Experiences of Family Communication and Cascade Genetic Testing for Hereditary Cancer in Medically Underserved Populations-A Qualitative Study.

We sought to explore the intrafamilial communication and cascade genetic testing (CGT) experiences of patients with hereditary cancer from diverse, medically underserved populations and their relatives. Participants included patients receiving oncology care at an urban, safety net hospital in Texas or comprehensive cancer center in Alabama and their first-degree relatives. In-depth semi-structured qualitative interviews were completed wherein patients shared their experiences with genetic counseling (GC), genetic testing (GT), and communicating their results to relatives. Relatives shared their experiences receiving information from the patient and considering CGT. Interviews were transcribed, coded, and themes were identified. Of 25 participating patients, most recalled key aspects of GC and their GT results. Most (80%) patients shared their results with relatives, but only some relatives underwent CGT; patients reported low perceived susceptibility to hereditary cancer as a common barrier to CGT for their relatives. Of 16 participating relatives, most reported feeling distress upon learning the patient's GT results. Relatives were fearful of learning their own CGT results but identified prevention and early detection as CGT benefits. Interviews identified opportunities during family communication to improve relatives' perceived susceptibility to hereditary cancer. Tailored resources may support patients and relatives experiencing distress and fear during GT. PREVENTION RELEVANCE: This study of intrafamilial communication and cascade genetic testing experiences of patients with hereditary cancer and their relatives from diverse, medically underserved populations identified relatives' perceived susceptibility to hereditary cancer risks, distress, and fear as frequent reactions and barriers to testing. These results may inform future hereditary cancer prevention efforts.

Nutritional Parameters, Biomass Production, and Antioxidant Activity of Festuca arundinacea Schreb. Conditioned with Selenium Nanoparticles.

Festuca arundinacea Schreb. is a widely used type of forage due to its great ecological breadth and adaptability. An agricultural intervention that improves the selenium content in cultivated plants has been defined as bio-fortification, a complementary strategy to improve human and non-human animals' nutrition. The advancement of science has led to an increased number of studies based on nanotechnologies, such as the development of nanoparticles (NPs) and their application in crop plants. Studies show that NPs have different physicochemical properties compared to bulk materials. The objectives of this study were (1) to determine the behavior of F. arundinacea Schreb. plants cultivated with Se nanoparticles, (2) to identify the specific behavior of the agronomic and productive variables of the F. arundinacea Schreb. plants, and (3) to quantify the production and quality of the forage produced from the plant (the bioactive compounds' concentrations, antioxidant activity, and the concentration of selenium). Three different treatments of SeNPs were established (0, 1.5, 3.0, and 4.5 mg/mL). The effects of a foliar fertilization with SeNPs on the morphological parameters such as the root size, plant height, and biomass production were recorded, as well as the effects on the physicochemical parameters such as the crude protein (CP), lipids (L), crude fiber (CF), neutral detergent fiber (NDF), acid detergent fiber (ADF), carbohydrates (CH), the content of total phenols, total flavonoids, tannins, quantification of selenium and antioxidant activity 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and 2,2-diphenyl-1-picrylhydrazyl (DPPH). Significant differences (p < 0.05) were found between treatments in all the response variables. The best results were obtained with foliar application treatments with 3.0 and 4.5 mg/mL with respect to the root size (12.79 and 15.59 cm) and plant height (26.18 and 29.34 cm). The F. arundinacea Schreb. plants fertilized with 4.5 mg/L had selenium contents of 0.3215, 0.3191, and 0.3218 mg/Kg MS; total phenols of 249.56, 280.02, and 274 mg EAG/100 g DM; and total flavonoids of 63.56, 64.96, and 61.16 mg QE/100 g DM. The foliar biofortified treatment with a concentration of 4.5 mg/mL Se NPs had the highest antioxidant capacities (284.26, 278.35, and 289.96 mg/AAE/100 g).

Sarcopenia in people living with HIV. A review.

