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1.
Sci Rep ; 14(1): 12876, 2024 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-38834612

RESUMO

This study investigates quercetin complexes as potential synergistic agents against the important respiratory pathogen Streptococcus pneumoniae. Six quercetin complexes (QCX1-6) were synthesized by reacting quercetin with various metal salts and boronic acids and characterized using FTIR spectroscopy. Their antibacterial activity alone and in synergism with antibiotics was evaluated against S. pneumoniae ATCC 49619 using disc diffusion screening, broth microdilution MIC determination, and checkerboard assays. Complexes QCX-3 and QCX-4 demonstrated synergy when combined with levofloxacin via fractional inhibitory concentration indices ≤ 0.5 as confirmed by time-kill kinetics. Molecular docking elucidated interactions of these combinations with virulence enzymes sortase A and sialidase. A biofilm inhibition assay found the synergistic combinations more potently reduced biofilm formation versus monotherapy. Additionally, gene-gene interaction networks, biological activity predictions and in-silico toxicity profiling provided insights into potential mechanisms of action and safety.


Assuntos
Antibacterianos , Biofilmes , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Quercetina , Streptococcus pneumoniae , Streptococcus pneumoniae/efeitos dos fármacos , Quercetina/farmacologia , Quercetina/química , Antibacterianos/farmacologia , Antibacterianos/química , Biofilmes/efeitos dos fármacos , Sinergismo Farmacológico , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/antagonistas & inibidores , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/química , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/metabolismo , Neuraminidase/antagonistas & inibidores , Neuraminidase/metabolismo
2.
Microb Pathog ; 189: 106603, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38417686

RESUMO

Isolation of novel bioactive metabolites from Streptomyces strains is a promising source for drug discovery. However, conventional screening approaches have limitations in identifying new leads due to redundant discoveries. Optimization of culture conditions is important but traditionally optimized one factor at a time, failing to consider interactions. This study addressed these gaps by enhancing metabolite production from Streptomyces thinghirensis WAE1 through statistical optimization. Various chemical and physical factors impacting metabolite production were identified. Response surface methodology with a central composite design was applied to optimize significant factors like carbon source, nitrogen source, inoculum size, pH, temperature and incubation period. This optimized production against Streptococcus pneumoniae, increasing antibacterial activity by 74.92%. Gas chromatography-mass spectrometry revealed 19 bioactive compounds, including 1,25-dihydroxyvitamin D3 inhibiting cell wall development. This highlights S. thinghirensis WAE1's potential as a bioresource and emphasizes studying metabolite production from novel Streptomyces strains to discover new antibacterial drugs.


Assuntos
Antibacterianos , Streptomyces , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Temperatura
3.
Environ Sci Pollut Res Int ; 30(53): 114610-114624, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37863854

RESUMO

Microalgae have the potential as a source of biofuels due to their high biomass productivity and ability to grow in a wide range of conditions, including wastewater. This study investigated cultivating two microalgae species, Oocystis pusilla and Chlorococcus infusionum, in wastewater for biodiesel production. Compared to Kühl medium, KC medium resulted in a significant fold increase in cellular dry weight production for both O. pusilla and C. infusionum, with an increase of 1.66 and 1.39, respectively. A concentration of 100% wastewater resulted in the highest growth for O. pusilla, with an increase in biomass and lipid content compared to the KC medium. C. infusionum could not survive in these conditions. For further increase in biomass and lipid yield of O. pusilla, different total dissolved solids (TDS) levels were used. Maximum biomass and lipid productivities were achieved at 3000 ppm TDS, resulting in a 28% increase in biomass (2.50 g/L) and a 158% increase in lipid yield (536.88 mg/g) compared to KC medium. The fatty acid profile of O. pusilla cultivated on aerated wastewater at 3000 ppm TDS showed a high proportion of desirable saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) for biodiesel production. Cultivating microalgae in wastewater for biodiesel production can be cost-effective, especially for microalgae adapted to harsh conditions. It could be concluded that O. pusilla is a promising candidate for biodiesel production using wastewater as a growth medium, as it has high biomass productivity and lipid yield, and its fatty acid profile meets the standard values of American and European biodiesel standards. This approach offers a sustainable and environmentally friendly solution for producing biofuels while reducing the environmental impact of wastewater disposal.


