analyzed considering the sow as the experimental unit. Feeding level, diet type, parity and their interactions were included as fixed effects, whereas the day of weaning was considered as a random effect. The feed intake of MFL and HFL groups averaged 2.5 ± 0.02 and 3.8 ± 0.02 kg/day, respectively. There was an interaction between feeding level and parity for daily feed intake. Within HFL, multiparous sows consumed 181 g/day more than primiparous sows (P < 0.01), but no difference was observed within MFL (P > 0.05). Both primiparous and multiparous sows lost proportionally less weight when fed HFL than MFL gestation diet during WEI. The percentage of weight loss was lower in HFL than in the MFL group in multiparous sows fed the lactation diet. The WEI was not affected by feeding level, diet type or its interaction (P > 0.05), but it was longer in primiparous than in multiparous sows (P = 0.001). There was no effect of feeding level, diet type, parity or their interactions on anestrus and farrowing rates. Multiparous sows showed greater follicular size, and greater numbers of total born and born alive piglets in the subsequent cycle than primiparous sows (P < 0.05). In conclusion, feeding weaned primiparous and multiparous sows with 4.3 kg/day of a gestation (58.78 MJ ME and 26.66 g SID Lys) or a lactation diet (61.66 MJ ME and 51.60 g SID Lys) does not improve follicular size and reproductive performance in the subsequent cycle.

A novel splicing mutation in GALT gene causing Galactosemia in Ecuadorian family.

Classic Galactosemia (OMIM 230400) is an autosomal recessive disorder of galactose metabolism caused by mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. This disease caused by the inability to metabolize galactose is potentially life-threatening but its pathophysiology has not been clearly defined. GALT gene presents high allelic heterogeneity and around 336 variations have been identified. Here, we report the case of a patient with Classic Galactosemia who was detected during a neonatal screening in Ecuador. Molecular study revealed a mutation in GALT gene intron 1, c.82+3A>G in homozygous condition, this mutation has not been previously reported. This gene variation was not found in any of the 119 healthy Ecuadorian individuals used as control. Furthermore, the mutation was the only alteration detected in the propositus's GALT after sequencing all exons and introns of this gene. In silico modeling predicted that the mutation was pathogenic.

The tubular compartment and the spermatogenic dynamics of the wild rodent Oxymycterus nasutus (Rodentia: Cricetidae).

Despite the order Rodentia present worldwide distribution and large number of species in the Brazilian fauna, detailed studies on testicular morphophysiology are still scarce. Therefore, this study aimed to analyze the dynamics of the spermatogenic process of Oxymycterus nasutus using morphometrical and stereological tools. Testicles from ten sexually mature males were used, showing a gonadosomatic index of 0.89%. The testicular parenchyma showed one of the highest tubulesomatic indexes reported among wild rodents - 0.82% - from which 65.12% was allocated into seminiferous epithelium. The average tubular diameter was 249.89 µm, whereas the epithelium height was 62.47 µm and the total length was 18.62 m per gram of testis. Eight different stages of the seminiferous epithelium cycle were described. Stage 1 was used for counting the germ cell population as well as the Sertoli cells. On average, 3.47 type-A spermatogonia, 24.39 primary spermatocytes in preleptotene/leptotene, 24.13 primary spermatocytes in pachytene, 68.38 round spermatids and 7.33 Sertoli cells were found per tubular cross section. There were 91.02 × 10(6) Sertoli cells per gram of testis and each cell was able to support 9.33 spermatids and 16.43 germ cells. The coefficient of spermatogonial mitosis was 7.02, while 2.83 spermatids were produced for each primary spermatocyte in pachytene. The overall efficiency of spermatogenesis was 19.70 cells, whereas the sperm reserve per gram of testis totalized 849.63 × 10(6) spermatids. Therefore, the presented data showed that O. nasutus shows a high energetic investment in reproduction, corroborating the findings for other species of the Cricetidae family.

Characterization of two pathogenic mutations in cystathionine beta-synthase: different intracellular locations for wild-type and mutant proteins.