The aim of this review is to know the current status of sarcopenia in people living with acquired immunodeficiency virus, as well as predictors, prevalence, and associated factors. Searches were done in PubMed, Scielo, and ScienceDirect databases (January 2010 to August 2021), using predefined search terms. Prevalence, intervention, and meta-analysis studies investigating sarcopenia or muscle mass and function in people living with Human immunodeficiency virus (PLHIV) were selected. We identified reports of high prevalence and increased risk for sarcopenia due to factors such as prolonged exposure to antiretroviral drugs, lack of physical activity, central obesity, drug use, and other sociodemographic factors, as well as disease duration. HIV should be considered a risk factor for sarcopenia, and evaluation of sarcopenia should be included as part of the comprehensive medical care of PLHIV. Forceful actions are required to prevent muscle weakness, especially in stages before old age with actions aimed at preserving strength and function.

Assembly of nuclear dimers of PI3K regulatory subunits is regulated by the Cdc42-activated tyrosine kinase ACK.

Activated Cdc42-associated kinase (ACK) is an oncogenic nonreceptor tyrosine kinase associated with poor prognosis in several human cancers. ACK promotes proliferation, in part by contributing to the activation of Akt, the major effector of class 1A phosphoinositide 3-kinases (PI3Ks), which transduce signals via membrane phosphoinositol lipids. We now show that ACK also interacts with other key components of class 1A PI3K signaling, the PI3K regulatory subunits. We demonstrate ACK binds to all five PI3K regulatory subunit isoforms and directly phosphorylates p85α, p85ß, p50α, and p55α on Tyr607 (or analogous residues). We found that phosphorylation of p85ß promotes cell proliferation in HEK293T cells. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also resides, and identify an interaction between pTyr607 and the N-terminal SH2 domain that supports dimerization of the regulatory subunits. We infer from this that ACK targets p110-independent p85 and further postulate that these regulatory subunit dimers undertake novel nuclear functions underpinning ACK activity. We conclude that these dimers represent a previously undescribed mode of regulation for the class1A PI3K regulatory subunits and potentially reveal additional avenues for therapeutic intervention.

Familial hypercholesterolemia in Mexico: Initial insights from the national registry.

BACKGROUND: Familial hypercholesterolemia (FH) remains underdiagnosed and undertreated. OBJECTIVE: Report the results of the first years (2017-2019) of the Mexican FH registry. METHODS: There are 60 investigators, representing 28 federal states, participating in the registry. The variables included are in accordance with the European Atherosclerosis Society (EAS) FH recommendations. RESULTS: To date, 709 patients have been registered, only 336 patients with complete data fields are presented. The mean age is 50 (36-62) years and the average time since diagnosis is 4 (IQR: 2-16) years. Genetic testing is recorded in 26.9%. Tendon xanthomas are present in 43.2%. The prevalence of type 2 diabetes is 11.3% and that of premature CAD is 9.8%. Index cases, male gender, hypertension and smoking were associated with premature CAD. The median lipoprotein (a) level is 30.5 (IQR 10.8-80.7) mg/dl. Statins and co-administration with ezetimibe were recorded in 88.1% and 35.7% respectively. A combined treatment target (50% reduction in LDL-C and an LDL-C <100 mg/dl) was achieved by 13.7%. Associated factors were index case (OR 3.6, 95%CI 1.69-8.73, P = .002), combination therapy (OR 2.4, 95%CI 1.23-4.90, P = .011), type 2 diabetes (OR 2.8, 95%CI 1.03-7.59, P = .036) and age (OR 1.023, 95%CI 1.01-1.05, P = .033). CONCLUSION: The results confirm late diagnosis, a lower than expected prevalence and risk of ASCVD, a higher than expected prevalence of type 2 diabetes and undertreatment, with relatively few patients reaching goals. Recommendations include, the use of combination lipid lowering therapy, control of comorbid conditions and more frequent genetic testing in the future.

Three-dimensional nonlinear finite element model to estimate backflow during flow-controlled infusions into the brain.

