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1.
J Chem Phys ; 141(12): 124505, 2014 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-25273449

RESUMO

Using the Rogers-Young (RY) integral equation scheme for the static pair correlation functions combined with the liquid-phase Hansen-Verlet freezing rule, we study the generic behavior of the radial distribution function and static structure factor of monodisperse charge-stabilized suspensions with Yukawa-type repulsive particle interactions at freezing. In a related article, labeled Paper I [J. Gapinski, G. Nägele, and A. Patkowski, J. Chem. Phys. 136, 024507 (2012)], this hybrid method was used to determine two-parameter freezing lines for experimentally controllable parameters, characteristic of suspensions of charged silica spheres in dimethylformamide. A universal scaling of the RY radial distribution function maximum is shown to apply to the liquid-bcc and liquid-fcc segments of the universal freezing line. A thorough analysis is made of the behavior of characteristic distances and wavenumbers, next-neighbor particle coordination numbers, osmotic compressibility factor, and the Ravaché-Mountain-Streett minimum-maximum radial distribution function ratio.


Assuntos
Coloides/química , Congelamento , Algoritmos , Modelos Químicos , Tamanho da Partícula
2.
J Vis Exp ; (85)2014 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-24686247

RESUMO

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1(nu)). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.


Assuntos
Técnicas Bacteriológicas/métodos , Mycobacterium leprae/citologia , Animais , Modelos Animais de Doenças , Congelamento , Hanseníase/microbiologia , Camundongos , Camundongos Nus , Mycobacterium leprae/crescimento & desenvolvimento , Mycobacterium leprae/isolamento & purificação , Suspensões
3.
s.l; s.n; 2014. 7 p. ilus, tab.
Não convencional em Inglês | SES-SP, HANSEN, SESSP-ILSLPROD, SES-SP, SESSP-ILSLACERVO, SES-SP | ID: biblio-1095887

RESUMO

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1(nu)). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.


Assuntos
Animais , Camundongos , Suspensões , Técnicas Bacteriológicas/métodos , Modelos Animais de Doenças , Congelamento , Hanseníase/microbiologia , Camundongos , Camundongos Nus , Mycobacterium leprae/isolamento & purificação , Mycobacterium leprae/citologia , Mycobacterium leprae/crescimento & desenvolvimento
4.
J Chem Phys ; 136(2): 024507, 2012 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-22260603

RESUMO

Using the Rogers-Young (RY) integral equation scheme for the static structure factor combined with the one-phase Hansen-Verlet (HV) freezing rule, we study the equilibrium structure and two-parameter freezing lines of colloidal particles with Yukawa-type pair interactions representing charge-stabilized silica spheres suspended in dimethylformamide (DMF). Results are presented for a vast range of concentrations, salinities and effective charges covering particles with masked excluded-volume interactions. The freezing lines were obtained for the low-charge and high-charge solutions of the static structure factor, for various two-parameter sets of experimentally accessible system parameters. All RY-HV based freezing lines can be mapped on a universal fluid-solid coexistence line in good agreement with computer simulation predictions. The RY-HV calculations extend the freezing lines obtained in earlier simulations to a broader parameter range. The experimentally observed fluid-bcc-fluid reentrant transition of charged silica spheres in DMF can be explained using the freezing lines obtained in this work.


Assuntos
Algoritmos , Dimetilformamida/química , Congelamento , Dióxido de Silício/química , Coloides/química , Simulação por Computador
5.
Int J Lepr Other Mycobact Dis ; 69(1): 1-12, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11480310