Assuntos
Microalgas , Águas Residuárias , Biocombustíveis , Ácidos Graxos , Estresse Salino , Biomassa
4.
J Fungi (Basel) ; 8(11)2022 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-36354915

RESUMO

Chaetomiaceae fungi are ascosporulating fungi whose importance as human pathogens has been frequently ignored. In the current study, a new isolate of the genus Subramaniula was described. The fungus was isolated from the soil of Wadi Om Nefa'a, Hurghada in the Red Sea Governorate, Egypt. Previously, Subramaniula were misidentified as Papulaspora spp. According to molecular analysis, the fungus was identified as Subramaniula asteroids OP484336. Remarkably, this species has been found among other fungi responsible for keratitis in humans and has been recorded for the first time in Egypt. Analysing the Subramaniula asteroids' metabolic profile was one of the objectives of the current study because little is known about this family's metabolome. The fungal extract's untargeted metabolic profiling was carried out by gas chromatography-mass spectroscopy (GC/MS), 1H and 1H-HSQC nuclear magnetic resonance (NMR) data, and their corresponding databases. In total, fifty-nine metabolites have been reported in the polar and non-polar extracts. The majority of polar metabolites are amino acids and carbohydrates. The non-polar extract's main components were 1-dodecanamine, N,N-dimethyl-, 1-tetradecanamine, N,N-dimethyl-, and 9-octadecenoic acid ethyl ester. The current study is the first to provide a metabolic profile of Subramaniula asteroids, which can be used in chemotaxonomical classification, antifungal drug development, and biological activity investigation of the studied species.

5.
ACS Omega ; 7(24): 21346-21356, 2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35755350

RESUMO

In the present study, a series of K-modified CoMoS catalysts with compositions of 10% K, 3.6% Co, and 12 wt % Mo supported over novel commercial activated carbons such as powder materials (DAC and OBC-1) and fiber materials (fabric active sorption (TCA) and nonwoven activated material (AHM)) were prepared and characterized by Brunauer-Emmett-Teller (BET), X-ray fluorescence (XRF), scanning electron microscopy (SEM), SEM-energy dispersive X-ray (EDX), and transmission electron microscopy (TEM). The catalytic activities for higher alcohol synthesis from syngas, conducted at T = 300-360 °C, P = 5 MPa, GHSV = 760 L h-1 (kg cat)-1, and H2/CO = 1.0, were investigated. Cat-TCA and Cat-AHM have shown a filamentous morphology with a strip axial arrangement and that a few longitudinal grooves and many irregular particles are distributed on the fiber surfaces. The degree of entanglement of the strip axial arrangement in AHM was found to be more than that in TCA, thus leading to form tangled MoS2 slabs on AHM and long linear slabs on TCA with long rim sites. The obtained results revealed that the CO conversion increases in the order Cat-TCA < Cat-OBC-1 < Cat-DAC < Cat-AHM. Ethanol, propanol-1, and methanol are the most predominant alcohol products in the collected liquid products, with the byproducts containing mainly butanol-1, isobutanol, amyl alcohol, and isoamyl alcohol. Cat-DAC and Cat-OBC-1 show higher selectivity toward C3+, C4+, propanol-1, butanol-1, isobutanol, and amyl alcohol-1 than Cat-TCA and Cat-AHM. For powdered activated carbons, microporous catalysts inhibited isomerization because the catalyst that contains the highest micropores (Cat-DAC) produced a considerable amount of linear alcohols compared with Cat-OBC-1.