Convection-enhanced delivery is a technique to bypass the blood-brain barrier and deliver therapeutic drugs into the brain tissue. However, animal investigations and preliminary clinical trials have reported reduced efficacy to transport the infused drug in specific zones, attributed mainly to backflow, in which an annular gap is formed outside the catheter and the fluid preferentially flows toward the surface of the brain rather than through the tissue in front of the cannula tip. In this study, a three-dimensional human brain finite element model of backflow was developed to study the influence of anatomical structures during flow-controlled infusions. Predictions of backflow length were compared under the influence of ventricular pressure and the distance between the cannula and the ventricles. Simulations with zero relative ventricle pressure displayed similar backflow length predictions for larger cannula-ventricle distances. In addition, infusions near the ventricles revealed smaller backflow length and the liquid was observed to escape to the longitudinal fissure and ventricular cavities. Simulations with larger cannula-ventricle distances and nonzero relative ventricular pressure showed an increase of fluid flow through the tissue and away from the ventricles. These results reveal the importance of considering both the subject-specific anatomical details and the nonlinear effects in models focused on analyzing current and potential treatment options associated with convection-enhanced delivery optimization for future clinical trials.

Immunologic Consequences of Sequencing Cancer Radiotherapy and Surgery.

PURPOSE: Early-stage cancers are routinely treated with surgery followed by radiotherapy (SR). Radiotherapy before surgery (RS) has been widely ignored for some cancers. We evaluate overall survival (OS) and disease-free survival (DFS) with SR and RS for different cancer types and simulate the plausibility of RS- and SR-induced antitumor immunity contributing to outcomes. MATERIALS AND METHODS: We analyzed a SEER data set of early-stage cancers treated with SR or RS. OS and DFS were calculated for cancers with sufficient numbers for statistical power (cancers of lung and bronchus, esophagus, rectum, cervix uteri, corpus uteri, and breast). We simulated the immunologic consequences of SR, RS, and radiotherapy alone in a mathematical model of tumor-immune interactions. RESULTS: RS improved OS for cancers with low 20-year survival rates (lung: hazard ratio [HR], 0.88; P = .046) and improved DFS for cancers with higher survival (breast: HR = 0.64; P < .001). For rectal cancer, with intermediate 20-year survival, RS improved both OS (HR = 0.89; P = .006) and DFS (HR = 0.86; P = .04). Model simulations suggested that RS could increase OS by eliminating cancer for a broader range of model parameters and radiotherapy-induced antitumor immunity compared with SR for selected parameter combinations. This could create an immune memory that may explain increased DFS after RS for certain cancers. CONCLUSION: Study results suggest plausibility that radiation to the bulk of the tumor could induce a more robust immune response and better harness the synergy of radiotherapy and antitumor immunity than postsurgical radiation to the tumor bed. This exploratory study provides motivation for prospective evaluation of immune activation of RS versus SR in controlled clinical studies.

Mineral trioxide aggregate enriched with iron disulfide nanostructures: an evaluation of their physical and biological properties.

The purpose of this study was to characterize mineral trioxide aggregates (MTA) enriched with iron disulfide (FeS2 ) nanostructures at different concentrations, and to investigate their storage modulus, radiopacity, setting time, pH, cytotoxicity, and antimicrobial activity. Iron disulfide nanostructures [with particle size of 0.357 ± 0.156 µm (mean ± SD)] at weight ratios of 0.2, 0.4, 0.6, 0.8, and 1.0 wt% were added to white MTA (wMTA). The radiopacity, rheological properties, setting time, and pH, as well as the cytotoxicity (assessed using the MTT assay) and antibacterial activity (assessed using the broth microdilution test) were determined for MTA/FeS2 nanostructures. The nanostructures did not modify the radiopacity values of wMTA (~6 mm of aluminium); however, they reduced the setting time from 18.2 ± 3.20 min to 13.7 ± 1.8 min, and the storage modulus was indicative of a good stiffness. Whereas the wMTA/FeS2 nanostructures did not induce cytotoxicity when in contact with human pulp cells (HPCs) and human gingival fibroblasts (HGFs), they showed bacteriostatic activity against Staphylococcus aureus, Escherichia coli, and Enterococcus faecalis. Adding FeS2 nanostructures to MTA might be an option for improving the root canal sealing and antibacterial effects of wMTA in endodontic treatments.

