RESUMO
Cultured fibroblasts derived from patients with late infantile metachromatic leukodystrophy incorporated arylsulfatase A from the growth medium. Upon exposure to cerebroside sulfate, they exhibited patterns of uptake and hydrolysis indistinguishable from cells derived from control subjects. Furthermore, inclusion granules formed in the metachromatic leukodystrophy fibroblasts upon exposure to sulfatides were cleared by subsequent supplementation of the growth medium with arylsulfatase A.
Assuntos
Cerebrosídeos/metabolismo , Esclerose Cerebral Difusa de Schilder/metabolismo , Fibroblastos/metabolismo , Sulfatos/metabolismo , Meios de Cultura , Técnicas de Cultura , Hidrólise , Sulfatases/metabolismo , Isótopos de EnxofreRESUMO
OBJECTIVE: Severe congenital neutropenia (SCN), also known as Kostmann syndrome (SCN3, OMIM 610738), includes a variety of haematological disorders caused by different genetic abnormalities. Mutations in ELA2 are most often the cause in autosomal dominant or sporadic forms. Recently, mutations in HAX1 have been identified as the cause of some autosomal recessive forms of SCN, including those present in the original pedigree first reported by Kostmann. We sought to determine the relationship between HAX1 gene mutations and the clinical characteristics of Japanese cases of SCN. METHODS: The genes implicated in SCN (ELA2, HAX1, Gfi-1, WAS, and P14) were analysed in 18 Japanese patients with SCN. The clinical features of these patients were obtained from medical records. Immunoblotting of HAX1 was performed on cell extracts from peripheral blood leucocytes from patients and/or their parents. RESULTS: We found five patients with HAX1 deficiency and 11 patients with mutations in the ELA2 gene. In HAX1 deficiency, a homozygous single base pair substitution (256C>T), which causes the nonsense change R86X, was identified in three affected individuals. Two sibling patients showed a compound heterozygous mutation consisting of a single base pair substitution (256C>T) and a 59 bp deletion at nucleotides 376-434. There was no detectable phenotype in any heterozygous carrier. All patients with HAX1 deficiency had experienced developmental delay. Three patients carrying R86X also suffered from epileptic seizures. In contrast, no SCN patient with heterozygous mutations in the ELA2 gene suffered from any neurodevelopmental abnormality. CONCLUSIONS: These findings suggest that the R86X mutation in the HAX1 gene is an abnormality in Japanese SCN patients with HAX1 deficiency and may lead to neurodevelopmental abnormalities and severe myelopoietic defects.
Assuntos
Deficiências do Desenvolvimento/genética , Mutação , Neutropenia/congênito , Neutropenia/genética , Proteínas/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Feminino , Homozigoto , Humanos , Lactente , Masculino , Dados de Sequência Molecular , LinhagemRESUMO
Nutritional supplement foods containing antioxidant vitamins and minerals and fish oil (mainly docosahexaenoic acid, DHA, C22:6n-3), referred to as capsules, were administered to seven smokers every day for 34 days. Concentrations of antioxidant vitamins and minerals in serum, activity of superoxide dismutase in plasma and the concentration of 8-isoprostane (8-epi-prostaglandin F(2) alpha) in the urine showed an increase or a tendency to increase after the end of administration. The frequency of subjects showing poor state of psychological health evidenced by a total score of 8 points or more on the General Health Questionnaire (30-item edition) scale was 42.9%, although there was a significant decrease to 14.3% upon completion of administration of the capsules. These biochemical and psychological changes were mostly returned to the basal level one month after the end of administration of the capsules. The results suggest that administration of antioxidant vitamins and minerals and fish oil to smokers resulted in an increase in antioxidant capacity. Effectiveness in alleviating psychosocial stress likely to be attributable to DHA was also observed.
