Hear us! Accounts of people treated with injectables for drug-resistant TB.

BACKGROUND: WHO drug-resistant TB (DR-TB) treatment recommendations now emphasize all-oral regimens, recommending against certain injectable agents and deprioritizing others due to inferior safety and efficacy. Despite increasing focus on patient-centered care, we are not aware of systematic attempts to qualitatively document patients' perspectives on injectable agents. This may inform implementation of WHO guidelines, emphasizing the importance of consultation with affected communities. METHODS: Testimonies were provided by TB survivors who experienced hearing loss from treatment with injectable agents. Testimonies were submitted in writing in response to minimal, standardized, open-ended prompts. Participants provided a signed consent form (with options to participate anonymously or as a named co-author), and later gave input into the overall shape and recommendations of the article. RESULTS: Fourteen TB survivors in 12 countries contributed testimonies. The following common themes emerged: lack of access to appropriate testing, information, treatment, or a collaborative treatment environment; the power of supportive care and social environments; stigma and isolation from TB treatment itself and resultant disability; and inaccessibility of cochlear implants. CONCLUSIONS: Survivor testimonies indicate strong preferences for avoidance of injectable agents, supporting rapid implementation of revised WHO guidelines, as well as for quality and supportive care for both TB and disabilities.

CONTEXTE: Les recommandations de l'OMS pour le traitement de la TB pharmacorésistante (DR-TB) mettent désormais l'accent sur les schémas thérapeutiques entièrement par voie orale, préconisant de ne pas utiliser certains agents injectables et de ne plus donner la priorité à d'autres en raison d'une innocuité et d'une efficacité inférieures. Malgré l'attention accrue portée aux soins centrés sur le patient, nous ne connaissons aucune étude systématique ayant cherché à documenter de manière qualitative le point de vue des patients sur les agents injectables. Ce travail pourrait guider la mise en place des directives de l'OMS, en mettant l'accent sur l'importance de consulter les communautés concernées. MÉTHODES: Des personnes ayant survécu à une TB et ayant connu une perte d'audition due à un traitement par agents injectables ont apporté leurs témoignages. Les témoignages ont été soumis par écrit en réponse à des questions courtes, ouvertes et standardisées. Les participants ont signé un formulaire de consentement (avec possibilité de participer de manière anonyme ou en tant que coauteur nommé) et ont ensuite contribué au format général et aux recommandations de l'article. RÉSULTATS: Quatorze personnes ayant survécu à une TB provenant de 12 pays ont apporté leur témoignage. Les thématiques suivantes ont été fréquemment mentionnées : manque d'accès aux tests, informations et traitements appropriés ou à un environnement thérapeutique collaboratif ; importance des soins de soutien et de l'environnement social ; stigmatisation et isolement dus au traitement antituberculeux et handicaps qui en résultent ; et inaccessibilité aux implants cochléaires. CONCLUSIONS: Le témoignage des personnes ayant survécu à une TB indique qu'elles préfèrent nettement éviter les agents injectables, allant ainsi dans le sens d'une mise en place rapide des directives révisées de l'OMS, et qu'elles préfèrent des soins de qualité et de soutien pour la TB mais aussi pour les handicaps qui en résultent.

A genome-wide association study identifies novel gene associations with facial skin wrinkling and mole count in Latin Americans.

BACKGROUND: Genome-wide association studies (GWASs) have identified genes influencing skin ageing and mole count in Europeans, but little is known about the relevance of these (or other genes) in non-Europeans. OBJECTIVES: To conduct a GWAS for facial skin ageing and mole count in adults < 40 years old, of mixed European, Native American and African ancestry, recruited in Latin America. METHODS: Skin ageing and mole count scores were obtained from facial photographs of over 6000 individuals. After quality control checks, three wrinkling traits and mole count were retained for genetic analyses. DNA samples were genotyped with Illumina's HumanOmniExpress chip. Association testing was performed on around 8 703 729 single-nucleotide polymorphisms (SNPs) across the autosomal genome. RESULTS: Genome-wide significant association was observed at four genome regions: two were associated with wrinkling (in 1p13·3 and 21q21·2), one with mole count (in 1q32·3) and one with both wrinkling and mole count (in 5p13·2). Associated SNPs in 5p13·2 and in 1p13·3 are intronic within SLC45A2 and VAV3, respectively, while SNPs in 1q32·3 are near the SLC30A1 gene, and those in 21q21·2 occur in a gene desert. Analyses of SNPs in IRF4 and MC1R are consistent with a role of these genes in skin ageing. CONCLUSIONS: We replicate the association of wrinkling with variants in SLC45A2, IRF4 and MC1R reported in Europeans. We identify VAV3 and SLC30A1 as two novel candidate genes impacting on wrinkling and mole count, respectively. We provide the first evidence that SLC45A2 influences mole count, in addition to variants in this gene affecting melanoma risk in Europeans.