PARAMETRIC ANALYSIS OF DRUG DISTRIBUTION DURING INFUSIONS INTO THE BRAIN USING AN AXISYMMETRIC MODEL WITH BACKFLOW / ANÁLISIS PARAMÉTRICO DE LA DISTRIBUCIÓN DE DROGA DURANTE INFUSIONES EN EL CEREBRO CON UN MODELO AXISIMÉTRICO CON REFLUJO / A ANÁLISE PARAMÉTRICA DE DISTRIBUIÇÃO DE DROGAS NO CÉREBRO DURANTE INFUSÕES COM UM MODELO AXISYMMETRIC COM REFLUXO

Convection-enhanced delivery as a means to deliver therapeutic drugs directly to the brain has shown limited clinical efficacy, primarily attributed to the phenomena of backflow, in which the infused fluid flows preferentially along the shaft catheter rather than forward into the tissue. We have previously developed a finite element model of backflow that includes both material and geometric nonlinearities and the free boundary conditions associated with the displacement of the tissue away from the external surface of the catheter. However, that study was limited to predictions of the tissue deformation and resulting convective fluid velocity in the interstitial space. In this study, we use results from that model to solve for the distribution of the infused therapeutic agent. We demonstrate that a significant percentage of the infused drug is not transported into the region of tissue located forward from the catheter tip, but instead is transported into the region along the lateral sides of the catheter. For lower flow rates, this study suggests that the use of a catheter with a larger radius may be preferable since it will provide the higher amount of drug to be transported to the tissue in front of the catheter. In contrast, for higher flow rates consistent with clinical infusions, the radius of the infusion catheter had minimal effect on the distribution of the infused drug, with most being transported into the tissue around the shaft of the catheter.

Convection-enhanced delivery es una técnica que permite transportar drogas directamente en el cerebro para el tratamiento de enfermedades del sistema nervioso central. Este método ha mostrado una eficacia limitada debido principalmente al fenómeno de reflujo (backflow), según el cual, el fluido inyectado fluye preferiblemente a lo largo del catéter y no hacia el tejido delante de la punta. Previamente desarrollamos un modelo de elementos finitos para representar el reflujo, el cual incluye las no linealidades geométricas y del material y las condiciones de borde libre asociadas con el desplazamiento del tejido en la superficie externa del catéter. Sin embargo, ese modelo solo predice la deformación del tejido y el campo de velocidades en el espacio intersticial. En este estudio, hemos utilizado los resultados provenientes del mencionado modelo bifásico para resolver la ecuación de transporte de masa y predecir la distribución de droga suministrada. Se pudo demostrar que un porcentaje significativo de droga no penetra en el tejido ubicado delante de la punta del catéter, sino que es transportado hacia el tejido ubicado alrededor del catéter. Para bajo caudales, este estudio sugiere que el uso de un catéter con un radio mayor permitiría transportar una mayor cantidad de droga hacia el tejido al frente de la punta. Por otro lado, para los mayores caudales usados en la práctica clínica, el radio del catéter tiene un efecto marginal en la distribución del fármaco, y la mayor cantidad de droga se transporta hacia el tejido ubicado alrededor del catéter.

Convection-enhanced delivery é uma técnica para o transporte de drogas directamente no cérebro para tratar doenças do sistema nervoso central. Este método tem demonstrado eficácia limitada devido, principalmente, ao fenómeno de refluxo (refluxo), através do qual, de preferência, o fluido injectado flui através do cateter para o tecido e não à frente da ponta. Anteriormente desenvolvido um modelo de elementos finitos para representar a refluxo, que inclui geométricas e não-linearidades do material e as condições associadas com a extremidade livre de deslocamento da trama na superfície exterior do cateter. No entanto, este modelo apenas prevê deformação do tecido e campo de velocidades no espaço intersticial. Neste estudo, foram utilizados os resultados do modelo de duas fases acima referidas, para resolver a equação de transporte e prever a distribuição de massa de medicamentos fornecidos. Demonstrou-se que uma percentagem significativa da droga não penetra no tecido localizado em frente da ponta do cateter, que é transportado para o tecido que rodeia o cateter. Para as taxas de fluxo baixas, este estudo sugere que o uso de um cateter com um raio maior do que transportar uma maior quantidade de droga para o tecido em frente da ponta. Além disso, para taxas de fluxo mais elevadas utilizadas na prática clínica, o raio do cateter tem um efeito marginal sobre a distribuição da droga, e tanto fármaco é transportado para o tecido que rodeia o cateter.