Cystathionine ß-synthase (CBS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the condensation of homocysteine with serine to generate cystathionine. Homocystinuria is an autosomal recessive disorder commonly caused by a deficiency of CBS activity. Here, we characterized a novel CBS mutation (c.260C>A (p.T87N)) and a previously reported variant (c.700G>A (p.D234N)) found in Venezuelan homocystinuric patients, one nonresponsive and one responsive to vitamin B6. Both mutant proteins were expressed in vitro in prokaryotic and eukaryotic cells, finding lower soluble expression in HEK-293 cells (19% T87N and 23% D234N) compared to wild-type CBS. Residual activities obtained for the mutant proteins were 3.5% T87N and 43% D234N. Gel exclusion chromatography demonstrated a tendency of the T87N mutant to aggregate while the distribution of the D234N mutant was similar to wild-type enzyme. Using immunofluorescence microscopy, an unexpected difference in intracellular localization was observed between the wild-type and mutant proteins. While the T87N mutant exhibited a punctate appearance, the wild-type protein was homogeneously distributed inside the cell. Interestingly, the D234N protein showed both distributions. This study demonstrates that the pathogenic CBS mutations generate unstable proteins that are unable (T87N) or partially unable (D234N) to assemble into a functional enzyme, implying that these mutations might be responsible for the homocystinuria phenotype.

[Clinical findings and mutational spectrum in Venezuelan patients with delayed diagnosis of phenylketonuria]. / Hallazgos clínicos y espectro mutacional en pacientes venezolanos con diagnóstico tardío de fenilcetonuria.

INTRODUCTION: Massive neonatal screening for phenylketonuria (PKU) began in developed countries in 1963, and eventually disappeared as a cause of mental retardation. Yet, this is not the case in most developing countries. AIM: To describe the phenotype and the genotype of PKU patients with a delayed diagnosis in order to draw attention to the importance of neonatal studies and molecular diagnosis. PATIENTS AND METHODS: Clinical data were collected from five unrelated patients by means of a medical assessment. The molecular study was conducted using the DGGE, sequencing and/or restriction analysis techniques to search for mutations in the PAH gene. RESULTS. Owing to the delayed diagnosis all the patients presented severe clinical manifestations, such as psychomotor retardation, atypical behaviours and language disorders. Four of them presented epilepsy and there were two cases of microcephaly. The phenotype was as expected, given the genotype. Seven different mutations were detected in the 10 alleles that were studied. The IVS10nt +5 g>t mutation was the most frequent, followed by the Venezuelan mutation S349L. Furthermore, two patients presented mutated proteins with residual activity, and good results were obtained using BH4 therapy. CONCLUSIONS: In our country, as in most developing countries, PKU neonatal studies are performed but the programme does not cover the whole neonatal population. In this work, we want to stress the importance of neonatal studies in the welfare of children, as well as the use of molecular diagnosis to improve the therapeutic orientation and genetic counselling of the families involved.

Hallazgos clínicos y espectro mutacional en pacientes venezolanos con diagnóstico tardío de fenilcetonuria / Clincial findings and mutational spectrum in Venezuelan patients with delayed diagnosis of phenylketonuria

Introducción. En 1963 comenzó el cribado neonatal masivo de fenilcetonuria (PKU) en países desarrollados, queacabó desapareciendo como causa de retraso mental. Sin embargo, éste no es el caso en la mayoría de los países en vías de desarrollo. Objetivo. Describir el fenotipo y el genotipo de pacientes con diagnóstico tardío de PKU, con el fin de resaltar la importancia del estudio neonatal y el diagnóstico molecular. Pacientes y métodos. Se recogieron datos clínicos de cinco pacientes no relacionados mediante evaluación médica. El estudio molecular se realizó empleando las técnicas de DGGE, secuenciación y/o análisis de restricción para la búsqueda de mutaciones en el gen PAH. Resultados. Todos los pacientes presentaronmanifestaciones clínicas graves debidas al diagnóstico tardío, como retraso psicomotor, conductas atípicas y trastornos del lenguaje. Cuatro de ellos presentaron epilepsia y dos, microcefalia. El fenotipo fue el esperado de acuerdo con el genotipo. Se detectaron siete mutaciones diferentes en los 10 alelos estudiados. La mutación IVS10nt+5g>t fue la más frecuente,seguida de la mutación venezolana S349L. Por otra parte, dos pacientes presentan proteínas mutadas con actividadresidual, y pudieron verse beneficiados de la terapia con BH4. Conclusiones. En Venezuela, al igual que en gran parte de los países en vías de desarrollo, se realiza el estudio neonatal de PKU pero el programa no cubre toda la población neonatal. En este trabajo se quiere destacar la importancia del estudio neonatal en el bienestar de los niños, y el uso del diagnóstico molecular para mejorar la orientación terapéutica y la asesoría genética de la familia

