Functional characterization of the oxaloacetase encoding gene and elimination of oxalate formation in the beta-lactam producer Penicillium chrysogenum

Penicillium chrysogenum is widely used as an industrial antibiotic producer, in particular in the synthesis of g-lactam antibiotics such as penicillins and cephalosporins. In industrial processes, oxalic acid formation leads to reduced product yields. Moreover, precipitation of calcium oxalate complicates product recovery. We observed oxalate production in glucose-limited chemostat cultures of P. chrysogenum grown with or without addition of adipic acid, side-chain of the cephalosporin precursor adipoyl-6-aminopenicillinic acid (ad-6-APA). Oxalate accounted for up to 5% of the consumed carbon source. In filamentous fungi, oxaloacetate hydrolase (OAH; EC3.7.1.1) is generally responsible for oxalate production. The P. chrysogenum genome harbours four orthologs of the A. niger oahA gene. Chemostat-based transcriptome analyses revealed a significant correlation between extracellular oxalate titers and expression level of the genes Pc18g05100 and Pc22g24830. To assess their possible involvement in oxalate production, both genes were cloned in Saccharomyces cerevisiae, yeast that does not produce oxalate. Only the expression of Pc22g24830 led to production of oxalic acid in S. cerevisiae. Subsequent deletion of Pc22g28430 in P. chrysogenum led to complete elimination of oxalate production, whilst improving yields of the cephalosporin precursor ad-6-APA. (C) 2011 Elsevier Inc. All rights reserved.

Effects of Commonly Used Solubilizing Agents on a Model Nerve-Muscle Synapse

Solubility represents a limiting factor when testing new compounds in animal experiments, since solubilizing agents generally have pharmacological effects that can interfere with the studied substance. Vehicles are commonly used for solubilizing certain substances including apolar and polar extracts obtained from medicinal plants. In this study, fifteen vehicles were investigated on mice neuromuscular preparations. A known in vitro neuroblocker myotoxin from Bothrops jararacussu venom, bothropstoxin-I, was used as a pharmacological tool for testing the medicinal potential of apolar and polar extracts (hexane, dichloromethane, ethyl acetate and methanol) obtained from Casearia sylvestris Sw. leaves, which in turn were used for testing their solubility and concomitantly to produce no change on basal response of indirectly stimulated mouse phrenic nerve-diaphragm preparations. Taken together in vitro biological system and extracts solubility, our results showed that dimethyl sulphoxide and polyethylene glycol 400 were the better vehicles, and methanol extract solubilized on PEG 400 was the only one able to act against the paralysis induced by the myotoxin. Thus, this study points out to the relevant role that vehicles exhibit for extracting the potential pharmacological activity of plants in a given test system.

Kinetics of elimination and distribution in blood and liver of biocompatible ferrofluids based on Fe(3)O(4) nanoparticles: An EPR and XRF study

In this study, we evaluated the biodistribution and the elimination kinetics of a biocompatible magnetic fluid, Endorem (TM), based on dextrancoated Fe(3)O(4) nanoparticles endovenously injected into Winstar rats. The iron content in blood and liver samples was recorded using electron paramagnetic resonance (EPR) and X-ray fluorescence (XRF) techniques. The EPR line intensity at g=2.1 was found to be proportional to the concentration of magnetic nanoparticles and the best temperature for spectra acquisition was 298 K. Both EPR and XRF analysis indicated that the maximum concentration of iron in the liver occurred 95 min after the ferrofluid administration. The half-life of the magnetic nanoparticles (MNP) in the blood was (11.6 +/- 0.6) min measured by EPR and (12.6 +/- 0.6) min determined by XRF. These results indicate that both EPR and XRF are very useful and appropriate techniques for the study of kinetics of ferrofluid elimination and biodistribution after its administration into the organism. (c) 2007 Elsevier B.V. All rights reserved.