Celsior versus University of Wisconsin preserving solutions for liver transplantation: postreperfusion syndrome and outcome of a 5-year prospective randomized controlled study.

BACKGROUND: Celsior solution (CS) is a high-sodium, low-potassium, low-viscosity extracellular solution that has been used for liver graft preservation in recent years, although experience with it is still limited. We performed an open-label randomized active-controlled trial comparing CS with the University of Wisconsin solution (UW) for liver transplantation (LT), with a follow-up period of 5 years. METHODS: Adult transplant recipients (n=102) were prospectively randomized to receive either CS (n=51) or UW (n=51). The two groups were comparable with respect to donor and recipient characteristics. The primary outcome measure was the incidence of postreperfusion syndrome (PRS). Secondary outcome measures included primary nonfunction (PNF) or primary dysfunction (PDF), liver retransplantation, and graft and patient survival. Other secondary outcome measures were days in the intensive care unit (ICU) and the rates of acute rejection, chronic rejection, infectious complications, postoperative reoperations, and vascular and biliary complications. RESULTS: In all, 14 posttransplant variables revealed no significant differences between the groups. There were no cases of PNF or PDF. The incidence of PRS was 5.9% in the CS group and 21.6% in the UW group (P=0.041). After reperfusion, CS revealed greater control of serum potassium (P=0.015), magnesium levels (P=0.005), and plasma glucose (P=0.042) than UW. Respective patient survivals at 3, 12, and 60 months were 95.7, 87.2, and 82.0% for the CS group and 95.7, 83.3, and 66.6% for the UW group (P=0.123). CONCLUSIONS: While retaining the same degree of safety and effectiveness as UW for LT, CS may yield postliver graft reperfusion benefits, as shown in this study by a significant reduction in the incidence of PRS and greater metabolic control.

Lack of correlation between minimal erythema dose and skin phototype in a Colombian scholar population.

BACKGROUND: Sun exposure and skin phototype are the most relevant risk factors for skin cancer. Colombia has high levels of ultraviolet radiation during the whole year, therefore, both, high UVI's and outdoor worker's daily activities, in our country are very important risk factors for the development of cutaneous cancer. To date no study has evaluated the usefulness of Fitzpatrick's skin phototype classification in Colombians and its correlation with the minimal erythema dose (MED) and constitutional skin color. Such information is gaining importance in other nations due to the fact that several country's population is becoming more ethnically diverse. OBJECTIVES: To determine the skin phototype, accumulated sun exposure, sun protection behavior, MED and phenotype in a Colombian school population. METHODS: Last year high school students from the western Antioquia were invited to participate by phone and letter through their respective school directors. A self-questionnaire was handled to each student. A representative sample of the universe was selected for a medical examination by a dermatologist in order to validate the results of the self-questionnaire. The constitutional skin color was determined with the chromameter CR 300 Minolta. The MED was defined as the minimal dose of UVB being able to induce erythema 24 h later. RESULTS: Eight schools of the area agreed to participate in the study, and a total of 911 students (58% girls and 42% boys) filled-out the self-questionnaire. Sun exposure in the majority of individuals was in a level between moderate and very high. Ninety percent of students do not use any sun protection device or cream. Only a 50% of concordance between self-assessed skin phototype vs. medical skin phototype was found, and the highest concordance corresponded to skin phototype II (82%). There was a marked difference in skin photosensitivity of Colombians compared with reports in Caucasians. We observed a marked overlapping in MED's and L* values in phototypes II and III. CONCLUSIONS: The Fitzpatrick's classification was not useful in Hispanic populations such as ours. Therefore, a new skin-phototype classification system is required. In our population the constitutional color was a good predictor of the MED but it did not correlate with skin phototype. The self-assessed questionnaire method was not useful to determine skin cancer risk in our population. The majority of this population has light skin phototypes and is highly exposed to solar UV radiation without proper protection.