6.
J Genet Eng Biotechnol ; 20(1): 32, 2022 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-35190920

RESUMO

BACKGROUND: The increased demand for oil and fats to satisfy the ever-increasing human needs has enhanced the research in this field. Single-cell oils or microbial lipids produced by oleaginous microorganisms are being utilized as an alternative to traditional oil sources. Oleaginous yeasts can accumulate lipids above 20% of their biomass when they are grown under controlled conditions. RESULTS: In the present study, sixty-five yeasts were isolated from different sources. Using Sudan Black B staining technique, five yeast isolates were selected. Under nitrogen-limited cultivation conditions, the Co1 isolate was the best lipid accumulation potential of 39.79%. Isolate (Co1) was characterized morphologically and identified using the ribosomal DNA internal transcribed spacers regions (rDNA-ITS) from their genomic DNA. The sequence alignment revealed a 99.2% similarity with Rhodotorula diobovata. Under the optimized conditions, Rhodotorula diobovata accumulated lipids up to 45.85% on a dry biomass basis. R. diobovata, when grown on different raw materials, accumulated lipid up to 46.68% on sugar beet molasses medium, and the lipid had a high degree of monounsaturated fatty acids which gives biodiesel better quality. CONCLUSIONS: The data suggest that the potent oleaginous yeast, R. diobovata, together with the use of cheap feedstock raw materials such as sugar beet molasses, can be considered as a promising feedstock for biodiesel production.

7.
Sci Total Environ ; 792: 148359, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34147795

RESUMO

The high demand for sufficient and safe food, and continuous damage of environment by conventional agriculture are major challenges facing the globe. The necessity of smart alternatives and more sustainable practices in food production is crucial to confront the steady increase in human population and careless depletion of global resources. Nanotechnology implementation in agriculture offers smart delivery systems of nutrients, pesticides, and genetic materials for enhanced soil fertility and protection, along with improved traits for better stress tolerance. Additionally, nano-based sensors are the ideal approach towards precision farming for monitoring all factors that impact on agricultural productivity. Furthermore, nanotechnology can play a significant role in post-harvest food processing and packaging to reduce food contamination and wastage. In this review, nanotechnology applications in the agriculture and food sector are reviewed. Implementations of nanotechnology in agriculture have included nano- remediation of wastewater for land irrigation, nanofertilizers, nanopesticides, and nanosensors, while the beneficial effects of nanomaterials (NMs) in promoting genetic traits, germination, and stress tolerance of plants are discussed. Furthermore, the article highlights the efficiency of nanoparticles (NPs) and nanozymes in food processing and packaging. To this end, the potential risks and impacts of NMs on soil, plants, and human tissues and organs are emphasized in order to unravel the complex bio-nano interactions. Finally, the strengths, weaknesses, opportunities, and threats of nanotechnology are evaluated and discussed to provide a broad and clear view of the nanotechnology potentials, as well as future directions for nano-based agri-food applications towards sustainability.


Assuntos
Nanoestruturas , Praguicidas , Agricultura , Indústria Alimentícia , Humanos , Nanotecnologia
8.
Environ Sci Pollut Res Int ; 27(26): 32481-32493, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32506400

RESUMO

The present study evaluated the sequential biodiesel-bioethanol production from seaweeds. A total of 22 macroalgal species were collected at different seasons and screened based on lipid and carbohydrate contents as well as biomass production. The promising species was selected, based on the relative increase in energy compounds (REEC, %), for further energy conversion. Seasonal and annual biomass yields of the studied species showed significant variations. The rhodophyte Amphiroa compressa and the chlorophyte Ulva intestinalis showed the highest annual biomass yield of 75.2 and 61.5 g m-2 year-1, respectively. However, the highest annual carbohydrate productivity (ACP) and annual lipid productivity (ALP) were recorded for Ulva fasciata and Ulva intestinalis (17.0 and 3.0 g m-2 year-1, respectively). The later was selected for further studies because it showed 14.8% higher REEC value than Ulva fasciata. Saturated fatty acids (SAFs) showed 73.4%, with palmitic acid as a dominant fatty acid (43.8%). Therefore, biodiesel showed high saturation degree, with average degree of unsaturation (ADU) of 0.508. All the measured biodiesel characteristics complied the international standards. The first route of biodiesel production (R1) from Ulva intestinalis showed biodiesel recovery of 32.3 mg g-1 dw. The hydrolysate obtained after saccharification of the whole biomass (R2) and lipid-free biomass (R3) contained 1.22 and 1.15 g L-1, respectively, reducing sugars. However, bioethanol yield from R3 was 0.081 g g-1 dw, which represented 14.1% higher than that of R2. Therefore, application of sequential biofuel production using R3 resulted in gross energy output of 3.44 GJ ton-1 dw, which was 170.9% and 82.0% higher than R1 and R2, respectively. The present study recommended the naturally-grown Ulva intestinalis as a potential feedstock for enhanced energy recovery through sequential biodiesel-bioethanol production.