RESUMO

Mycobacterium leprae remain a rare research resource. They cannot be cultivated on artificial media, and the only established means to quantify viability of M. leprae has been by its relative growth in the foot pads of conventional mice (MFP). The MFP method is technically difficult and requires several months to yield results. More effective methods are needed. We examined the association between M. leprae's ability to oxidize 14C-palmitate in axenic culture and the MFP growth results of a large number of suspensions. Oxidative activity was assessed by radiorespirometry (RR) using the Buddemeyer-type biphasic culture vessels containing 7H12 liquid medium and 14C-palmitate, or with commercially prepared BACTEC 12B vessels containing the same medium. The RR results were highly correlated (r = 0.71) with the growth level that each M. leprae suspension achieved by the MFP technique. In using this technique to examine the effects that many common laboratory practices have on M. leprae viability, we found that viability varies markedly between bacillary suspensions derived from different hosts and tissues. The highest viabilities were obtained with bacilli from moderately enlarged nude MFP (< 1 g). Viability tended to be lower among very large nude MFP or long-duration infections and from armadillo tissues. After their harvest from host tissues, leprosy bacilli lost viability quickly. Suspensions stored in 7H12 liquid medium retained < 1% of their viability within 3 weeks of harvest, and freezing bacillary preparations or incubating them at 37 degrees C resulted in nearly an immediate equivalent loss in metabolic activity and viability. M. leprae viability is maintained best when bacilli are stored for only short periods of time at 4 degrees C-33 degrees C. Palmitate oxidation is a rapid, reliable and objective means by which to estimate the viability of M. leprae and can be used effectively as a surrogate for the conventional MFP technique in many studies.


Assuntos
Tatus/microbiologia , Mycobacterium leprae/metabolismo , Palmitatos/metabolismo , Animais , Radioisótopos de Carbono , Meios de Cultura , Modelos Animais de Doenças , Congelamento , Humanos , Hanseníase/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mycobacterium leprae/crescimento & desenvolvimento , Contagem de Cintilação , Temperatura , Fatores de Tempo
6.
Int J Lepr Other Mycobact Dis ; 60(2): 234-43, 1992 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1381740

RESUMO

The sensitivity of the polymerase chain reaction (PCR) on the DNA coding for the species-specific fragment of 16S rRNA of Mycobacterium leprae studied on mouse foot pad harvests and human skin biopsies varies widely between 1 and 3 x 10(4) organisms. This is probably the result of variations in the proportions of organisms with sufficiently intact DNA suitable for PCR. Preserving human skin biopsies for 3 weeks at an ambient temperature even after boiling for 6 minutes gives rise to a 10-fold decrease in sensitivity. Fixation of tissues in formol 10% or Lowy fixative or preserving in Dubos OAA broth is very harmful to the PCR, mainly due to the enhancement of an inhibitory effect on the PCR reaction. For preservation, the best choice at the moment seems to be alcohol 70%. Sample preparation of five cycles of freeze-boiling is simple and generally more efficient than proteinase K treatment and DNA extraction.


Assuntos
DNA Bacteriano/análise , DNA Ribossômico/análise , Hanseníase/microbiologia , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Animais , Sequência de Bases , Biópsia , Meios de Cultura , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Endopeptidase K , Congelamento , Temperatura Alta , Humanos , Camundongos , Dados de Sequência Molecular , Mycobacterium leprae/isolamento & purificação , Preservação Biológica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Ribossômico 16S/química , Sensibilidade e Especificidade , Serina Endopeptidases/metabolismo , Pele/microbiologia , Especificidade da Espécie
7.
Int J Lepr Other Mycobact Dis ; 57(1): 54-64, 1989 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2471759

RESUMO

In previous reports on the ultrastructure of Mycobacterium leprae, we described the occurrence of symmetric membranes in normal-looking bacilli from fresh or frozen samples primarily fixed with aldehydes. In those reports we admitted that such a symmetric profile, which is not found in the other normal mycobacteria, would not represent the structure of the normal membrane of the leprosy bacillus. We, therefore, re-analyzed the ultrastructure of the membrane of M. leprae. In the present work the micromorphology of the M. leprae membrane was studied by transmission electronmicroscopy after the fixation of fresh samples by OsO4 plus calcium followed by glutaraldehyde plus formaldehyde and calcium followed by uranyl acetate. The study of samples from two patients with lepromatous (LL) leprosy, three armadillos with natural leprosy, and one nude mouse with experimental leprosy showed that normal-looking bacilli present in lead-stained sections had asymmetric membranes with a thickness of 6.49 +/- 0.36 nm. These membranes showed periodic acid-Schiff (PAS)-positive components exclusively located in the outer half of the bilayer. We demonstrated that the symmetric profile of the M. leprae membrane described in our previous reports corresponds, as admitted in those reports, to an abnormal membrane structure. Such an abnormality was now found to result from the use of primary fixation with aldehydes or of samples stored frozen before fixation. These results indicate that, although ultrastructurally similar to that of the other mycobacteria, the membrane of M. leprae has a peculiar sensitivity to fixation by aldehydes. Such a characteristic, which was not found in M. lepraemurium, M. aurum, M. avium, and M. tuberculosis H37Ra, must reflect a unique membrane molecular structure, which is presently unknown.