Model for Differential Nursing Diagnosis of Alterations in Urinary Elimination Based on Fuzzy Logic

Nursing diagnoses associated with alterations of urinary elimination require different interventions, Nurses, who are not specialists, require support to diagnose and manage patients with disturbances of urine elimination. The aim of this study was to present a model based on fuzzy logic for differential diagnosis of alterations in urinary elimination, considering nursing diagnosis approved by the North American Nursing Diagnosis Association, 2001-2002. Fuzzy relations and the maximum-minimum composition approach were used to develop the system. The model performance was evaluated with 195 cases from the database of a previous study, resulting in 79.0% of total concordance and 19.5% of partial concordance, when compared with the panel of experts. Total discordance was observed in only three cases (1.5%). The agreement between model and experts was excellent (kappa = 0.98, P < .0001) or substantial (kappa = 0.69, P < .0001) when considering the overestimative accordance (accordance was considered when at least one diagnosis was equal) and the underestimative discordance (discordance was considered when at least one diagnosis was different), respectively. The model herein presented showed good performance and a simple theoretical structure, therefore demanding few computational resources.

Role of Rab27 in synaptic transmission at the squid giant synapse

Small GTPase Rab is a member of a large family of Ras-related proteins, highly conserved in eukaryotic cells, and thought to regulate specific type(s) and/or specific step(s) in intracellular membrane trafficking. Given our interest in synaptic transmission, we addressed the possibility that Rab27 (a close isoform of Rab3) could be involved in cytosolic synaptic vesicle mobilization. Indeed, preterminal injection of a specific antibody against squid Rab27 (anti-sqRab27 antibody) combined with confocal microscopy demonstrated that Rab27 is present on squid synaptic vesicles. Electrophysiological study of injected synapses showed that the anti-sqRab27 antibody inhibited synaptic release in a stimulation-dependent manner without affecting presynaptic action potentials or inward Ca2+ current. This result was confirmed in in vitro synaptosomes by using total internal reflection fluorescence microscopy. Thus, synaptosomal Ca2+-stimulated release of FM1-43 dye was greatly impaired by intraterminal anti-sqRab27 antibody. Ultrastructural analysis of the injected giant preterminal further showed a reduced number of docked synaptic vesicles and an increase in nondocked vesicular profiles distant from the active zone. These results, taken together, indicate that Rab27 is primarily involved in the maturation of recycled vesicles and/or their transport to the presynaptic active zone in the squid giant synapse.

Single Synapse Information Coding in Intraburst Spike Patterns of Central Pattern Generator Motor Neurons

Burst firing is ubiquitous in nervous systems and has been intensively studied in central pattern generators (CPGs). Previous works have described subtle intraburst spike patterns (IBSPs) that, despite being traditionally neglected for their lack of relation to CPG motor function, were shown to be cell-type specific and sensitive to CPG connectivity. Here we address this matter by investigating how a bursting motor neuron expresses information about other neurons in the network. We performed experiments on the crustacean stomatogastric pyloric CPG, both in control conditions and interacting in real-time with computer model neurons. The sensitivity of postsynaptic to presynaptic IBSPs was inferred by computing their average mutual information along each neuron burst. We found that details of input patterns are nonlinearly and inhomogeneously coded through a single synapse into the fine IBSPs structure of the postsynaptic neuron following burst. In this way, motor neurons are able to use different time scales to convey two types of information simultaneously: muscle contraction (related to bursting rhythm) and the behavior of other CPG neurons (at a much shorter timescale by using IBSPs as information carriers). Moreover, the analysis revealed that the coding mechanism described takes part in a previously unsuspected information pathway from a CPG motor neuron to a nerve that projects to sensory brain areas, thus providing evidence of the general physiological role of information coding through IBSPs in the regulation of neuronal firing patterns in remote circuits by the CNS.