Assuntos
Antioxidantes/metabolismo , Ácido Ascórbico/administração & dosagem , Óleos de Peixe/administração & dosagem , Minerais/administração & dosagem , Fumar/metabolismo , Estresse Psicológico/terapia , Vitamina E/administração & dosagem , Antioxidantes/administração & dosagem , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/administração & dosagem , Feminino , Humanos , Masculino , Estresse Psicológico/metabolismoRESUMO
The aim of this study was to evaluate the barrier function of platelet-induced epithelial sheets on titanium surfaces. The lack of functional peri-implant epithelial sealing with basal lamina (BL) attachment at the interface of the implant and the adjacent epithelium allows for bacterial invasion, which may lead to peri-implantitis. Although various approaches have been reported to combat bacterial infection by surface modifications to titanium, none of these have been successful in a clinical application. In our previous study, surface modification with protease-activated receptor 4-activating peptide (PAR4-AP), which induced platelet activation and aggregation, was successful in demonstrating epithelial attachment via BL and epithelial sheet formation on the titanium surface. We hypothesized that the platelet-induced epithelial sheet on PAR4-AP-modified titanium surfaces would reduce bacterial attachment, penetration, and invasion. Titanium surface was modified with PAR4-AP and incubated with platelet-rich plasma (PRP). The aggregated platelets released collagen IV, a critical BL component, onto the PAR4-AP-modified titanium surface. Then, human gingival epithelial cells were seeded on the modified titanium surface and formed epithelial sheets. Green fluorescent protein (GFP)-expressing Escherichia coli was cultured onto PAR4-AP-modified titanium with and without epithelial sheet formation. While Escherichia coli accumulated densely onto the PAR4-AP titanium lacking epithelial sheet, few Escherichia coli were observed on the epithelial sheet on the PAR4-AP surface. No bacterial invasion into the interface of the epithelial sheet and the titanium surface was observed. These in vitro results indicate the efficacy of a platelet-induced epithelial barrier that functions to prevent bacterial attachment, penetration, and invasion on PAR4-AP-modified titanium.
Assuntos
Plaquetas/fisiologia , Implantes Dentários , Materiais Dentários/química , Inserção Epitelial , Peri-Implantite/prevenção & controle , Receptores de Trombina/química , Titânio/química , Aderência Bacteriana/efeitos dos fármacos , Dente Suporte , Escherichia coli , Humanos , Técnicas In Vitro , Peri-Implantite/etiologia , Plasma Rico em Plaquetas , Propriedades de Superfície , CicatrizaçãoRESUMO
The structure of native and modified uracil DNA glycosylase from E. coli in solution was studied by synchrotron small-angle X-ray scattering. The modified enzyme (6His-uracyl DNA glycosylase) differs from the native one by the presence of an additional N-terminal 11-meric sequence amino acid residues including a block of six His residues. It was found that the conformations of these enzymes in solution at moderate ionic strength (60 mM NaCI) substantially differ in spite of minimal differences in the amino acid sequences and functional activity. The structure of native uracil DNA glycosylase in solution is close to that in crystal, showing a tendency for association. The interaction of this enzyme with nonhydrolyzable analogues of DNA ligands causes a partial dissociation of associates and a compactization of protein structure. At the same time, 6His-uracyl DNA glycosylase has a compact structure essentially different from the crystal one. A decrease in the ionic strength of solution results in a partial disruption of compact structure of the modified protein, without changes in its functional activity.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Uracila-DNA Glicosidase/química , Histidina/química , Hidrólise , Oligopeptídeos/química , Conformação Proteica , Soluções/química , Especificidade por Substrato , Difração de Raios XRESUMO
BACKGROUND: Reduced gray matter volumes in the superior temporal gyrus (STG) have been reported in patients with schizophrenia. Such volumetric abnormalities might denote alterations in cortical thickness, surface area, local gyrification or all of these factors. The STG can be anatomically divided into five subregions using automatic parcellation in FreeSurfer: lateral aspect of the STG, anterior transverse temporal gyrus of Heschl gyrus (HG), planum polare (PP) of the STG, planum temporale (PT) of the STG and transverse temporal sulcus. METHODS: We acquired magnetic resonance imaging (MRI) 3T scans from 40 age- and sex-matched patients with schizophrenia and 40 healthy subjects, and the scans were automatically processed using FreeSurfer. General linear models were used to assess group differences in regional volumes and detailed thickness, surface area and local gyrification. RESULTS: As expected, patients with schizophrenia had significantly smaller bilateral STG volumes than healthy subjects. Of the five subregions in the STG, patients with schizophrenia showed significantly and marginally reduced volumes in the lateral aspect of the STG and PT of the STG bilaterally compared with healthy subjects. The volumetric alteration in bilateral lateral STG was derived from both the cortical thickness and surface area but not local gyrification. There was no significant laterality of the alteration in the lateral STG between patients and controls and no correlation among the structures and clinical characteristics. CONCLUSIONS: These findings suggest that of five anatomical subregions in the STG, the lateral STG is one of the most meaningful regions for brain pathophysiology in schizophrenia.