An unusual presentation of human papillomavirus (HPV) infection in a black epidermodysplasia verruciformis (EV) patient.

A 25-year-old black man had a 20-year history of disseminated plaques on his body. Two years before consultation he noticed several tumors on the genitalia. Physical examination revealed generalized, coalescing, hypopigmented plaques with a very defined and keratotic border that resembled actinic porokeratosis. (Fig. 1). Multiple verrucous and moist tumors were observed on the scrotum (Fig. 2). His past medical history was unremarkable, and no family member had the disease. Histology of the body lesions revealed hyperkeratosis with a horny-layer, basket-weave appearance, large and clear blue-gray keratinocytes with finely granular cytoplasm and vacuolized nuclei, and abundant keratohyaline granules located in the upper epidermal layer (Fig. 3). Scrotum biopsy showed hyperkeratosis, irregular acanthosis with papillomatosis, and koilocytic figures. The genital lesions were treated with monthly cryotherapy plus surgical excision with complete disappearance of the lesions. A decrease in ultraviolet exposure and daily sun-block were encouraged for epidermodysplasia verruciformis lesions. After genital wart clearance, follow-ups are being scheduled every 4 months, with no new lesions to date.

ATP6 homoplasmic mutations inhibit and destabilize the human F1F0-ATP synthase without preventing enzyme assembly and oligomerization.

The molecular pathogenic mechanism of the human mitochondrial diseases neurogenic ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome was determined in cultured human cells harboring homoplasmic T8993G/T8993C point mutations in the mitochondrial ATP6 gene, which encodes subunit 6 of the F1F0-ATP synthase. Immunoprecipitation and blue native electrophoresis showed that F1F0-ATP synthase assembles correctly in homoplasmic mutant mitochondria. The mutants exhibited a tendency to have an increased sensitivity to subsaturating amounts of oligomycin; this provided further evidence for complete assembly and tight coupling between the F1 and F0 sectors. Furthermore, human ATP synthase dimers and higher homo-oligomers were observed for the first time, and it was demonstrated that the mutant enzymes retain enough structural integrity to oligomerize. A reproducible increase in the proportion of oligomeric-to-monomeric enzyme was found for the T8993G mutant suggesting that F1F0 oligomerization is regulated in vivo and that it can be modified in pathological conditions. Despite correct assembly, the T8993G mutation produced a 60% inhibition in ATP synthesis turnover. In vitro denaturing conditions showed F1F0 instability conferred by the mutations, although this instability did not produce enzyme disassembly in the conditions used for determination of ATP synthesis. Taken together, the data show that the primary molecular pathogenic mechanism of these deleterious human mitochondrial mutations is functional inhibition in a correctly assembled ATP synthase. Structural instability may play a role in the progression of the disease under potentially denaturing conditions, as discussed.

The inhibitor protein (IF1) promotes dimerization of the mitochondrial F1F0-ATP synthase.

The effect of increased expression or reconstitution of the mitochondrial inhibitor protein (IF1) on the dimer/monomer ratio (D/M) of the rat liver and bovine heart F1F0-ATP synthase was studied. The 2-fold increased expression of IF1 in AS-30D hepatoma mitochondria correlated with a 1.4-fold increase in the D/M ratio of the ATP synthase extracted with digitonin as determined by blue native electrophoresis and averaged densitometry analyses. Removal of IF1 from rat liver or bovine heart submitochondrial particles increased the F1F0-ATPase activity and decreased the D/M ratio of the ATP synthase. Reconstitution of recombinant IF1 into submitochondrial particles devoid of IF1 inhibited the F1F0-ATPase activity by 90% and restored partially the D/M ratio of the whole F1F0 complex as revealed by blue native electrophoresis and subsequent SDS-PAGE or glycerol density gradient centrifugation. Thus, the inhibitor protein promotes or stabilizes the dimeric form of the intact F1F0-ATP synthase. A possible location of the IF1 protein in the dimeric structure of the rat liver F1F0 complex is proposed. According to crystallographic and electron microscopy analyses, dimeric IF1 could bridge the F1-F1 part of the dimeric F1F0-ATP synthase in the inner mitochondrial membrane.