Assuntos
Alga Marinha , Ulva , Biocombustíveis , Biomassa , Ácidos Graxos
10.
J Eval Clin Pract ; 24(1): 206-211, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29285849

RESUMO

RATIONALE, AIMS, AND OBJECTIVES: Implementation of clinical practice guidelines (CPGs) has been shown to reduce variation in practice and improve health care quality and patients' safety. There is a limited experience of CPG implementation (CPGI) in the Middle East. The CPG program in our institution was launched in 2009. The Quality Management department conducted a Failure Mode and Effect Analysis (FMEA) for further improvement of CPGI. METHODS: This is a prospective study of a qualitative/quantitative design. Our FMEA included (1) process review and recording of the steps and activities of CPGI; (2) hazard analysis by recording activity-related failure modes and their effects, identification of actions required, assigned severity, occurrence, and detection scores for each failure mode and calculated the risk priority number (RPN) by using an online interactive FMEA tool; (3) planning: RPNs were prioritized, recommendations, and further planning for new interventions were identified; and (4) monitoring: after reduction or elimination of the failure mode. The calculated RPN will be compared with subsequent analysis in post-implementation phase. RESULTS: The data were scrutinized from a feedback of quality team members using a FMEA framework to enhance the implementation of 29 adapted CPGs. The identified potential common failure modes with the highest RPN (≥ 80) included awareness/training activities, accessibility of CPGs, fewer advocates from clinical champions, and CPGs auditing. Actions included (1) organizing regular awareness activities, (2) making CPGs printed and electronic copies accessible, (3) encouraging senior practitioners to get involved in CPGI, and (4) enhancing CPGs auditing as part of the quality sustainability plan. CONCLUSION: In our experience, FMEA could be a useful tool to enhance CPGI. It helped us to identify potential barriers and prepare relevant solutions.


Assuntos
Fidelidade a Diretrizes/normas , Análise do Modo e do Efeito de Falhas na Assistência à Saúde/métodos , Padrões de Prática Médica/normas , Gestão de Riscos/organização & administração , Humanos , Guias de Prática Clínica como Assunto , Estudos Prospectivos , Melhoria de Qualidade , Arábia Saudita
11.
Mycologia ; 108(4): 646-56, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27091386

RESUMO

Lichen secondary metabolites (polyketides) are produced by the fungal partner, but the role of algal carbohydrates in polyketide biosynthesis is not clear. This study examined whether the type and concentration of algal carbohydrate explained differences in polyketide production and gene transcription by a lichen fungus (Cladonia rangiferina). The carbohydrates identified from a free-living cyanobacterium (Spirulina platensis; glucose), a lichen-forming alga (Diplosphaera chodatii; sorbitol) and the lichen alga that associates with C. rangiferina (Asterochloris sp.; ribitol) were used in each of 1%, 5% and 10% concentrations to enrich malt yeast extract media for culturing the mycobiont. Polyketides were determined by high performance liquid chromatography (HPLC), and polyketide synthase (PKS) gene transcription was measured by quantitative PCR of the ketosynthase domain of four PKS genes. The lower concentrations of carbohydrates induced the PKS gene expression where ribitol up-regulated CrPKS1 and CrPKS16 gene transcription and sorbitol up-regulated CrPKS3 and CrPKS7 gene transcription. The HPLC results revealed that lower concentrations of carbon sources increased polyketide production for three carbohydrates. One polyketide from the natural lichen thallus (fumarprotocetraric acid) also was produced by the fungal culture in ribitol supplemented media only. This study provides a better understanding of the role of the type and concentration of the carbon source in fungal polyketide biosynthesis in the lichen Cladonia rangiferina.