Assuntos
Esquizofrenia/patologia , Lobo Temporal/patologia , Adulto , Córtex Auditivo/patologia , Estudos de Casos e Controles , Feminino , Lateralidade Funcional , Substância Cinzenta/patologia , Humanos , Imageamento Tridimensional , Imageamento por Ressonância Magnética/métodos , MasculinoRESUMO
The dynamic behavior of various types of cytochromes c in the redox reaction with iron hexacyanides was studied using a temperature-jump method in order to elucidate the molecular mechanism of the redox reaction of cytochromes with their oxidoreductants. Transmittance after the temperature jump changed through a single exponential decay for all cytochromes investigated. Under a constant concentration of anion, the redox reaction of various types of cytochrome c with iron hexacyanides was analyzed according to the scheme: (see formula in text) where C(III) and C(II) are ferric and ferrous cytochromes, respectively, Fe(III) and Fe(II) are ferri- and ferrocyanides, respectively, C(III) . Fe(II) is the ferricytochrome-ferrocyanide complex and C(II) . Fe(III) is the ferrocytochrome-ferricyanide complex. When step B is slower than the other two steps A and C, tau-1 can be represented approximately as (see formula in text) where the bar over the variables denotes the equilibrium value. In a large excess of ferrocyanide against cytochrome, we can estimate kappa 2, kappa-2, K1 and K3 independently. In the case of horse cytochrome c at 18 degrees C in 0.1 M phosphate buffer at pH 7 with 0.3 M KNO3, the estimated parameters are kappa 2 = 100 +/- 50 S-1, kappa-2 = (3.5 +/- 1.0) . 10(3) S-1, K1 = 15 +/- 7 M-1 and K3 = (8.5 +/- 1.5). 10(-4) M. From the same experiments for seven cytochromes (cytochrome c from horse, tuna, Candida krusei, Saccharomyces oviformis, Rhodospirillum rubrum cytochrome c2, Spirulina platensis cytochrome c-554 and Thermus thermophilus cytochrome c-552), the following results can be deduced. (1) Each parameter defined in the scheme above (kappa 2, kappa-2, K1, K3) diverged beyond the error range. Above all, kappa 2 values of cytochromes c-554 and c-552 are as large as 1 . 10(4) S-1 and much larger than those for the other cytochromes (to 50 approx. 700 S-1). (2) The variance of kappa 2K1 and kappa-2/K3 are relatively less than the variances of individual parameters (kappa 2, kappa-2, K1 and K3), which suggests that the values of kappa 2K1 and kappa-2/K3 have been conserved during the course of evolution.
Assuntos
Grupo dos Citocromos c , Ferricianetos , Ferrocianetos , Animais , Cinética , Matemática , Miocárdio , Oxirredução , TemperaturaRESUMO
The structure of the thermoresistant cytochrome c (552, Thermus thermophilus) has been investigated at neutral and alkaline pH by absorption and resonance Raman spectroscopy and compared with that of horse heart cytochrome c. The ligands of the ferricytochrome c-552 at neutral pH are considered to be histidine and methionine, whereas the ligands of ferrocytochrome c-552 are histidine and another nitrogen base, histidine or lysine. Ferric cytochrome c-552 undergoes an alkaline isomerization with a pK of 12.3 (25 degrees C), accompanied by a ligand exchange. Horse heart cytochrome c has at least three isomerization states at alkaline pH (pK 9.3, 12.9 and greater than 13.5 at 25 degrees C). The replacement of the sixth ligand may not be involved in the second isomerization. The thermodynamic parameters for the isomerization were also estimated. The entropy change upon isomerization of cytochrome c-552 is negative, whereas for that of horse heart cytochrome c the entropy change is positive.
