<p>The purification and biochemical characterization of the substance P (SP) hydrolyzing enzyme, substance P endopeptidase (SPE), have been carried out; with subsequent orientation in neurobiological fundamental processes involved in opioid dependence, withdrawal, and heat-stress.</p><p>SPE was purified from rat spinal cord, human spinal cord and cerebrospinal fluid (CSF), rat ventral tegemental area (VTA), and rat hippocampus. The enzyme activity was found to release the biologically active fragments SP(1-7) and SP(1-8) as major products. The purified enzymes were characterized with regard to their biochemical and kinetic properties. The typical SPE is neither inhibited by phosphoramidon nor captopril nor phenylmethanesulfonylflourid (PMSF). In comparison to other known proteases SPE differed in characteristics regarding substrate specificity, inhibition-profile, cleavage pattern, and other kinetic parameters. The technically very delicate approach of micro purification of SPE from the rat ventral tegemental area (VTA) (this is a very small tissue), turned out to be possible with the ÄKTA™-purifier system. Studies revealed a crucial role of SPE in a series of clinically important neuropathological conditions, such as opioid tolerance, and withdrawal (SPE, increased); and heat-stress (SPE, increased). These findings emerged from assessment of enzyme activity in hypothalamus, nucleus accumbens (NAc) periaqueductal gray (PAG), pituitary, striatum, substantia nigra (SN), VTA, spinal cord. Viewing the role of SPE in morphine tolerance, it was possible to note regional differences with a decrease in PAG, and striatum, whereas an increase was seen in SN, and VTA. After heat-stress treatment, SPE was raised in several regions (cerebral cortex, hippocampus, diencephalon, cerebellum, spinal cord), and the most precise observation of this was located to the hippocampus structure.</p>

The purification and biochemical characterization of the substance P (SP) hydrolyzing enzyme, substance P endopeptidase (SPE), have been carried out; with subsequent orientation in neurobiological fundamental processes involved in opioid dependence, withdrawal, and heat-stress. SPE was purified from rat spinal cord, human spinal cord and cerebrospinal fluid (CSF), rat ventral tegemental area (VTA), and rat hippocampus. The enzyme activity was found to release the biologically active fragments SP(1-7) and SP(1-8) as major products. The purified enzymes were characterized with regard to their biochemical and kinetic properties. The typical SPE is neither inhibited by phosphoramidon nor captopril nor phenylmethanesulfonylflourid (PMSF). In comparison to other known proteases SPE differed in characteristics regarding substrate specificity, inhibition-profile, cleavage pattern, and other kinetic parameters. The technically very delicate approach of micro purification of SPE from the rat ventral tegemental area (VTA) (this is a very small tissue), turned out to be possible with the ÄKTA™-purifier system. Studies revealed a crucial role of SPE in a series of clinically important neuropathological conditions, such as opioid tolerance, and withdrawal (SPE, increased); and heat-stress (SPE, increased). These findings emerged from assessment of enzyme activity in hypothalamus, nucleus accumbens (NAc) periaqueductal gray (PAG), pituitary, striatum, substantia nigra (SN), VTA, spinal cord. Viewing the role of SPE in morphine tolerance, it was possible to note regional differences with a decrease in PAG, and striatum, whereas an increase was seen in SN, and VTA. After heat-stress treatment, SPE was raised in several regions (cerebral cortex, hippocampus, diencephalon, cerebellum, spinal cord), and the most precise observation of this was located to the hippocampus structure.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 120-126.

Celiac Disease (CD) is an autoimmune disorder that emerges due to the ingestion of gluten, a protein found in a variety of common grains such as wheat, rye, and barley. Approximately 1 in 100 individuals in the US suffer from CD, making it the most commonly diagnosed gastrointestinal disorder (Ciclitira et. al., 2005). These proline-rich gluten peptides are resistant to proteolysis and accumulate in the duodenum of the small intestine. Once in the duodenum, these peptides illicit an autoimmune response resulting in villous atrophy. Current treatment for CD requires a rigorous adherence to a gluten-free diet. Nevertheless, gluten-containing grains are ubiquitous in the western diet, so accidental exposure to gluten remains as a persistent threat. The approach of this project centers on genetically engineering a strain Lactobacillus reuteri to secrete a Myxococcus xanthus prolyl endopeptidase (PEP), an enzyme that hydrolyzes a peptide bond adjacent to an internal proline residue. The data from this study revealed that recombinant M. xanthus PEP purified from E. coli was effective in degrading Suc-Ala-Pro-pNA, a chromogenic substrate containing an internal proline residue. When introduced into a L. reuteri expression vector, mutations accumulated in the vector over the course of 5 days. These data suggested that toxicity was possibly associated with M. xanthus PEP and the amyl signal peptide.