Assuntos
Ascomicetos/metabolismo , Metabolismo dos Carboidratos , Carboidratos/análise , Policetídeos/metabolismo , Ascomicetos/crescimento & desenvolvimento , Carbono/metabolismo , Clorófitas/química , Cromatografia Líquida de Alta Pressão , Meios de Cultura/química , Perfilação da Expressão Gênica , Líquens/microbiologia , Policetídeo Sintases/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Spirulina/química
12.
Sudan J Paediatr ; 12(2): 60-4, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-27493347

RESUMO

Immune thrombocytopenia (ITP) is an acquired hematological disease in which the body produces antibodies against its own platelets leading to platelet destruction resulting in isolated thrombocytopenia. Childhood ITP may enter complete remission in the majority of cases within six months from diagnosis. However, 20-30% of affected children may develop chronic ITP (lasting for more than 12 months). First line treatment includes intravenous immunoglobulin (IVIG), corticosteroids or anti-D immunoglobulin. Second line treatment includes splenectomy, immunosuppressive therapy or Rituximab. Recently two thrombopoietin (TPO) receptor agonists (Romiplostim and Eltrombopag) are used to increase platelet count in refractory chronic ITP by increasing platelet production in bone marrow. Here is a case report on an 8 ½ -year-old boy with refractory chronic ITP who failed therapy with IVIG, corticosteroids, splenectomy and Rituximab. He showed excellent response to treatment with TPO receptor agonist (Romiplostim). His platelet count increased from less than 10 x10(3)/dl and maintained between 100x10(3)/dl to 200x10(3)/dl after few weeks of starting Romiplostim therapy.

13.
Environ Toxicol Pharmacol ; 21(1): 42-50, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21783637

RESUMO

In this investigation, the cyanobacterium Nostoc muscorum exhibited antagonistic activity against Gram-positive and Gram-negative bacteria and filamentous fungi. The results indicated that the active substance produced maximally after 12 days of incubation in shaken culture at 35°C, at pH 8.0 in BG-11 medium. The increase in nitrate concentration of the medium led to an increase in the antimicrobial production. Chloroform was the best solvent for extracting the active material. The antagonistic material was purified using thin layer chromatography. The compound showed maximum absorption at 240nm. Infrared (IR) and nuclear magnetic resonance (NMR) indicated presence of γOH, γCH aromatic, γCH aliphatic, γCN, γCO, γCC and CO. Mass spectroscopy indicated that its molecular weight is 279. The results also indicated that the compound is phenolic compound.

14.
Microbiol Res ; 160(4): 343-52, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16255138

RESUMO

Xanthomonas campestris pv. vesicatoria strain 2 was isolated from infected tomato seedlings grown in open field in Egypt. This strain produced irregular yellow-necrotic areas on tomato leaves and spotting of the stem. In an attempt to control this disease biologically, four experiments were conducted and tomato seedlings were pretreated, before the pathogen, with either of two antagonistic strains of Rahnella aquatilis through leaves, roots, soil or seeds. In all experiments, seedlings pretreated with R. aquatilis showed reduced susceptibility toward X. c. pv. vesicatoria. They also contained reduced protein concentration and showed reduced number of protein bands in SDS-PAGE analysis as well as increased fresh and dry weight relative to control seedlings inoculated with the pathogen only. This indicates that R. aquatilis reduced the deleterious effect and the stress exerted by X. c. pv. vesicatoria on tomato seedlings. Foliar application of R. aquatilis was the most effective method in disease reduction which could be attributed to the direct effect of the antagonistic bacteria on the pathogen. The highest amounts of fresh and dry weight were obtained from seed treatment, which might suggest that bacterial seed inoculation provides earlier protection than could be achieved with foliar, soil or root treatment.