Assuntos
Grupo dos Citocromos c , Miocárdio/metabolismo , Thermus/metabolismo , Animais , Histidina , Cavalos , Concentração de Íons de Hidrogênio , Isomerismo , Metionina , Espectrofotometria , Análise Espectral Raman , TermodinâmicaRESUMO
Human arylsulfatase A (cerebroside-3-sulfate 3-sulfohydrolase, EC 3.1.6.8) exhibited microheterogeneity on isoelectric focusing in polyacrylamide gels. Pure urinary enzyme gave 3 bands of activity with pI values of 4.7, 4.8 and 4.9, whereas purified liver enzyme yielded six equally spaced bands from pI 4.4 to 4.9. Detection of enzyme in the gel was made by either methylumbelliferyl sulfate or nitrocatechol sulfate. Crude enzyme preparations from human liver, kindey, placenta, brain and testis showed the six-banded pattern with varying amounts of activity in the different bands. The banding pattern of cultured human fibroblast extracts was distinctive: in addition to activity in the area of Bands 1-6 a sharp band at pI 5.1 was observed with both enzyme stains. This latter band was also present in metachromatic leukodystrophy fibroblast extracts. However, in this case the band did not appear when the specific aryl-sulfatase A stain was used. Enzyme Bands 1, 2 and 3 from urine were isolated by extraction of the gel. The three bands refocused in their initial positions; showed nearly identical enzymatic activities toward methylumbelliferyl sulfate, mitrocatechol sulfate, cerebroside sulfate and ascorbic acid 2-sulfate; and demonstrated equivalent immunological competence by antibody titration. The banding pattern of urinary arylsulfatase A was unchanged with neuraminidase treatment, whereas Bands 4-6 of the liver enzyme were converted to Bands 1-3 by this treatment. It appears that Bands 4-6 are due to sialylation of aryl-sulfatase A but that Bands 1-3 are probably due to some other type of post-ribosomal protein modification.
Assuntos
Cerebrosídeo Sulfatase/metabolismo , Isoenzimas/metabolismo , Sulfatases/metabolismo , Encéfalo/enzimologia , Cerebrosídeo Sulfatase/isolamento & purificação , Cerebrosídeo Sulfatase/urina , Feminino , Fibroblastos/enzimologia , Humanos , Focalização Isoelétrica , Isoenzimas/isolamento & purificação , Isoenzimas/urina , Rim/enzimologia , Fígado/enzimologia , Masculino , Especificidade de Órgãos , Placenta/enzimologia , Gravidez , Testículo/enzimologiaRESUMO
The oxidation-reduction reaction of horse heart cytochrome c and cytochrome c (552, Thermus thermophilus), which is highly thermoresistant, was studied by temperature-jump method. Ferrohexacyanide was used as reductant. (Formula: see text.) Thermodynamic and activation parameters of the reaction obtained for both cytochromes were compared with each other. The results of this showed that (1) the redox potential of cytochrome c-552, + 0.19 V, is markedly less than that of horse heart cytochrome c. (2) deltaHox of cytochrome c-552 is considerably lower than that of horse heart cytochrome c. (3) deltaSox and deltaSred of cytochrome c-552 are more negative than those of horse heart cytochrome c. (4) kred of cytochrome c-552 is much lower than that of horse heart cytochrome c at room temperature.
Assuntos
Grupo dos Citocromos c , Ferrocianetos , Animais , Bactérias , Cavalos , Cinética , Matemática , Oxirredução , Espectrofotometria , Espectrofotometria Ultravioleta , Temperatura , TermodinâmicaRESUMO
L-Tyrosine O-sulfate was hydrolyzed by pure human arylsulfatase A (arylsufate sulfohydrolase, EC 3.1.6.1). The rate of hydrolysis was 1/20 of the rate with nitrocatechol sulfate, but was comparable to the rate with cerebroside sulfate. The reaction was optimal at pH 5.3--5.5 and displayed zero order kinetics with time and enzyme concentration. The Km was about 35 mM. The enzyme showed no stereospecificity and hydrolyzed D-tyrosine O-sulfate with Km and V similar to those for the L-isomer. Arylsulfatase B was less than 5% as effective as arylsulfatase A in catalyzing the hydrolysis of the tyrosine sulfates. The daily urinary excretion of tyrosine sulfate by a patient with metachromatic leukodystrophy (arylsulfatase A deficiency) was comparable to the excretion by control subjects. The biological relevance of the tyrosine sulfatase activity of arylsulfatase A remains uncertain.