The inhibitor protein of the F1F0-ATP synthase is associated to the external surface of endothelial cells.

The ATPase inhibitor protein (IP) of mitochondria was detected in the plasma membrane of living endothelial cells by flow cytometry, competition assays, and confocal microscopy of cells exposed to IP antibodies. The plasma membranes of endothelial cells also possess beta-subunits of the mitochondrial ATPase. Plasma membranes have the capacity to bind exogenous IP. TNF-alpha decreases the level of beta-subunits and increases the amount of IP, indicating that the ratio of IP to beta-subunit exhibits significant variations. Therefore, it is probable that the function of IP in the plasma membrane of endothelial cells is not limited to regulation of catalysis.

Overexpression of the inhibitor protein IF(1) in AS-30D hepatoma produces a higher association with mitochondrial F(1)F(0) ATP synthase compared to normal rat liver: functional and cross-linking studies.

According to functional studies, the higher IF(1) content reported in mitochondria of cancerous cells is supposed to induce a higher association with the F(1)F(0) complex than in normal cells and therefore a better inhibition of its ATPase activity. The first structural evidence supporting this prediction is here presented. Densitometric analyses of Western blotting experiments indicated a 2-fold increase in IF(1) content of AS-30D submitochondrial particles compared to normal rat liver controls. The ratio of IF(1)/F(1) alpha subunit increased similarly as judged by Westernblot analyses. This IF(1) overexpression correlated with a slower rate of IF(1) release (F(1)F(0)-ATPase activation) from the F(1)F(0) complex in AS-30D than in normal rat liver submitochondrial particles. The IF(1)-IF(1), gamma-IF(1), and alpha-IF(1) cross-linkages previously formed with dithiobis(succinimidylpropionate) in bovine F(1)F(0)I and IF(1) complexes were reproduced in the F(1)F(0)I-ATP synthase of hepatoma AS-30D cells. However, a much lower yield of IF(1) cross-linkages was found in normal rat liver particles which made them almost undetectable in SMP as well as in the immunoprecipitated F(1)F(0)I complex. Modeling in vivo IF(1) overexpression of cancerous cells by in vitro reconstitution of excess recombinant IF(1) with rat liver submitochondrial particles devoid of IF(1) reproduced the same IF(1) cross-linkages observed in AS-30D particles.

Sulfite inhibits the F1F0-ATP synthase and activates the F1F0-ATPase of Paracoccus denitrificans.

The F1F0 complex of Paracoccus denitrificans (PdF1F0) is the fastest ATP synthase but the slowest ATPase. Sulfite exerts maximal activation of the PdF1F0-ATPase (Pacheco-Moisés, F., García, J. J., Rodríguez-Zavala, J. S., and Moreno-Sánchez, R. (2000). Eur J. Biochem. 267, 993-1000) but its effect on the PdF1F0-ATP synthase activity remains unknown. Therefore, we studied the effect of sulfite on ATP synthesis and 32Pi <--> ATP exchange reactions of inside-out membrane vesicles of P. denitrificans. Sulfite inhibited both reactions under conditions of maximal delta pH and normal sensitivity to dicyclohexylcarbodiimide. Sulfite increased by 10- and 5-fold the K0.5 for Mg2+-ADP and Pi during ATP synthesis, respectively, and by 4-fold the IC50 of Mg2+-ADP for inhibition of the PdF1F0-ATPase activity. Thus, sulfite exerts opposite effects on the forward and reverse functioning of the PdF1F0 complex. These effects are not due to membrane or PdF1F0 uncoupling. Kinetic and structural modifications that could account for these results are discussed.

A functionally active human F1F0 ATPase can be purified by immunocapture from heart tissue and fibroblast cell lines. Subunit structure and activity studies.