Assuntos
Antibiose , Controle Biológico de Vetores/métodos , Doenças das Plantas/microbiologia , Rahnella/crescimento & desenvolvimento , Solanum lycopersicum/microbiologia , Xanthomonas campestris/crescimento & desenvolvimento , Biomassa , Eletroforese em Gel de Poliacrilamida , Folhas de Planta/microbiologia , Proteínas de Plantas/análise , Proteínas de Plantas/isolamento & purificação , Raízes de Plantas/microbiologia , Caules de Planta/microbiologia , Rahnella/metabolismo , Sementes/microbiologia , Xanthomonas campestris/metabolismo
15.
Environ Toxicol Pharmacol ; 19(2): 357-65, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21783496

RESUMO

The growth of Nostoc muscorum and Anabaena subcylindrica in sterilized sewage wastewater and N. muscorum in sterilized wastewater of El-Soda Company was higher than those grown in Allen synthetic medium. Whereas, the growth of A. subcylindrica in El-Soda Company sterilized wastewater and N. muscorum as well as A. subcylindrica grown in Verta Company sterilized wastewater was slightly lower than that grown in the standard synthetic medium. The contents of chlorophyll a, carotenoids and protein of N. muscorum and A. subcylindrica grown in sterilized sewage wastewater were higher than those grown in the standard medium. Similarly, N. muscorum and the bio-mixture of N. muscorum and A. subcylindrica grown in the sterilized wastewater of El-Soda Company showed high pigments and protein contents more than those reared in Allen medium. On the other hand, the bio-mixture of N. muscorum and A. subcylindrica grown in the sterilized sewage wastewater, A. subcylindrica grown in El-Soda Company and Verta Company sterilized wastewater showed lower contents of pigments and protein compared to synthetic medium. Heavy metals, copper, cobalt, lead and manganese were removed by 12.5-81.8, 11.8-33.7, 26.4-100 and 32.7-100%, respectively, from wastewater by using cyanobacterial cultures. The metal sorption efficiency depended on the type of biosorbent, the physiological state of the cells, availability of heavy metal, concentration of heavy metal and chemical composition of wastewater. It was observed also that the single cultures in most cases was better than the mixed cultures in heavy metal removal, this may be due to the cyanobacterial competition for nutrients in mixed cultures.

16.
Environ Toxicol Pharmacol ; 16(3): 169-78, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21782704

RESUMO

Growth, morphological changes, amino acid composition, total soluble protein, and protein electrophoretic pattern were monitored for Scenedesmus obliquus and Nitzschia perminuta grown in the presence of different concentrations of Co(2+) and Ni(2+). Lower concentrations of cobalt stimulated the dry mass production and total soluble protein content of the two algae, whereas higher concentrations were inhibitory. Generally, N. perminuta showed more tolerance to the phytotoxicity of the two metals than S. obliquus and more tolerance to nickel than cobalt. However, S. obliquus seems to be more tolerant to cobalt than nickel. Cobalt and nickel have induced an increase in cell volume, change and disorder in cell shape. The increase in cell volume was much observed in Ni(2+) treated cells. At the same time, the two metals did not induce any distinct morphological abnormalities in N. perminuta. Co(2+) has stimulated the biosynthesis of all free amino acids in S. obliquus, except aspartic acid and phenylalanine, whereas Ni(2+) caused 22% inhibition in the content of total free amino acids, except cystine and arginine. On the other hand, Co(2+) has reduced the content of free amino acids in N. perminuta, except cystine, methionine, valine, and lysine. On the other hand, Ni(2+) stimulated the biosynthesis of glycine, alanine and histidine and highly stimulated valine and sulphur containing amino acids (cystine and methionine) in N. perminuta. High cobalt concentration (4ppm) resulted in the disappearance of 28.7kDa protein, 3.5ppm Ni(2+) stimulated the appearance of 18 and 20kDa proteins in S. Obliquus, while 37kDa proteins disappeared from N. perminuta treated with high doses of Co(2+) and Ni(2+).

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