Assuntos
Cerebrosídeo Sulfatase/urina , Condro-4-Sulfatase/metabolismo , Placenta/enzimologia , Sulfatases/metabolismo , Sulfatases/urina , Tirosina/análogos & derivados , Cerebrosídeo Sulfatase/deficiência , Criança , Feminino , Humanos , Cinética , Leucodistrofia Metacromática/enzimologia , Leucodistrofia Metacromática/urina , Gravidez , Especificidade por Substrato , Tirosina/metabolismo , Tirosina/urinaRESUMO
Pure human arylsulfatase A (EC 3.1.6.1) was found to hydrolyze ascorbic acid 2-sulfate to ascorbic acid and inorganic sulfate at rates from 200 to 2000 mumol/mg per h depending on the method of assay. This rate was lower than that observed with the synthetic substrate 4-nitrocatechol sulfate, but higher than that seen with the physiological substrate cerebroside sulfate. Extracts of cultured fibroblasts from normal subjects were also shown to hydrolyze ascorbic acid 2-sulfate; extracts of fibroblasts from patients with metachromatic leukodystrophy, known to be deficient in arylsulfatase A, did not. Similarly, hydrolysis of ascorbic acid 2-sulfate was not observed when a partially purified preparation of human arylsulfatase B was tested under a variety of conditions. Thus, in the human, arylsulfatase A appears to be the major, if not the only, ascorbic acid-2-sulfate sulfohydrolase.
Assuntos
Ácido Ascórbico/metabolismo , Cerebrosídeo Sulfatase/metabolismo , Pele/enzimologia , Sulfatases/metabolismo , Células Cultivadas , Fibroblastos/enzimologia , Humanos , Cinética , Relação Estrutura-AtividadeRESUMO
We investigate the refolding of ubiquitin Phe45Trp/Ile61Ala (Ub(*)I61A) in a low-temperature, high-viscosity buffer, where folding is slowed so that apparent two-state and three-state mechanisms are readily distinguishable. Ub(*)I61A forms a compact ensemble rapidly (as judged from stopped-flow, small-angle X-ray scattering) with a secondary structure signature similar to that of the native state (as judged from stopped-flow circular dichroism from 215 nm to 250 nm), but the fluorescence signature still resembles the guanidinium-denatured state. The compact ensemble forms over a range of solvent and temperature conditions. The native fluorescence signature, which requires the tryptophan residue to be packed tightly, is acquired at least 500 times more slowly. Molecular dynamics simulations at 495 K show no contraction of the backbone in ethylene glycol buffer compared to pure aqueous buffer, and no significant effect on the local backbone structure of the unfolded protein. Only at higher simulation temperature does a backbone contraction appear. Thus, it appears unlikely that the aqueous ethylene glycol buffer fundamentally changes the folding mechanism of ubiquitin. We suggest that ubiquitin forms a compact ensemble with native-like secondary structure, but without tight packing, long before the native state.
Assuntos
Ubiquitina/química , Dicroísmo Circular , Modelos Moleculares , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Espalhamento de Radiação , TemperaturaRESUMO
Adenosine triphosphatase from the thermophilic bacterium PS3(TF1) has been studied by solution X-ray scattering. A structural change in TF1 caused by the binding of ADP was observed by examining the difference between the radii of gyration of the unligated and ligated forms. The radius of gyration of the unligated TF1 was found to be 49.5 +/- 0.3 A, and it decreased by approximately 3% after ligation with ADP. The positions and the amplitudes of a subsidiary maximum and a shoulder in the scattering profile showed subtle change on nucleotide binding. The lower limit of the maximum length of TF1 was determined to be 165 A for the unligated form and 150 A for the ligated form. The shape analysis of TF1 was performed by model calculations for simple triaxial bodies or their complexes. Among the various models tested, the one that gave the best fit with the experimental data consisted of seven ellipsoids of revolution; six identical ellipsoids with semi-axes: a = b = 18.5 A and c = 74 A. arranged hexagonally, and the other with a = b = 28 A and c = 45 A, located below the other six on the 6-fold axis. On the basis of this model it was suggested that there is a structural change on ligation with nucleotides, consisting of a shrinkage of the six long ellipsoids by 6% along their major axes.