Thesis (Ph.D. in Experimental Pathology, Program in Cancer Biology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 144-158). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

A L-Asparaginase II de Escherichia coli (EcA II) é uma enzima amplamente utilizada no tratamento da Leucemia Linfoblástica Aguda (LLA), atuando na depleção do aminoácido L-asparagina, o qual é fundamental para a multiplicação das células cancerosas. Contudo, o tratamento com a EcA II está associado a altos índices de hipersensibilidade, devido à formação de anticorpos anti-L-asparaginase e à clivagem da enzima pelas proteases sanguíneas asparagina endopeptidase (AEP) e catepsina B (CTSB). Também ocorre neurotoxicidade associada ao efeito L-glutaminase da enzima. O principal objetivo do presente trabalho é a obtenção de mutantes da EcA II (gene ansB) com equivalente eficiência catalítica, maior resistência à clivagem proteolítica e menor atividade glutaminase. Para este propósito, através da reação em cadeia da polimerase propensa a erro (epPCR) do gene ansB, foi construída uma biblioteca de 1128 clones expressos no vetor pET15b em BL21(DE3). Nenhum mutante com atividade asparaginásica equivalente à EcA II selvagem apresentou atividade glutaminásica inferior à esta. Dentre os clones triados obtivemos um mutante (T161I) resistente à clivagem proteolítica pela CTSB e dois mutantes (Q190L e P40S/S206C) resistentes à clivagem proteolítica por ambas AEP e CTSB. Estes três mutantes apresentaram atividade asparaginásica e glutaminásica equivalentes a EcA II selvagem. Nossos resultados mostram promissoras possibilidades de EcA II mutantes com maior estabilidade frente às proteases sanguíneas humanas e possivelmente menos imunogênicas. / Escherichia coli L-asparaginase (EcA II) is an enzyme widely used in the treatment of acute lymphoblastic leukemia (ALL), acting in the depletion of the amino acid L-asparagine, which is essential for cancer cells proliferation. However, treatment with L-asparaginase is associated with a high rate of hypersensitivity, due to formation of anti-L-asparaginase antibody and the enzyme cleavage by the serum proteases asparagine endopeptidase (AEP) and cathepsin B (CTSB). Furthermore, the neurotoxicity is associated with the effect of the enzyme L-glutaminase activity. The main aim of the current work is to obtain variants of EcA II (gene ansB) with an equivalent catalytic efficiency, greater resistance to proteolytic cleavage and a reduced glutaminase activity. For such purpose, through error-prone polymerase chain reaction (epPCR) of gene ansB, a library of 1128 clones was constructed in pET15b vector and expressed in BL21(DE3). None mutant with an asparaginase activity equivalent to EcA II wild type showed a reduced glutaminase activity. Among the screened clones, one mutant (T161I) was resistant to CTSB proteolytic cleavage and two mutants (Q190L e P40S/S206C) were resistant to both CTSB and AEP proteolytic cleavages. These three mutants were EcA II wild type equivalents in asparaginase and glutaminase activities. Our data show promising new possibilities of mutant EcA II presenting higher stability against human serum proteolytic cleavage and maybe lower immunogenicity.