Introduction. Massive neonatal screening for phenylketonuria (PKU) began in developed countries in 1963, and eventually disappeared as a cause of mental retardation. Yet, this is not the case in most developing countries. Aim. To describe the phenotype and the genotype of PKU patients with a delayed diagnosis in order to draw attention to the importance of neonatal studies and molecular diagnosis. Patients and methods. Clinical data were collected from five unrelated patients by means of a medical assessment. The molecular study was conducted using the DGGE, sequencing and/or restriction analysis techniques to search for mutations in the PAH gene. Results. Owing to the delayed diagnosis all thepatients presented severe clinical manifestations, such as psychomotor retardation, atypical behaviours and language disorders. Four of them presented epilepsy and there were two cases of microcephaly. The phenotype was as expected, given the genotype. Seven different mutations were detected in the 10 alleles that were studied. The IVS10nt+5g>t mutation was themost frequent, followed by the Venezuelan mutation S349L. Furthermore, two patients presented mutated proteins with residual activity, and good results were obtained using BH4 therapy. Conclusions. In our country, as in most developingcountries, PKU neonatal studies are performed but the programme does not cover the whole neonatal population. In this work, we want to stress the importance of neonatal studies in the welfare of children, as well as the use of molecular diagnosis to improve the therapeutic orientation and genetic counselling of the families involved

Intubation conditions following rocuronium: influence of induction agent and priming.

A small priming dose of rocuronium can shorten the onset time of neuromuscular blockade. Induction agents with less cardiovascular depression also reduce the onset time. We hypothesized that ketamine, compared to thiopentone, would reduce onset time and improve intubating conditions following priming. Sixty patients ASA I to II, randomized by computer-generated sequence to four groups were investigated in a double-blind controlled trial. In the two groups with priming, 0.04 mg/kg of rocuronium was followed by three minutes of priming interval. Induction was followed by an intubation dose of 0.4 mg/kg of rocuronium. After 30 seconds, intubation was attempted within a further 20 seconds. In the two control groups, the same sequence was repeated except sham priming (saline) was given. For induction, S-ketamine (1 mg/kg) or thiopentone (4 mg/kg) were administered. Intubating conditions were graded as excellent, good, poor, or not possible. Neuromuscular transmission was monitored by acceleromyography of the thumb. There were no measured differences in onset time of neuromuscular block or in haemodynamics between the groups. The proportion of good to excellent intubating conditions was higher when ketamine was preceded by priming compared to ketamine without priming (87% vs 20%; P<0.05). In both priming and control groups intubating conditions were improved when using ketamine compared to thiopentone (P<0.05). The mechanism of this effect was not clear from this study.

Effects of ephedrine on intubating conditions following priming with rocuronium.

BACKGROUND: Onset of action of muscle relaxants is influenced by cardiac output and muscle blood flow. Ephedrine reduces the onset time of rocuronium. Onset is also shortened by priming. Accordingly, we hypothesized that priming combined with ephedrine is superior to either technique used separately. METHODS: Four groups of randomly allocated patients (n = 31), ASA I - II, were induced with propofol 2.5 mg kg(-1). In groups I and II, 0.04 mg kg(-1) of rocuronium was followed by a 3-min priming interval. Induction was followed by an intubation dose of 0.04 mg kg(-1). Then a 30-s intubation was attempted. In groups III and IV the same sequence was repeated except for sham priming and an intubation dose of 0.44 mg kg(-1). In groups I and II, ephedrine (210 microg kg(-1)) was injected before propofol. In groups II and V, an equivalent volume of normal saline was injected. Jaw relaxation, vocal cord position, and diaphragmatic response were used to assess intubating conditions. RESULTS: All patients of group I were intubated 30 s after the intubating dose and within a 20-s interval compared with 74% of patients in groups II and III, and 84% of patients in group IV. Intubating conditions were graded good to excellent in all patients in group I compared with 42% of those in group II, 35% in group III and 52% in group IV (P < 0.01 vs. group I). During the priming interval, no adverse effects were observed or reported. CONCLUSIONS: Ephedrine in combination with propofol significantly improved clinical intubating conditions at 30 s following priming with rocuronium compared with priming with ephedrine without priming.

pH control of the production of recombinant glucose oxidase in Aspergillus nidulans.