Gas-Phase Nucleophilic and Elimination Reactions in Simple Alkyl Nitrates

There has been increasing interest in the gas-phase reactivity of alkyl nitrates because of their well-known applications as explosives and because of then role in atmospheric and in marine processes This manuscript describes an experimental study by FT-ICR techniques of the gas-phase reactions of OH(-) and F(-) with methyl and ethyl Innate For methyl nitrate, the main reaction channel is found to be an elimination process promoted by abstraction of an a proton from the methyl group. Nucleophilic displacement of nitrate anion through an S(N)2 process at the carbon center Is also found to he an important reaction channel with methyl nitrate In ethyl nitrate, Ruination of NO(3)(-) is greatly enhanced and this is attributed to the ease of an E2-type elimination process promoted by proton abstraction at the beta position of the ethyl group. Theoretical calculations at the MP2/6-311+G(3df,2p)//MP2/6-31+G(d) level of theory ale consistent with the relative importance of the reaction channels and suggest that these reactions proceed through a double well potential The calculations also predict that nucleophilic attack by OH(-) at the nitrogen center (Sn2@N) is energetically the rueful ad pathway but experiments with (18)OH(-) showed no evidence for this channel. Single-point calculations reveal a strong preference for approach to the emboli center and may explain the lack of reactivity at the nitrogen center. Calculations were also carried out or NH(2)(-) and SH(-) to establish the reactivity pattern to provide a better understanding of environmentally relevant nitrate esters.

Could leprosy reaction episodes be exacerbated by oral infections?

INTRODUCTION: This study evaluated whether leprosy reactions could be associated with oral infection. METHODS: Leprosy patients (n = 38) with (Group I) and without (Group II) oral infections were selected. Reactions were identified from the clinical and histopathological features associated with serum C-reactive protein (CRP) and10kDa interferon-gamma-induced protein (IP-10) levels, determined before and after elimination of the foci of infection. RESULTS: Group I presented more reactions than group II did, and improvement of the reactions after dental treatment. Serum CRP and IP-10 did not differ before and after the dental treatment, but differed between the groups. CONCLUSIONS: Oral infection could be an exacerbating factor in leprosy reactions.

The recurrence of leprosy reactional episodes could be associated with oral chronic infections and expression of serum IL-1, TNF-α, IL-6, IFN-γ and IL-10

The aim of this study was to determine whether the presence of leprosy reactional episodes could be associated with chronic oral infection. Thirty-eight leprosy patients were selected and divided into 2 groups: group I - 19 leprosy patients with oral infections, and group II - 19 leprosy patients without oral infections. Ten patients without leprosy, but presenting oral infections, were assigned to the control group. Leprosy patients were classified according to Ridley and Jopling classification and reactional episodes of the erythema nodosum type or reversal reaction were identified by clinical and histopathological features associated with serum IL-1, TNF-α, IL-6, IFN-γ and IL-10 levels. These analyses were performed immediately before and 7 days after the oral infection elimination. Patients from group I presenting oral infections reported clinical improvement of the symptoms of reactional episodes after dental treatment. Serum IL-1, TNF-α, IL-6, IFN-γ and IL-10 levels did not differ significantly before and after dental treatment as determined by the Wilcoxon test (p>0.05). Comparison of the 2 groups showed statistically significant differences in IL-1 and IL-6 at baseline and in IL-1, IL-6 and IL-10 on the occasion of both collections 7 days after therapy. Serum IL-6 and IL-10 levels in group I differed significantly at baseline compared to control (Mann-Whitney test; p<0.05). These results suggest that oral infection could be involved as a maintenance factor in the pathogenesis of leprosy reactional episodes.

Immediate implants placed into infected sockets: a case report with 3-year follow-up

The esthetics and functional integrity of the periodontal tissue may be compromised by dental loss. Immediate implants became a viable option to maintain the periodontal architecture because of their anatomic compatibility with the dental socket and the possibility of eliminating local contamination. This article describes the procedure of immediate implant placement in the anterior maxilla replacing teeth with chronic periapical lesions, which were condemned due to endodontic lesions persisting after failed endodontic treatment and endodontic surgery, and discusses the relationship between the procedure and periapical lesions. Surgical removal of hopeless teeth 11, 12 and 21 was performed conservatively in such a way to preserve the anatomy and gingival esthetics. A second surgical access was gained at the apical level, allowing the debridement of the surgical chamber for elimination of the periapical lesion, visual orientation for setting of the implants and filling of the surgical chamber with xenogenous bovine bone graft. After this procedure, the bone chamber was covered with an absorbent membrane and the healing screws were positioned on the implants. Later, a provisional partial removable denture was installed and the implants were inserted after 6 months. After 3 years of rehabilitation, the implants present satisfactory functional and esthetic conditions, suggesting that immediate implant placement combined with guided bone regeneration may be indicated for replacing teeth lost due to chronic periapical lesions with endodontic failure history in the anterior maxilla.