Introdução: raquitismo e osteomalácia hipofosfatêmicos de origem genética mediados por FGF23 (RQ/OM-FGF23) são caracterizados pelo aumento patológico dos níveis séricos de FGF23 com consequentes hiperfosfatúria e hipofosfatemia. A forma hereditária mais comum é a ligada ao X dominante (XLHR) ocasionada por mutações inativadoras no gene PHEX. Objetivos: identificar a etiologia molecular; avaliar a densidade mineral óssea (DMO) e a microarquitetura óssea e, determinar a prevalência de nefrocalcinose (NC), nefrolitíase (NL) e de alterações metabólicas urinárias em 47 pacientes com RQ/OM-FGF23 (16 crianças e 31 adultos). Métodos: as análises dos genes PHEX e FGF23 foram realizadas pelos métodos de Sanger e MLPA. A DMO areal (DMOa) foi avaliada por densitometria óssea (DXA), enquanto a DMO volumétrica (DMOv) e os parâmetros de microarquitetura óssea foram analisados por HR-pQCT. A NC foi classificada segundo uma escala de 0-3 (0 = ausência de NC; 3 = NC grave) pelas ultrassonografia (US) e tomografia computadorizada (TC) renais. A presença de NL foi analisada pela TC renal. Fatores de risco para NC e NL foram avaliados pela urina de 24 horas. Resultados: foram identificadas mutações no PHEX em 41 pacientes (87,2%). A avaliação óssea foi realizada em 38 pacientes com XLHR que foram comparados a controles saudáveis. Os pacientes tiveram maior DMOa em L1-L4 (p=0,03) e menor DMOa em 1/3 distal do rádio (p < 0,01). Em rádio distal, a DMOv total (Total.vBMD) e os componentes trabecular (Tb.vBMD) e cortical (Ct.vBMD) foram semelhantes entre os grupos. Na tíbia distal, os pacientes apresentaram menor Total.vBMD em relação aos controles devido ao déficit no Tb.vBMD (p < 0,01). Além do mais, ao separarmos por status metabólico, os pacientes descompensados tiveram menor Ct.vBMD em tíbia distal comparados aos controles (p=0,02). Quanto aos parâmetros estruturais, em rádio distal, os pacientes apresentaram menor número de trabéculas (Tb.N; p=0,01), maior espessura trabecular (Tb.Th; p < 0,01) e maior falta da homogeneidade trabecular (SD.1/Tb.N; p=0,02). Na tíbia distal, eles tiveram menor Tb.N (p < 0,01), maior separação trabecular (Tb.Sp; p < 0,01) e maior SD.1/Tb.N (p < 0,01). A avaliação renal foi feita em 39 pacientes com XLHR. A NC foi diagnosticada em 15 (38,5%) pacientes pelas US e TC, principalmente no grupo pediátrico em uso intensivo de fosfato. A US detectou NC em 37 (94,8%), majoritariamente como grau 1 (97%), enquanto a TC identificou NC medular em 15 (38,5%): 10 (66,7%) como grau 1 e cinco (33,3%) como grau 2. Quatro (10,2%) pacientes adultos tinham NL determinada pela CT. Além da hiperfosfatúria presente em todos os pacientes, a hipocitratúria foi a alteração metabólica mais comum (30,7%); somente dois pacientes apresentaram hipercalciúria (5,1%) e nenhum apresentou hiperoxalúria. Conclusões: nesta casuística, a XLHR foi a principal forma hereditária de RQ/OM-FGF23. A HR-pQCT foi mais informativa do que a DXA e o compartimento ósseo trabecular foi mais afetado pela doença, particularmente na tíbia distal. Finalmente, a NC foi mais prevalente que a NL; o principal fator de risco metabólico foi a hiperfosfatúria e o tratamento intensivo com fosfato parece ser um agravante na formação da NC / Background: FGF23-mediated hypophosphatemic rickets is a group of diseases characterized by a pathological increase of FGF23 serum levels, resulting in hyperphosphaturia and hypophosphatemia. In this group, the most common form of inheritance is the X-linked dominant (XLHR) caused by inactivating mutations in the PHEX gene. Aims: to identify the molecular basis; to evaluate the bone mineral density and bone microarchitecture; to determinate the prevalence of nephrocalcinosis (NC), nephrolithiasis (NL) and their related metabolic factors in 47 patients with FGF23-mediated hypophosphatemic rickets (16 children and 31 adults). Methods: PHEX and FGF23 were analyzed by conventional Sanger sequencing and MLPA. The areal BMD (aBMD) was evaluated by dual-energy x-ray absorptiometry (DXA), while the volumetric BMD (vBMD) and the bone microarchitecture were analyzed by high-resolution peripheral quantitative computed tomography (HR-pQCT). NC was investigated by renal ultrasonography (US) and computed tomography (CT) and classified using a 0-3 scale (0= no NC and 3= severe NC). The presence of NL was determined by renal CT. Risk factors for NC and NL were evaluated by 24-hour urinary samples. Results: 41 patients (87.2%) presented mutations in PHEX. The bone analysis was made in 38 XLHR patients compared to healthy controls. XLHR patients presented higher aBMD at L1-L4 (p=0.03) and lower aBMD at the distal third of the radius (p < 0.01). At the distal radius, HR-pQCT showed no differences in the vBMD neither in its trabecular (Tb.vBMD) and