Pedigree analysis of pre-breeding efforts in Trifolium spp. germplasm in New Zealand.

BACKGROUND: Prebreeding in plants is the activity designed to identify useful characteristics from wild germplasm and its integration in breeding programs. Prebreeding aims to introduce new variation into the populations of a species of interest. Pedigree analysis is a valuable tool for evaluation of variation in genebanks where pedigree maps are used to visualize and describe population structure and variation within these populations. Margot Forde Germplasm Centre (MFGC) is New Zealand's national forage genebank and holds a collection of ~ 75 species of the genus Trifolium, of which only a dozen have been taken through prebreeding programs. The main objective of this study was to construct pedigree maps and analyse patterns of relatedness for seven minor Trifolium species accessions contained at the MFGC. These species are Trifolium ambiguum, Trifolium arvense, Trifolium dubium, Trifolium hybridum, Trifolium medium, Trifolium subterraneum and the Trifolium repens x Trifolium occidentale interspecific hybrids. We present a history of Trifolium spp. prebreeding in New Zealand and inform breeders of possible alternative forage species to use. RESULTS: Pedigree data from accessions introduced between 1950 and 2016 were used and filtered based on breeding activity. Kinship levels among Trifolium spp. remained below 8% and no inbreeding was found. Influential ancestors that contributed largely to populations structure were identified. The Australian cultivar 'Monaro' had a strong influence over the whole population of accessions in T. ambiguum. T. subterraneum and T. repens x T. occidentale had the largest number of generations (3). T. ambiguum and T. medium had the highest cumulative kinship across the decades. CONCLUSIONS: We conclude that there are high levels of diversity in the seven Trifolium spp. studied. However, collection and prebreeding efforts must be strengthened to maximize utilization and bring useful genetic variation.

Relationship between tumor-associated immune infiltrate and p16 staining over clinicopathological features in acral lentiginous melanoma.

PURPOSE: This study aims to evaluate the association between composition of tumor-infiltrating lymphocytes (TIL) and expression of p16 in acral lentiginous melanoma (ALM), and their impact on prognosis. MATERIALS AND METHODS: A cohort of 148 surgical pathology specimens of ALM was studied. TIL were evaluated by immunohistochemical detection of CD3 and CD8, along with CD20, CD4, CD68, and CD163 in a subset of 43 cases. p16 protein expression was also investigated in all the cases. RESULTS: The median age was 66 years, median Breslow thickness was 6.0 mm, grade III TIL was found in 28.4% and lymph nodes were involved in 54.2%. Breslow thickness (p < 0.001), stage I-II (p < 0.001), negative lymph nodes (p < 0.001) and < 10% p16 (p = 0.01) were associated with longer survival. Grade III of TIL was associated with thinner Breslow thickness (p = 0.008) and lower mitosis (p = 0.047). A higher density of CD3 TIL was associated with male gender (p = 0.008), thinner Breslow thickness (p = 0.047), negative lymph node (p = 0.031), early stage (p = 0.046), and p16 nuclear expression of > 10% (p = 0.045). Higher CD8 TIL was associated with > p16 (p = 0.03). Survival analysis found that longer survival had a trend to be associated with high TIL (p = 0.090). Levels of CD3+ and CD8+ cells were correlated with those of CD4+, CD20+, CD68+ and CD163+ immune cells. CONCLUSIONS: Higher levels of TIL tend to be associated with better overall survival in ALM. Loss of expression of p16 is associated with lower levels of CD3+ and CD8+ TIL, indicating a probable relationship between p16 and TIL immune response in ALM .