Assuntos
Adenosina Trifosfatases , Bactérias/enzimologia , Difosfato de Adenosina/metabolismo , Sítios de Ligação , ATPase de Ca(2+) e Mg(2+) , Modelos Químicos , Conformação Proteica , Difração de Raios XRESUMO
Using a chemical quench device, the rate of synthesis of carbamyl aspartate from the substrates aspartate and carbamyl phosphate was followed as a function of the time between mixing the enzyme with substrates and quenching with trichloroacetic acid. This function, which is linear at long times, shows (at 4 degrees C) a transient lag phase of product of roughly 10 ms. However, when the catalytic subunit (in which the enzymatic activity is desensitized) is used instead of the enzyme, the lag disappears. Therefore the lag seems to be associated with the control functions of the enzyme, i.e. to represent the allosteric transition involved in substrate-substrate (homotropic) co-operativity. Thus the relaxation time for the activation process is roughly 10 ms. The implications of these results are examined.
Assuntos
Aspartato Carbamoiltransferase/metabolismo , Regulação Alostérica , Ácido Aspártico/análogos & derivados , Ácido Aspártico/biossíntese , Sítios de Ligação , Escherichia coli/enzimologia , Cinética , Fatores de TempoRESUMO
beta-Lactoglobulin (beta LG) is a predominantly beta-sheet protein with a markedly high helical propensity and forms non-native alpha-helical intermediate in the refolding process. We measured the refolding reaction of beta LG with various techniques and characterized the folding kinetics and the structure of the intermediate formed within the burst phase of measurements, i.e. the burst-phase intermediate. Time-resolved stopped-flow X-ray scattering measurements using the integral intensity of scattering show that beta LG forms a compact, globular structure within 30 ms of refolding. The averaged radius of gyration within 100 ms is only 1.1 times larger than that in the native state, ensuring that the burst-phase intermediate is compact. The presence of a maximum peak in a Kratky plot shows a globular shape attained within 100 ms of refolding. Stopped-flow circular dichroism, tryptophan absorption and fluorescence spectroscopy show that pronounced secondary structure regains rapidly in the burst phase with concurrent non-native alpha-helix formation, and that the subsequent compaction process is accompanied by annealing of non-native secondary structure and slow acquisition of tertiary structure. These findings strongly suggest that both compaction and secondary structure formation in protein folding are quite rapid processes, taking place within a millisecond time-scale. The structure of the burst-phase intermediate in beta LG refolding was characterized as having a compact size, a globular shape, a hydrophobic core, substantial beta-sheets and remarkable non-native alpha-helical structure, but little tertiary structure. These results suggest that both local interactions and non-local hydrophobic interactions are dominant forces early in protein folding. The interplay of local and non-local interactions throughout folding processes is important in understanding the mechanisms of protein folding.
Assuntos
Lactoglobulinas/metabolismo , Dobramento de Proteína , Animais , Bovinos , Dicroísmo Circular , Cinética , Modelos Moleculares , Espalhamento de Radiação , Espectrometria de Fluorescência , Síncrotrons , Raios XRESUMO
For the first time, the structural change associated with an allosteric transition has been monitored by X-ray solution scattering. The kinetics of the quaternary structure change of aspartate transcarbamylase were first slowed by using acetyl phosphate instead of carbamyl phosphate, and by the presence of 10% or 30% ethylene glycol. At 6.5 degrees C, the quaternary structure change was found to have a time constant of about 11 seconds. This appears to be larger than that obtained for the switching of homotropic co-operativity, measured by chemical quench under the same conditions.