cortical (Ct.vBMD) components. At the distal tibia, the XLHR patients showed lower Total.vBMD (p < 0.01) compared to controls due to decreased Tb.vBMD (p < 0.01). Moreover, after XLHR patients were sorted by metabolic status, the noncompensated ones revealed lower Ct.vBMD at the distal tibia compared to their respective controls (p=0.02). Regarding to the microarchitectural parameters, at the distal radius, XLHR patients showed lower trabecular number (Tb.N; p=0.01), greater trabecular thickness (Tb.Th; p < 0.01) and more inhomogeneous trabecular network (SD.1/Tb.N; p=0.02). At the distal tibia, they had lower Tb.N (p < 0.01), larger trabecular separation (Tb.Sp; p < 0.01) and greater SD.1/Tb.N (p < 0.01). The renal assessment was done in 39 XLHR patients. NC was diagnosed in 15 (38.5%) patients by US and CT, mainly in the pediatric group that was in phosphate treatment. US identified NC in 37 (94.8%), mostly as grade 1 (97%), meanwhile CT determined medullary NC in 15 (38.5%) patients: 10 (66.7%) as grade 1 and five (33.3%) as grade 2. Four (10.2%) adults patients had NL determined by CT. Besides hyperphosphaturia present in all XLHR patients, hypocitraturia was the most common metabolic factor (30.7%); hypercalciuria occurred in only two patients (5.1%) and none had hyperoxaluria. Conclusions: in our cohort, XLHR was the most prevalent form of FGF23-mediated inherited hypophosphatemic rickets. HR-pQCT was more informative than DXA and the cancellous bone compartment was the most affected by the disease particularly at the distal tibia. Finally, NC was more prevalent than NL; the main metabolic risk factor was hyperphosphaturia and the intensive treatment with phosphate seems to be an aggravating factor in the formation of NC

A L-Asparaginase II de Escherichia coli (EcA II) é uma enzima amplamente utilizada no tratamento da Leucemia Linfoblástica Aguda (LLA), atuando na depleção do aminoácido L-asparagina, o qual é fundamental para a multiplicação das células cancerosas. Contudo, o tratamento com a EcA II está associado a altos índices de hipersensibilidade, devido à formação de anticorpos anti-L-asparaginase e à clivagem da enzima pelas proteases sanguíneas asparagina endopeptidase (AEP) e catepsina B (CTSB). Também ocorre neurotoxicidade associada ao efeito L-glutaminase da enzima. O principal objetivo do presente trabalho é a obtenção de mutantes da EcA II (gene ansB) com equivalente eficiência catalítica, maior resistência à clivagem proteolítica e menor atividade glutaminase. Para este propósito, através da reação em cadeia da polimerase propensa a erro (epPCR) do gene ansB, foi construída uma biblioteca de 1128 clones expressos no vetor pET15b em BL21(DE3). Nenhum mutante com atividade asparaginásica equivalente à EcA II selvagem apresentou atividade glutaminásica inferior à esta. Dentre os clones triados obtivemos um mutante (T161I) resistente à clivagem proteolítica pela CTSB e dois mutantes (Q190L e P40S/S206C) resistentes à clivagem proteolítica por ambas AEP e CTSB. Estes três mutantes apresentaram atividade asparaginásica e glutaminásica equivalentes a EcA II selvagem. Nossos resultados mostram promissoras possibilidades de EcA II mutantes com maior estabilidade frente às proteases sanguíneas humanas e possivelmente menos imunogênicas. / Escherichia coli L-asparaginase (EcA II) is an enzyme widely used in the treatment of acute lymphoblastic leukemia (ALL), acting in the depletion of the amino acid L-asparagine, which is essential for cancer cells proliferation. However, treatment with L-asparaginase is associated with a high rate of hypersensitivity, due to formation of anti-L-asparaginase antibody and the enzyme cleavage by the serum proteases asparagine endopeptidase (AEP) and cathepsin B (CTSB). Furthermore, the neurotoxicity is associated with the effect of the enzyme L-glutaminase activity. The main aim of the current work is to obtain variants of EcA II (gene ansB) with an equivalent catalytic efficiency, greater resistance to proteolytic cleavage and a reduced glutaminase activity. For such purpose, through error-prone polymerase chain reaction (epPCR) of gene ansB, a library of 1128 clones was constructed in pET15b vector and expressed in BL21(DE3). None mutant with an asparaginase activity equivalent to EcA II wild type showed a reduced glutaminase activity. Among the screened clones, one mutant (T161I) was resistant to CTSB proteolytic cleavage and two mutants (Q190L e P40S/S206C) were resistant to both CTSB and AEP proteolytic cleavages. These three mutants were EcA II wild type equivalents in asparaginase and glutaminase activities. Our data show promising new possibilities of mutant EcA II presenting higher stability against human serum proteolytic cleavage and maybe lower immunogenicity.