Tumor infiltrating lymphocytes in acral lentiginous melanoma: a study of a large cohort of cases from Latin America

Purpose. Acral lentiginous melanoma (ALM) is a poor prognosis subtype and is the most prevalent in non-Caucasian populations. The presence of tumor infiltrating lymphocytes (TILs) has been associated with poor prognosis in melanoma. A large cohort of ALM cases was studied to determine status of TIL and its association with outcome. Methods. All patients with cutaneous melanoma presenting from 2005 to 2012 at Instituto Nacional de Enfermedades Neoplasicas in Peru were retrospectively identified. Clinicopathological information was obtained from the medical charts. A prospective evaluation of TIL was performed. Analysis of association between ALM and clinicopathological features including TIL as well as survival analysis compared the outcome of ALM to whole group and extremity NALM was performed. Results. 537 ALM from a total of 824 cutaneous melanoma cases were studied. Older age (p = 0.022), higher Breslow (p = 0.008) and ulceration (p < 0.001) were found to be more frequent in ALM. Acral had worse overall survival (OS) compared with the whole group (p = 0.04). Clinical stage (CS) I-II patients had a median OS of 5.3 (95% CI 4.3-6.2) for ALM and 9.2 (95% CI 5.0-7.0) for extremity NALM (p = 0.016). Grade 0 (absence of TIL), I, II and III were found in 7.5, 34.5, 32.1, and 25.9%, respectively. Lower TIL grade was associated with larger tumor size (p = 0.003), higher Breslow (p = 0.001), higher Clark level (p = 0.007), higher CS (p = 0.002), extremity location (p = 0.048), histological subtype ALM (p = 0.024) and better OS (p = 0.001). Conclusions. ALM is highly prevalent in Peru and carries poor outcome. Lower TIL levels were associated with poor outcome and ALM (AU)

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Tumor infiltrating lymphocytes in acral lentiginous melanoma: a study of a large cohort of cases from Latin America.

PURPOSE: Acral lentiginous melanoma (ALM) is a poor prognosis subtype and is the most prevalent in non-Caucasian populations. The presence of tumor infiltrating lymphocytes (TILs) has been associated with poor prognosis in melanoma. A large cohort of ALM cases was studied to determine status of TIL and its association with outcome. METHODS: All patients with cutaneous melanoma presenting from 2005 to 2012 at Instituto Nacional de Enfermedades Neoplasicas in Peru were retrospectively identified. Clinicopathological information was obtained from the medical charts. A prospective evaluation of TIL was performed. Analysis of association between ALM and clinicopathological features including TIL as well as survival analysis compared the outcome of ALM to whole group and extremity NALM was performed. RESULTS: 537 ALM from a total of 824 cutaneous melanoma cases were studied. Older age (p = 0.022), higher Breslow (p = 0.008) and ulceration (p < 0.001) were found to be more frequent in ALM. Acral had worse overall survival (OS) compared with the whole group (p = 0.04). Clinical stage (CS) I-II patients had a median OS of 5.3 (95% CI 4.3-6.2) for ALM and 9.2 (95% CI 5.0-7.0) for extremity NALM (p = 0.016). Grade 0 (absence of TIL), I, II and III were found in 7.5, 34.5, 32.1, and 25.9%, respectively. Lower TIL grade was associated with larger tumor size (p = 0.003), higher Breslow (p = 0.001), higher Clark level (p = 0.007), higher CS (p = 0.002), extremity location (p = 0.048), histological subtype ALM (p = 0.024) and better OS (p = 0.001). CONCLUSIONS: ALM is highly prevalent in Peru and carries poor outcome. Lower TIL levels were associated with poor outcome and ALM.

The role of hyperiid parasites as a trophic link between jellyfish and fishes.

The trophic interactions between the scyphozoan medusa Chrysaora plocamia and the palm ruff Seriolella violacea were investigated off northern Chile and showed that large numbers of hyperiid amphipods parasitizing the medusa may channel energy back to the fishes, which feed on the parasites. The biomass of hyperiids eaten by the fish was a function of the biomass of hyperiids parasitizing the medusa. This temporally available food supply may enhance fish recruitment. The large number of hyperiids parasitizing diverse jellyfish species represents a missing trophic link in current efforts to understand the effects of jellyfish blooms on marine food webs.