Assuntos
Mycobacterium leprae/ultraestrutura , Animais , Tatus/microbiologia , Membrana Celular/ultraestrutura , Densitometria , Fixadores , Congelamento , Humanos , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Mycobacterium avium/ultraestrutura , Mycobacterium lepraemurium/ultraestrutura , Preservação Biológica , Especificidade da Espécie , Coloração e Rotulagem
8.
Int J Lepr Other Mycobact Dis ; 56(4): 580-7, 1988 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-3065421

RESUMO

The influence of different frequencies of freezing-thawing cycles on the viability of in vivo grown mycobacteria was investigated. Pieces of armadillo tissues naturally or experimentally infected with Mycobacterium leprae were analyzed. The viability of M. leprae was determined by mouse foot pad titration. The viability of cultivable mycobacteria, sometimes present in armadillo tissues, was determined by culture. Electron-microscopic studies were performed on fresh or frozen-thawed armadillo tissues with natural leprosy and on livers of C57BL/6 mice experimentally infected with M. avium or M. lepraemurium. We found that the percentage of viable M. leprae bacilli is identical for naturally infected and experimentally infected tissues, frozen and thawed once. When the tissues were subjected to a second freezing-thawing cycle, a considerable loss of viability was observed (65%-97%). A third freezing-thawing cycle was lethal for most of the M. leprae cells, and after four freezing-thawing cycles no viable bacilli were found. The cultivable mycobacteria present in some armadillo tissues were found to be more resistant than M. leprae to freezing-thawing since these mycobacteria could still be cultivated after four freezing-thawing cycles. The results of the electron-microscopy study support the conclusion that M. leprae is more sensitive to freezing-thawing than the cultivable mycobacteria and show that the cytoplasmic membrane appears to be the target for the lethal action of freezing-thawing on mycobacterial cells. These results emphasize the importance of avoiding repeated thawing and refreezing of M. leprae-infected tissues when viable M. leprae cells need to be studied.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Mycobacterium leprae/fisiologia , Animais , Tatus , Membrana Celular/ultraestrutura , Congelamento , Camundongos , Mycobacterium leprae/crescimento & desenvolvimento , Mycobacterium leprae/ultraestrutura
9.
Int J Lepr Other Mycobact Dis ; 54(3): 403-8, 1986 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-2427626

RESUMO

In a comparison of the estimation of Mycobacterium leprae viability by morphology and the fluorescent vital dyes FDA/EB and R123/EB, the latter techniques were more satisfactory using suspensions and slit-skin smears of M. leprae bacilli. Both FDA/EB and R123/EB seem to more accurately reflect viability after freeze/thaw cycles and heating, and are able to detect lower percentages of viable bacilli. In addition, the fluorescent vital dye techniques are both simple and less open to subjective interpretation than the conventional estimation of the morphological index.


Assuntos
Mycobacterium leprae/crescimento & desenvolvimento , Pele/microbiologia , Etídio , Fluoresceínas , Congelamento , Temperatura Alta , Humanos , Hanseníase/tratamento farmacológico , Hanseníase/microbiologia , Mycobacterium leprae/citologia , Rodamina 123 , Rodaminas , Coloração e Rotulagem
10.
Int J Lepr Other Mycobact Dis ; 54(1): 1-10, 1986 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-3519794

RESUMO

A buoyant density centrifugation procedure using Percoll was developed for the isolation and purification of Mycobacterium leprae from experimentally infected armadillo liver tissue. The method separates the bacteria from host adenosine triphosphate (ATP) and tissue debris and recovers 20-25% of the bacteria within 2-2 1/2 hours under controlled conditions. The mean ATP content (585 pg/10(6] of the purified bacteria was similar to cultivable bacteria. The organisms did not leak intracellular ATP when exposed to phosphate buffer. Temperature-dependent ATP synthesis was observed within minutes and could be inhibited by 2,4-dinitrophenol. Freeze-thawing M. leprae as purified suspensions in buffer damaged the organisms, resulting in decreased ATP levels and an accelerated loss of ATP upon incubation under defined conditions. In vitro treatment with the antileprosy drug clofazimine increased the rate of ATP decay directly proportional to drug concentration.