Assuntos
Aspartato Carbamoiltransferase , Sítio Alostérico , Cinética , Substâncias Macromoleculares , Espalhamento de Radiação , Raios XRESUMO
Various conformational states of polypeptide chains were investigated by synchrotron small-angle X-ray scattering (SAXS). SAXS patterns of proteins and model polypeptides in globular states (native and "molten globule") and in non-globular states (unfolded protein as well as randomly coiled, partially alpha-helical and partially beta-structural synthetic polypeptides) were analyzed in terms of Guinier and Kratky plots. Large differences in the SAXS pattern have been found between globular and non-globular conformations of the polypeptide chains, and they have been interpreted in terms of differences in the shape and size of the globular and non-globular scatterers with the same molecular mass. The equilibrium and time-resolved unfolding curves of bovine carbonic anhydrase and yeast phosphoglycerate kinase were monitored by integrated SAXS intensity, and were found to be coincident with the curves measured by other physicochemical techniques, such as tryptophan fluorescence and peptide circular dichroism spectra. The intermolecular association of the protein "molten globule"-like intermediates accumulated during the guanidine hydrochloride-induced unfolding of bovine carbonic anhydrase has been investigated by various SAXS parameters. It has been shown that the integrated SAXS intensity is much less sensitive to the protein intermolecular association than the zero angle intensity and the radius of gyration. We propose the integrated SAXS intensity as a global parameter which is particularly appropriate for fast kinetic studies of protein coil to globule transitions. Time-resolved refolding curves of the above proteins were monitored by the integrated SAXS intensity to investigate the globularization process in protein folding. Two fast kinetic processes for bovine carbonic anhydrase and two fast (each within two seconds) as well as two slow (within 500 seconds) kinetic processes for yeast phosphoglycerate kinase have been recorded. The kinetic processes reflect both protein intramolecular globularization and its intermolecular association.
Assuntos
Conformação Proteica , Síncrotrons , Animais , Bovinos , Peso Molecular , Ácido Poliglutâmico/química , Polilisina/química , Espalhamento de RadiaçãoRESUMO
Hepatic portal venous gas (HPVG) has been rarely described in chronic hemodialysis patients. We report a case of HPVG in a 59-year-old female patient with hemodialysis-dependent chronic renal failure due to diabetes who presented with acute onset of abdominal pain. Abdominal CT demonstrated the presence of gas in the portal veins. However, on laparotomy, no evidence of bowel necrosis or perforation could be found. HPVG seemed to be caused by nonocclusive mesenteric ischemia (NOMI), an increasingly recognized complication in hemodialysis patients. The patient responded favorably to intravenous hyperalimentation and antibiotics.
Assuntos
Embolia Aérea/etiologia , Isquemia/complicações , Mesentério/irrigação sanguínea , Veia Porta , Diálise Renal/efeitos adversos , Diagnóstico Diferencial , Embolia Aérea/diagnóstico por imagem , Feminino , Seguimentos , Humanos , Isquemia/diagnóstico por imagem , Isquemia/cirurgia , Falência Renal Crônica/complicações , Falência Renal Crônica/terapia , Laparotomia , Pessoa de Meia-Idade , Veia Porta/diagnóstico por imagem , Portografia , Tomografia Computadorizada por Raios XRESUMO
Aggregation of the high affinity IgE receptors on rat basophilic leukemia (RBL-2H3) cells results in protein tyrosine phosphorylation although the receptor has no intrinsic enzymatic activity. The Src related protein tyrosine kinase p53/56lyn present in RBL-2H3 cells could play a role in this reaction. Here we have isolated the cDNA for rat Lyn and found it to be very homologous at the amino acid level to both the human and mouse proteins. A bacterially expressed maltose binding protein-Lyn (MBP-Lyn) fusion protein was already tyrosine phosphorylated and had tyrosine kinase activity. In a filter-binding assay, MBP-Lyn fusion protein (at 0.1 microM) specifically bound to several proteins of RBL-2H3 cells. In lysates of IgE receptor-activated cells, there was increased binding of MBP-Lyn to 65, 72, 78 and 110 kDa tyrosine phosphorylated proteins. The 72, 78 and 110 kDa tyrosine phosphorylated proteins were precipitated by a fusion protein containing the Lyn Src Homology 2 (SH2) domain. The 72 kDa Lyn binding protein was different from p72syk. Furthermore, paxillin, a cytoskeletal protein, was identified as one of the Lyn binding proteins. Thus Fc epsilon RI mediated signal transduction in RBL-2H3 cells may result from the interaction of p53/56lyn with paxillin, pp72, pp110 and other proteins.