Human mitochondrial F(1)F(0) ATP synthase was isolated with a one-step immunological approach, using a monoclonal antibody against F(1) in a 96-well microplate activity assay system, to establish a method for fast high throughput screening of inhibitors, toxins, and drugs with very small amounts of enzyme. For preparative purification, mitochondria from human heart tissue as well as cultured fibroblasts were solubilized with dodecyl-beta-d-maltoside, and the F(1)F(0) was isolated with anti-F(1) monoclonal antibody coupled to protein G-agarose beads. The immunoprecipitated F(1)F(0) contained a full complement of subunits that were identified with specific antibodies against five of the subunits (alpha, beta, OSCP, d, and IF(1)) and by MALDI-TOF and/or LC/MS/MS for all subunits except subunit c, which could not be resolved by these methods because of the limits of detection. Microscale immunocapture of F(1)F(0) from detergent-solubilized mitochondria or whole cell fibroblast extracts was performed using anti-F(1) monoclonal antibody immobilized on 96-well microplates. The captured complex V displayed ATP hydrolysis activity that was fully oligomycin and inhibitor protein IF(1)-sensitive. Moreover, IF(1) could be co-isolated with F(1)F(0) when the immunocapture procedure was carried out at pH 6.5 but was absent when the ATP synthase was isolated at pH 8.0. Immunocaptured F(1)F(0) lacking IF(1) could be inhibited by more than 90% by addition of recombinant inhibitor protein, and conversely, F(1)F(0) containing IF(1) could be activated more than 10-fold by brief exposure to pH 8.0, inducing the release of inhibitor protein. With this microplate system an ATP hydrolysis assay of complex V could be carried out with as little as 10 ng of heart mitochondria/well and as few as 3 x 10(4) cells/well from fibroblast cultures. The system is therefore suitable to screen patient-derived samples for alterations in amount or functionality of both the F(1)F(0) ATPase and IF(1).

Cross-linking of the endogenous inhibitor protein (IF1) with rotor (gamma, epsilon) and stator (alpha) subunits of the mitochondrial ATP synthase.

The location of the endogenous inhibitor protein (IF1) in the rotor/stator architecture of the bovine mitochondrial ATP synthase was studied by reversible cross-linking with dithiobis(succinimidylpropionate) in soluble F1I and intact F1F0I complexes of submitochondrial particles. Reducing two-dimensional electrophoresis, Western blotting, and fluorescent cysteine labeling showed formation of alpha-IF1, IF1-IF1, gamma-IF1, and epsilon-IF1 cross-linkages in soluble F1I and in native F1F0I complexes. Cross-linking blocked the release of IF1 from its inhibitory site and therefore the activation of F1I and F1F0I complexes in a dithiothreitol-sensitive process. These results show that the endogenous IF1 is at a distance < or = 12 angstroms to gamma and epsilon subunits of the central rotor of the native mitochondrial ATP synthase. This finding strongly suggests that, without excluding the classical assumption that IF1 inhibits conformational changes of the catalytic beta subunits, the inhibitory mechanism of IF1 may involve the interference with rotation of the central stalk.

The H+ -ATP synthase: a biochemical challenge

ATP is a high energy compound that living cells utilize for driving most of their endergonic reactions. Directly or indirectly, ATP yields energy through the splitting of its terminal pyrophosphate bond. In cells, the ATP synthase of energy transducing membranes is responsible for forming from ADP and phosphate most of the ATP that cells need for survival and reproduction. The question of how the enzyme catalyzes ATP synthesis has been addressed by numerous workers for over thirty years. A fundamental discovery was that the enzyme is localized in membranes, and that the energy for ATP formation derives from electrochemical gradients built up by enzymes that catalyze electron transfer and that are localized in those membranes. However, the molecular events that take place in the H+ -ATP synthase during the transformation of the energy of electrochemical gradients into the chemical energy of ATP have not been entirely unveiled. Studies of its structure have shown that the H+ -ATP synthase is one of the most complex enzymes discovered. It has a H+ conducting multisubunit pathway and a multisubunit complex where the catalytic events in ATP synthesis take place. Moreover, it is an enzyme that is regulated by numerous and different factors, i.e., adenine nucleotides, electrochemical H+ gradients and protein-protein interactions. Studies on the mechanisms of energy transduction have shown that synthesis of ATP at the catalytic site of the enzyme is a spontaneous process; this indicates that depending on the environment ATP may be a high or a low energy compound. Thus, even though the enzyme presents many unknowns, it continues to be a source of fundamental and unsuspected aspects of basic biochemistry.