AIMS: Recombinant Aspergillus nidulans sVAL040, capable of synthesizing and secreting glucose oxidase derived from Aspergillus niger was used to study the influence of pH and carbon source on enzyme production. METHODS AND RESULTS: Glucose oxidase gene (goxC) was expressed under transcriptional regulation by using the promoter of A. nidulans xlnB gene (encoding an acidic xylanase). A maximum specific glucose oxidase activity of approx. 10 U mg(-1) protein and a maximum volumetric productivity of 29.9 U l(-1) h(-1) were obtained at pH 5.5, after 80 h of growth by using xylose as inducer. Enzyme volumetric productivity increased when xylans were used instead of xylose; however, specific glucose oxidase activity did not differ significantly. CONCLUSIONS: Specific GOX activity obtained at pH 5.5 are two to three times more than those previously described for goxC multicopy transformants of A. nidulans. Xylans were a more powerful inducer than xylose although fungal growth was lower when the polymers were used. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained results by using xlnB promoter in A. nidulans could be useful in improving heterologous enzyme production by using genetic- and process-engineering strategies.

Glycerol and arabitol production by an intergeneric hybrid, PB2, obtained by protoplast fusion between Saccharomyces cerevisiae and Torulaspora delbrueckii.

An intergeneric osmotolerant hybrid yeast, PB2, was used together with the parental strains to study glycerol and arabitol production in batch culture. This fusion product was previously obtained by protoplast fusion between Torulaspora delbrueckii and Saccharomyces cerevisiae. Polyols and biomass production were determined in batch culture under aerobic conditions. Under the conditions tested, using PB2 hybrid and both parental strains, the best results were obtained with the hybrid. Arabitol reached a final concentration of 70 g/l and glycerol was increased to up to 50 g/l.

[Severe and prolonged EEG depression after induction of general anesthesia for carotid endarterectomy. Report of a clinical case]. / Grave e prolungata depressione elettroencelografica all'induzione di un'anestesia generale per TEA carotidea. Descrizione di un caso clinico.

A large number of methods are available for intraoperative neurologic monitoring during endarterectomy, although no single method is infallible. Debate over choice of regional or general anesthesia for this surgery persists because of differing conclusions of various studies of risks and benefits. The case of patient undergoing left carotid endarterectomy under general anesthesia is described, in whom after the induction of anesthesia with: midazolam 2 mg, fentanyl 50 g, propofol (fractionate dose of 180 mg) and cisatracurium 12 mg, a total EEG depression occurred persisting for over 10 minutes and was followed by slow recovery, during the awakening of the patient. No significant hemodynamic changes were noted during the induction. No neurologic deficit was observed in the patient after arousal. It is suggested that the probable reason for the EEG response is the particular sensitivity of the patient to some of the induction drugs or to their association. Two weeks later surgery was successfully performed using cervical plexus block, without any anesthetic support. Regional anesthesia allows continuous neurologic assessment of the awaken patient, it is the most sensitive method for detecting inadequate cerebral perfusion and function.

[Complications related to epidural catheter in caesarean delivery]. / Complicanze correlate al catetere epidurale in taglio cesareo.

A review of complications related to epidural catheters in caesarean delivery is presented. Catheters for prolongation of nerve blocks were first used in 1940s. Thereafter, there has been steady development in the design and plastic material technology of the different catheters. In the last decade the regional anaesthesia for caesarean section became very popular, as well as continuous increase in the use of epidural catheters. The anatomical changes of pregnancy like marked distension of the epidural veins resulted in increased risk of the complications due to the epidural catheter placement. It is likely that permanent neurologic sequelae due to regional anaesthesia in obstetrics almost never occur, while minor self-limiting complications do occur. The possible complications of epidural catheter techniques are: trauma, malposition and migration of the catheter, knotting and breaking, radiculopathy, dural puncture, subdural injection, abscess and infection, haematoma and wrong solution injection. Most of the malpositions of the epidural catheter can be avoided by a careful technique, advancing the catheter with no forceful movement and not more than 3 to 4 cm into epidural space. Broken parts of the catheters should be left as a rule within the spinal space. Test dose should be always done for continuous epidural anaesthesia. Early diagnosis and prompt appropriate treatment will usually lead to complete resolution of the neurological deficit even in cases of epidural haematoma or abscess.

Molecular characterization of phenylalanine hydroxylase deficiency in Chile. Mutations in brief no. 243. Online.