Assuntos
Trifosfato de Adenosina/análise , Tatus/microbiologia , Mycobacterium leprae/análise , /microbiologia , 2,4-Dinitrofenol , Trifosfato de Adenosina/metabolismo , Animais , Centrifugação com Gradiente de Concentração , Dinitrofenóis/farmacologia , Congelamento , Fígado/microbiologia , Mycobacterium leprae/crescimento & desenvolvimento , Mycobacterium leprae/isolamento & purificação , Mycobacterium leprae/metabolismo , Fosforilação Oxidativa
11.
Indian J Lepr ; 57(1): 97-106, 1985.
Artigo em Inglês | MEDLINE | ID: mdl-3928774

RESUMO

Cell free extracts of a fast growing mycobacterium (M. phlei) and a slow growing mycobacterium (M. tuberculosis H37Ra) were analysed for lactate dehydrogenase (LDH) isoenzymes under different experimental conditions. It was observed that growth of M. phlei when taken from Lowenstein Jensen (LJ) as well as Sauton's medium showed identical band but for (M. tuberculosis H37Ra the number of bands observed were less when grown on LJ-medium. There was no difference in LDH isoenzyme patterns when the mycobacteria were incubated at 30 degrees C and 37 degrees C and under different pH conditions (6.2-8.2). Actively growing cultures of both the species showed distinct LDH isoenzyme patterns whereas the activity and bands became indistinct in old cultures. The LDH bands from lyophilized growth studied resembled to those of fresh growth. The treatment of growth with 1M NaOH for one hour resulted in marked diminution of LDH activity. Sonication with wet growth weight of 0.5 gm per ml of distilled water was found to give clearer bands as compared to phosphate buffer. No loss of LDH isoenzymes activity was noticed after storing the extracts at -80 degrees C for one month, treating to 58 degrees C for one hour or freezing and thawing for 2 times whereas these isoenzymes were quite unstable at other storage temperatures. Increasing the staining time was found helpful in getting clearer bands when activity was low. It is concluded that the factors studied have important bearing on LDH isoenzyme patterns of mycobacteria and must be kept in mind while studying the LDH zymograms for any taxonomic identification of mycobacteria or for studying the metabolic role. These are important both for sensitivity and reproducibility of LDH zymograms.


Assuntos
L-Lactato Desidrogenase/análise , Mycobacterium/enzimologia , Meios de Cultura , Congelamento , Concentração de Íons de Hidrogênio , Isoenzimas , Mycobacterium phlei/enzimologia , Mycobacterium tuberculosis/enzimologia , Temperatura
12.
Lepr India ; 54(3): 461-70, 1982 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-6757579

RESUMO

The effects of freezing tissues from M. leprae-infected armadillos as well as biopsy specimens from leprosy patients have been studied. No loss of viability of M. leprae was observed using mouse foot pad technique. Similarly, using bioluminescent determinations of bacterial ATP, it was shown that these bacilli retain all their metabolic activity during such freezing and thawing process. The practical implications of these findings have been discussed.


Assuntos
Congelamento , Mycobacterium leprae/crescimento & desenvolvimento , Trifosfato de Adenosina/metabolismo , Animais , Tatus , Biópsia , Humanos , Fígado/microbiologia , Camundongos , Mycobacterium leprae/metabolismo , Manejo de Espécimes
13.
J Med Microbiol ; 12(1): 137-42, 1979 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-372533

RESUMO

The effects of rapid and slow rates of freezing in liquid nitrogen, storage in liquid nitrogen for 12 months, and the rate of subsequent thawing on the viability and growth of M. leprae in the mouse footpad were studied. Some loss of viability of M. leprae was detected, and this was found to be associated with the freezing process, rather than with storage or thawing. Slow freezing was less deleterious than quick freezing, with a loss of viability of 90% compared with 98%. The growth pattern of M. leprae was unaffected except for a delay in the appearance of growth caused by the loss of viability, though there was some evidence of an increased lag phase of one strain, possibly due to the repair of sub-lethally damaged organisms.