Osteonecrose dos maxilares associada ao uso de bisfosfonatos: importante complicação do tratamento oncológico

Os bisfosfonatos são um grupo de medicamentos utilizados no tratamento de doenças malignas metastáticas e em outras doenças ósseas como osteoporose e doença de Paget. A despeito dos seus benefícios, uma importante complicação denominada de osteonecrose dos maxilares vem sendo observada nos pacientes usuários crônicos dos bisfosfonatos que se caracteriza clinicamente por exposições ósseas na região maxilofacial persistente, acompanhadas de osteomielite, geralmente sintomáticas e cujo tratamento é complexo. Este estudo tem por objetivo revisar a literatura sobre a osteonecrose associada ao uso dos bisfosfonatos, em especial, em oncologia, no período de 2003 a 2008. Serão apresentados e discutidos os fatores de risco, aspectos etiopatogênicos, clínicos, imagenológicos, terapêuticos e preventivos desta doença. Devido à dificuldade de tratamento da osteonecrose associada aos bisfosfonatos, o foco deve ser a prevenção, sendo o ideal a eliminação de quadros infecciosos orais antes da terapia com os bisfosfonatos ter sido iniciada e minimizar traumas em boca após o uso destes medicamentos.

A convenient method for lecithin purification from fresh eggs

The increasing demand for fatty acid-free lecithin required modifications in existing purification methods. In this technical note we describe a purification procedure with the following steps: a) homogenization and extraction of yolks obtained from fresh eggs with acetone, b) solubilization with ethanol and solvent elimination and c) repeated solubilization/precipitation with petroleum ether/acetone. This crude extract was chromatographed on neutral alumina, which was exhaustively washed with chloroform before elution with chloroform:methanol, allowing the sequential separation of fatty acids and lecithin. Chromatographic behavior and mass spectra of the product are presented. This fast procedure yields fatty acid-free lecithin at a competitive cost.

The effects of the experimental infection from a focus of transmission of Schistosoma mansoni in a population of Biomphalaria tenagophila (D'Orbigny, 1835) in the region of "Vale do Ribeira de Iguape", Brazil

To observe the effects of the parasitic infection on the biology of B. tenagophila, field and laboratory populations of this mollusk from Itariri, in Vale do Ribeira, Brazil, were experimentally infected. Each mollusk received 10 miracidia of Schistosoma mansoni (SJ lineage) and was observed throughout the parasite's development. The biological variables were compared according to the criteria "group" and "infectious phase". The main damage caused by the parasitic infection manifested itself in reproduction, longevity and lesions on the shell of the mollusks in the patent phase. An infection rate of 58.8% was observed. Microanatomical study of the mollusk's digestive gland and ovotestis revealed the presence of evolving larval forms and cercariae. It was concluded that the effects of the parasitic infection on both populations were moderate, despite the low survival rate of the infected mollusks, the damage did not prevent either reproduction or the elimination of cercariae, which continued for a long time.

Hipersensibilidade alimentar em cães

Realizou-se um estudo retro e prospectivo em 117 cães com prontuários suspeitos de apresentarem hipersensibilidade alimentar. Os animais foram distribuídos em dois grupos: os do grupo I (n=86) foram atendidos em 1993 e 1994 e os do grupo II (n=31) em 1995. Os cães de ambos os grupos foram caracterizados quanto aos aspectos epidemiológicos e clínicos. Os do grupo II foram submetidos a exames complementares para a diferenciação diagnóstica do prurido, incluindo: hemograma, micológico e parasitológico cutâneo, coproparasitológico, histológico de pele e sorológicos - RAST (radioimunoensaio) e ELISA (ensaio imunoenzimático) - ambos para determinação de IgE contra antígenos alimentares -, e ao exame da dieta de eliminação seguida pela exposição provocativa. Este último exame foi o mais confiável para o estabelecimento do diagnóstico, ao determinar que 20 cães, provenientes de ambos os grupos, eram alérgicos a alimentos. Pelos RAST e ELISA, não foi possível demonstrar resultados confiáveis quando comparados aos resultados com a dieta de eliminação. Os animais acometidos foram principalmente machos, com raça definida e na faixa etária de um a seis anos. Os principais alimentos incriminados foram a carne bovina, o arroz e a carne de frango.