Both the haplotype distribution and the mutational spectrum of the phenylalanine hydroxylase (PAH) gene has been defined for the Chilean phenylketonuria (PKU) population. Mutation analysis was performed using a combined approach of screening for common European and Oriental mutations and application of the DGGE scanning method in the remaining uncharacterized alleles. A total of 16 different mutations have been identified, including two novel ones, Q232X and IVS11nt5. The most frequent mutations were IVS10nt-11 and V388M present both in the 13% of the mutant chromosomes. The rest of the mutations are rare. The haplotype association including VNTR and STR alleles, was examined to investigated the origin and distribution of PAH alleles in Chile. Our results are consistent with Southern Europeans as the major source of PAH mutations in Latin America. However, we have also detected mutations from East and Central Europe, such IVS12nt1, R408W and R252W. It is clear that the PKU mutation present in each Latin American country varies with the demographic profile and specific mutation scanning is necessary in each population both for diagnostic and prognostic purposes. The correlation between the genotypes and the phenotypes is consistent with the emerging pattern of mutation severity deduced from previous studies in related populations.

Molecular basis of phenylketonuria in Venezuela: presence of two novel null mutations.

This report describes the mutational spectrum and linked haplotypes of the phenylalanine hydroxylase gene in Venezuela. In this study, we have detected European mutations such as IVS10nt-11, R243Q, and R408W on the same haplotype background (6.7, 1.8, and 2.3, respectively) as in Europe. In this sample, we have found two novel mutations: S349L detected in two homozygous siblings on the background of haplotype 6.7, and a small deletion, P314fsdelC, that results in a frameshift and a premature stop codon detected on the background of haplotype 4.3. The definite demonstration that mutation S349L results in a nonfunctional protein was shown by expression analysis in prokaryotic and eukaryotic systems. This mutation results in an unstable phenylalanine hydroxylase (PAH) protein completely devoid of enzymatic activity well correlated with the severe form of the disease exhibited by the homozygous patients.

Evidence in Latin America of recurrence of V388M, a phenylketonuria mutation with high in vitro residual activity.

Phenylketonuria mutation V388M is frequent in the Iberian Peninsula. In vitro, the V388M mutant enzyme has similar immunoreactive protein and phenylalanine hydroxylase mRNA and has 43% residual activity, which correlates well with the mild phenotype exhibited by the homozygous patients. In Spain it has been detected in 5.7% of the mutant alleles and is always associated with haplotype 1.7. This mutation is also present in high frequency in some Latin American countries (Brazil, 9%; Chile, 13%). It is interesting that in Chile most of the alleles bearing this mutation carry haplotype 4.3, although in Brazil it is found only on the background of haplotype 1.7. The origin of V388M in Spain on haplotype 1.7 and in Chile on haplotype 4.3 is clearly different. Recurrence is the most plausible explanation, because the mutation involves a CpG dinucleotide, and a recombination event transferring the mutation from haplotype 1 to 4 is unlikely.

Complications of warfarin therapy monitored by the International Normalized Ratio versus the prothrombin time ratio.

The objective of our study was to determine the rates of bleeding complications and thromboembolic events in patients receiving oral anticoagulant therapy monitored with the prothrombin time (PT) ratio versus therapy monitored with the International Normalized Ratio (INR) using a retrospective time-series study design. Over 650 patients enrolled in a large anticoagulation clinic were studied during two time periods corresponding to the use of the PT ratio versus the INR to guide anticoagulant therapy, with over 400 patient-years of follow-up for each time period. The rate of bleeding complications using the PT ratio to guide therapy was 6.7% (1.2% major, 5.5% minor) per patient-year, compared with 2.9% (0% major, 2.9% minor) using the INR (p = 0.02). The rate of thromboembolic complications was 1.0% using the PT ratio, compared with 0.2% using the INR (p = NS). Therapy monitored with the INR required 19.8 visits per year, compared with 20.7 visits per year using the PT ratio. We conclude that the INR should be used to monitor oral anticoagulant therapy in an effort to reduce bleeding complications while maintaining an acceptable rate of thromboembolic events.

Continuous culture of Candida utilis: influence of medium nitrogen concentration.

When Candida utilis was grown in continuous culture, decreasing the concentration of N in the medium affected cell composition, biomass yield, biomass productivity, maximal growth rate and cell morphology. When the dilution rate was low (0.1 h(-1)), reducing N from 1100 to 100 mg/l led to a 40% decrease in RNA content of the cells. Nitrogen-limited growth, which occurred when N<420 mg/l, was associated with significant changes in cell-wall carbohydrates and a significant reduction in the glycogen content of the cells. A set of culture conditions was established which permitted maximal consumption of the main nutrients in the medium and the production of yeast biomass suitable as a source of single-cell protein.