Assuntos
Congelamento , Mycobacterium leprae/fisiologia , Mycobacterium leprae/crescimento & desenvolvimento , Nitrogênio , Preservação Biológica/métodos , Fatores de Tempo
14.
Infect Immun ; 22(1): 87-93, 1978 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-365752

RESUMO

The protection provided to mice by vaccines administered intradermally was measured after footpad challenge with Mycobacterium leprae. The protection offered by M. leprae suspensions was not decreased when the vaccines were killed by 60 degrees C heat or at the higher temperatures tested, which included 215 degrees C (autoclave). Even highly purified suspensions retained their immunogenicity. In contrast, the vaccine protection provided by intradermal M. bovis (strain BCG) was markedly reduced when heated to 60 degrees C. The enlargement of the lymph nodes regional to the intradermal vaccines was measured and found generally to parallel the vaccine protection provided by M. leprae and by BCG.


Assuntos
Vacinas Bacterianas/normas , Imunidade Celular , Hanseníase/imunologia , Mycobacterium leprae/imunologia , Vacinas Atenuadas/normas , Animais , Vacina BCG , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Congelamento , Temperatura Alta , Injeções Intradérmicas , Injeções Subcutâneas , Linfonodos/imunologia , Camundongos , Mycobacterium bovis , Vacinas Atenuadas/imunologia
15.
s.l; s.n; oct. 1978. 7 p. tab, graf.
Não convencional em Inglês | SES-SP, HANSEN, SESSP-ILSLACERVO, SES-SP | ID: biblio-1240731

RESUMO

The protection provided to mice by vaccines administered intradermally was measured after footpad challenge with Mycobacterium leprae. The protection offered by M. leprae suspensions was not decreased when the vaccines were killed by 60 degrees C heat or at the higher temperatures tested, which included 215 degrees C (autoclave). Even highly purified suspensions retained their immunogenicity. In contrast, the vaccine protection provided by intradermal M. bovis (strain BCG) was markedly reduced when heated to 60 degrees C. The enlargement of the lymph nodes regional to the intradermal vaccines was measured and found generally to parallel the vaccine protection provided by M. leprae and by BCG.


Assuntos
Animais , Camundongos , Hanseníase/imunologia , Imunidade Celular , Injeções Intradérmicas , Injeções Subcutâneas , Mycobacterium bovis , Mycobacterium leprae/imunologia , Vacina BCG , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/normas , Congelamento , Temperatura Alta
16.
Biochim Biophys Acta ; 449(1): 84-94, 1976 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-974152

RESUMO

Light-induced absorption changes associated with the primary photochemical reaction and dark relaxation in Photosystem I were measured at various low temperatures. A possible temperature-dependent long-range electron tunneling process was suggested to account for the unique temperature dependence of the dark decay process. The kinetics of the light-induced absorption changes are in good agreement with the light-induced EPR changes reported earlier (Ke, B., Sugahara, K., Shaw, E.R., Hansen, R. E., Hamilton, W. D. and Beinert, H. (1974) Biochim, Biophys. Acta 368, 401--408) for the same Photosystem I subchloroplast fragment at comparable temperatures. All absorption changes between 400 and 725 nm at 86degreesK have identical kinetics. The light-minus-dark difference spectrum is very similar to that of P-700 at room temperature, with an additional prominent positive change at 690 nm. Possible contributions by P-430 to the blue and red spectral changes were discussed. It was demonstrated that the intensity of the measuring beam has a drastic effect on the light-induced absorption changes of Photosystem I at low temperatures. Various pretreatments of the Photosystem I fragments such as those that photochemically (or chemically) oxidize the primary donor or photoreduce the primary acceptor abolish the subsequent photochemical reaction. Continuous illumination of the Photosystem I fragments before and during freezing has the same effect. In the temperature range of --20 to --60degreesC, an unusual counter absorption change as well as a counter EPR change were observed.