Introdução: raquitismo e osteomalácia hipofosfatêmicos de origem genética mediados por FGF23 (RQ/OM-FGF23) são caracterizados pelo aumento patológico dos níveis séricos de FGF23 com consequentes hiperfosfatúria e hipofosfatemia. A forma hereditária mais comum é a ligada ao X dominante (XLHR) ocasionada por mutações inativadoras no gene PHEX. Objetivos: identificar a etiologia molecular; avaliar a densidade mineral óssea (DMO) e a microarquitetura óssea e, determinar a prevalência de nefrocalcinose (NC), nefrolitíase (NL) e de alterações metabólicas urinárias em 47 pacientes com RQ/OM-FGF23 (16 crianças e 31 adultos). Métodos: as análises dos genes PHEX e FGF23 foram realizadas pelos métodos de Sanger e MLPA. A DMO areal (DMOa) foi avaliada por densitometria óssea (DXA), enquanto a DMO volumétrica (DMOv) e os parâmetros de microarquitetura óssea foram analisados por HR-pQCT. A NC foi classificada segundo uma escala de 0-3 (0 = ausência de NC; 3 = NC grave) pelas ultrassonografia (US) e tomografia computadorizada (TC) renais. A presença de NL foi analisada pela TC renal. Fatores de risco para NC e NL foram avaliados pela urina de 24 horas. Resultados: foram identificadas mutações no PHEX em 41 pacientes (87,2%). A avaliação óssea foi realizada em 38 pacientes com XLHR que foram comparados a controles saudáveis. Os pacientes tiveram maior DMOa em L1-L4 (p=0,03) e menor DMOa em 1/3 distal do rádio (p < 0,01). Em rádio distal, a DMOv total (Total.vBMD) e os componentes trabecular (Tb.vBMD) e cortical (Ct.vBMD) foram semelhantes entre os grupos. Na tíbia distal, os pacientes apresentaram menor Total.vBMD em relação aos controles devido ao déficit no Tb.vBMD (p < 0,01). Além do mais, ao separarmos por status metabólico, os pacientes descompensados tiveram menor Ct.vBMD em tíbia distal comparados aos controles (p=0,02). Quanto aos parâmetros estruturais, em rádio distal, os pacientes apresentaram menor número de trabéculas (Tb.N; p=0,01), maior espessura trabecular (Tb.Th; p < 0,01) e maior falta da homogeneidade trabecular (SD.1/Tb.N; p=0,02). Na tíbia distal, eles tiveram menor Tb.N (p < 0,01), maior separação trabecular (Tb.Sp; p < 0,01) e maior SD.1/Tb.N (p < 0,01). A avaliação renal foi feita em 39 pacientes com XLHR. A NC foi diagnosticada em 15 (38,5%) pacientes pelas US e TC, principalmente no grupo pediátrico em uso intensivo de fosfato. A US detectou NC em 37 (94,8%), majoritariamente como grau 1 (97%), enquanto a TC identificou NC medular em 15 (38,5%): 10 (66,7%) como grau 1 e cinco (33,3%) como grau 2. Quatro (10,2%) pacientes adultos tinham NL determinada pela CT. Além da hiperfosfatúria presente em todos os pacientes, a hipocitratúria foi a alteração metabólica mais comum (30,7%); somente dois pacientes apresentaram hipercalciúria (5,1%) e nenhum apresentou hiperoxalúria. Conclusões: nesta casuística, a XLHR foi a principal forma hereditária de RQ/OM-FGF23. A HR-pQCT foi mais informativa do que a DXA e o compartimento ósseo trabecular foi mais afetado pela doença, particularmente na tíbia distal. Finalmente, a NC foi mais prevalente que a NL; o principal fator de risco metabólico foi a hiperfosfatúria e o tratamento intensivo com fosfato parece ser um agravante na formação da NC / Background: FGF23-mediated