Assuntos
Luz , Fotossíntese/efeitos da radiação , 2,6-Dicloroindofenol/farmacologia , Ácido Ascórbico/farmacologia , Cloroplastos/efeitos da radiação , Congelamento , Cinética , Espectrofotometria , Temperatura , Tetrametilfenilenodiamina/farmacologia
17.
Int J Lepr Other Mycobact Dis ; 44(3): 294-300, 1976.
Artigo em Inglês | MEDLINE | ID: mdl-789261

RESUMO

The radiometric method has been applied for studying the metabolism of M. lepraemurium and the conditions which might force or inhibit its metabolic activity in vitro. These organisms assimilate and oxidize (U-14C) glycerol, and (U-14C) acetate, but are unable to oxidize (U-14C) glucose, (U-14C) pyruvate, (U-14C) glycine and 14C-formate. When incubated at 30 degrees C M. lepraemurium oxidizes (U-14C) acetate to 14CO2 faster than 37 degrees C. The smae effect was observed with increasing concentrations of polysorbate 80 (Tween 80), or the 14C-substrate. No change in metabolic rate was observed when the organisms were kept at -20 degrees C for 12 days. Although tried several times, it was not possible to demonstrate any "inhibitors" of bacterial metabolism in the reaction system. The radiometric method seems to be an important tool for studying metabolic pathways and the influence of physical and biochemical factors on the metabolism of M. lepraemurium in vitro.


Assuntos
Mycobacterium lepraemurium/metabolismo , Acetatos/metabolismo , Animais , Dióxido de Carbono/biossíntese , Radioisótopos de Carbono , Feminino , Congelamento , Glucose/metabolismo , Glicerol/metabolismo , Glicina/metabolismo , Fígado/microbiologia , Camundongos , Camundongos Endogâmicos , Ácidos Oleicos/farmacologia , Polissorbatos/farmacologia , Piruvatos/metabolismo
18.
Biochim Biophys Acta ; 423(2): 339-55, 1976 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-2321

RESUMO

1. Techniques and experiments are described concerned with the millisecond kinetics of EPT-detectable changes brought about in cytochrome c oxidase by reduced cytochrome c and, after reduction with various agents, by reoxidation with O2 or ferricyanide. Some experiments in the presence of ligands are also reported. Light absorption was monitored by low-temperature reflectance spectroscopy. 2. In the rapid phase of reduction of cytochrome c oxidase by cytochrome c (less than 50 ms) approx. 0.5 electron equivalent per heme a is transferred mainly to the low-spin heme component of cytochrome c oxidase and partly to the EPR-detectable copper. In a slow phase (less than 1 s) the copper is reoxidized and high-spin ferric heme signals appear with a predominant rhombic component. Simultaneously the absorption band at 655 nm decreases and the Soret band at 444 nm appears between the split Soret band (442 and 447 nm) of reduced cytochrome a. 3. On reoxidation of reduced enzyme by oxygen all EPR and optical features are restored within 6 ms. On reoxidation by O2 in the presence of an excess of reduced cytochrome c, states can be observed where the low-spin heme and copper signals are largely absent but the absorption at 655 nm is maximal, indicating that the low-spin heme and copper components are at the substrate side and the component(s) represented in the 655 nm absorption at the O2 side of the system. On reoxidation with ferricyanide the 655 nm absorption is not readily restored but a ferric high-spin heme, represented by a strong rhombic signal, accumulates. 4. On reoxidation of partly reduced enzyme by oxygen, the rhombic high-spin signals disappear within 6 ms., whereas the axial signals disappear more slowly, indicating that these species are not in rapid equilibrium. Similar observations are made when partly reduced enzyme is mixed with CO. 5. The results of this and the accompanying paper are discussed and on this basis an assignment of the major EPR signals and of the 655 nm absorption is proposed, which in essence is that published previously (Hartzell, C.R., Hansen, R.E. and Beinert, H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2477-2481). Both the low-spin (g=o; 2.2; 1.5) and slowly appearing high-spin (g=6; 2) signals are attributed to ferric cytochrome a, whereas the 655 nm absorption is thought to arise from ferric cytochrome a3, when it is present in a state of interaction with EPR-undectectable copper. Alternative possibilities and possible inconsistencies with this proposal are discussed.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Anaerobiose , Cianetos/farmacologia , Grupo dos Citocromos c , Espectroscopia de Ressonância de Spin Eletrônica , Ferricianetos/farmacologia , Congelamento , Concentração de Íons de Hidrogênio , Cinética , Oxirredução , Conformação Proteica , Análise Espectral
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