hypophosphatemic rickets is a group of diseases characterized by a pathological increase of FGF23 serum levels, resulting in hyperphosphaturia and hypophosphatemia. In this group, the most common form of inheritance is the X-linked dominant (XLHR) caused by inactivating mutations in the PHEX gene. Aims: to identify the molecular basis; to evaluate the bone mineral density and bone microarchitecture; to determinate the prevalence of nephrocalcinosis (NC), nephrolithiasis (NL) and their related metabolic factors in 47 patients with FGF23-mediated hypophosphatemic rickets (16 children and 31 adults). Methods: PHEX and FGF23 were analyzed by conventional Sanger sequencing and MLPA. The areal BMD (aBMD) was evaluated by dual-energy x-ray absorptiometry (DXA), while the volumetric BMD (vBMD) and the bone microarchitecture were analyzed by high-resolution peripheral quantitative computed tomography (HR-pQCT). NC was investigated by renal ultrasonography (US) and computed tomography (CT) and classified using a 0-3 scale (0= no NC and 3= severe NC). The presence of NL was determined by renal CT. Risk factors for NC and NL were evaluated by 24-hour urinary samples. Results: 41 patients (87.2%) presented mutations in PHEX. The bone analysis was made in 38 XLHR patients compared to healthy controls. XLHR patients presented higher aBMD at L1-L4 (p=0.03) and lower aBMD at the distal third of the radius (p < 0.01). At the distal radius, HR-pQCT showed no differences in the vBMD neither in its trabecular (Tb.vBMD) and cortical (Ct.vBMD) components. At the distal tibia, the XLHR patients showed lower Total.vBMD (p < 0.01) compared to controls due to decreased Tb.vBMD (p < 0.01). Moreover, after XLHR patients were sorted by metabolic status, the noncompensated ones revealed lower Ct.vBMD at the distal tibia compared to their respective controls (p=0.02). Regarding to the microarchitectural parameters, at the distal radius, XLHR patients showed lower trabecular number (Tb.N; p=0.01), greater trabecular thickness (Tb.Th; p < 0.01) and more inhomogeneous trabecular network (SD.1/Tb.N; p=0.02). At the distal tibia, they had lower Tb.N (p < 0.01), larger trabecular separation (Tb.Sp; p < 0.01) and greater SD.1/Tb.N (p < 0.01). The renal assessment was done in 39 XLHR patients. NC was diagnosed in 15 (38.5%) patients by US and CT, mainly in the pediatric group that was in phosphate treatment. US identified NC in 37 (94.8%), mostly as grade 1 (97%), meanwhile CT determined medullary NC in 15 (38.5%) patients: 10 (66.7%) as grade 1 and five (33.3%) as grade 2. Four (10.2%) adults patients had NL determined by CT. Besides hyperphosphaturia present in all XLHR patients, hypocitraturia was the most common metabolic factor (30.7%); hypercalciuria occurred in only two patients (5.1%) and none had hyperoxaluria. Conclusions: in our cohort, XLHR was the most prevalent form of FGF23-mediated inherited hypophosphatemic rickets. HR-pQCT was more informative than DXA and the cancellous bone compartment was the most affected by the disease particularly at the distal tibia. Finally, NC was more prevalent than NL; the main metabolic risk factor was hyperphosphaturia and the intensive treatment with phosphate seems to be an aggravating factor in the formation of NC