Estudio clínico del primer molar permanente en niños de 6 años de edad de la comuna de calbuco, chile / Clinical study of the first permanent molar in children 6 years of age Calbuco commune, chile

Determinar la prevalencia de caries que presentan los Primeros Molares Permanentes (PMP) en niños chilenos de 6 años. Se realizó un estudio descriptivo, de corte transversal del estado de los PMP, en niños de seis años que asistieron al Hospital de Calbuco, X Región, Chile, durante el año 2008. Un operador calibrado examinó 166 niños de nivel socio-económico similar. La presencia de caries se estableció en base a los criterios propuestos por la OMS en el Oral Health Survey Basic Methods, para estudios epidemiológicos. El 65% (434/664) de los PMP se encontraban erupcionados, 47 (7%) semi- erupcionados y el 28% (183/664) aún no erupcionaban. De los PMP erupcionados y semierupcionados, el 69,02% (332/481) presenta caries. Al comparar la prevalencia de caries entre los PMP superiores y los inferiores se encontró una diferencia estadísticamente significativa (p<0,001). En los PMP superiores la mayor prevalencia de caries fue en la superficie oclusal, con un 56% (131/235), y en los PMP inferiores la superficie más afectadas fue la vestibular con un 68% (168/246).Conclusión: Los resultados demuestran la necesidad de incorporar estrategias de educativas y preventivas antes de la erupción del PMP

To determinate the prevalence of caries in first permanent molar (FPM) in six year-old Chilean children.Method: A Cross-sectional study was performed to state of FPM in six year-old children who attended in Calbuco Hospital, during 2008. A investigator previously calibrated to examined 166 Children belonging to the same socio-economic level. Caries diagnoses were carried out using standardized methods World Health Organization. 65% (434/664) of FPM were erupted and 47 (7%) partially erupted and 28% (183/664) were not erupted yet. From FPM erupted and partially erupted 69,02% (332/481) have caries. When comparing the prevalence of caries between upper and lower PFM, a significant statistical association was found (p<0,001). In the upper FPM the mayor prevalence of caries was found in the occlusal with 56% (131/235), in the lower FPM the most affected surface was the vestibular with a 68% (168/246). The results show the necessity of incorporating educative and preventive strategies before the eruption of FPM

Manejo del trauma penetrante cervical: serie de casos y revisión de literatura / Management of penetrating neck trauma: case series and review of the literature

Introduction: Penetrating neck trauma is a rare disease, but with high severity that affects the Emergency Room Services. Morbidity and mortality are due to injury of vascular structures and the aerodigestive tract. Conservative or surgical management depends on hemodynamic stability, the affected cervical zone and the presence of clinical signs of alert. Objective: To describe cervical penetrating trauma managed in the emergency unit of the Hospital Base Osorno, between January 2004 and April 2011 and review the literature. Material and Methods: We performed a retrospective review of all patients with penetrating cervical trauma seen at our center in this period. Results: Twelve patients were treated, nine were male. The average age was 29 years, ranging from 4 to 55 years. Zone II was the most affected with 9cases. Eight were stab injuries. The main surgical indications were active bleeding, subcutaneous emphysema and air exiting from the wound. The average length of hospital stay was 3,9 days. One patient died. Conclusions: In our center, penetrating neck trauma is a rare disease, which usually requires a surgical approach, forcing the emergency surgeon to be aware of warning signs to determine the need for surgical exploration or conservative management.

Central corneal thickness and intraocular pressure in Puerto Rican glaucoma patients

OBJECTIVE: To compare the variations in central corneal thickness and intraocular pressure measurements according to race, gender, and age. METHODS: A non-concurrent prospective study of 372 (744 eyes) glaucoma patients was conducted. Central corneal thickness was measured with ultrasound pachymeter and intraocular pressure with Goldmann tonometer. The relationship between CCT, race, gender, and age was evaluated using both descriptive and statistical analysis. RESULTS: The population age was 64 +/- 19.52 years. The mean central corneal thickness was 546 +/- 43.84 microm. The mean corrected intraocular pressure was 17 +/- 5.26 mm Hg. Central corneal thickness of male patients (549 +/- 43.43 pm) was thicker than that of female patients (546 +/- 41.83 microm). The mean corrected intraocular pressure in male patients (16 +/- 5.41 mm Hg) was less than that of female patients (17 +/- 5.18 mm Hg). The mean central corneal thickness in patients 0 to 9 years-of-age was 548 +/- 36.08 microm; 10 to 19 years-of-age was 606 +/- 82.30 microm; 20 to 29 years-of-age was 564 +/- 29.23 microm; 30 to 39 years-of-age was 579 +/- 15.32 microm; 40 to 49 years-of-age was 546 +/- 48.29 microm; 50 to 59 years-of-age was 550 +/- 38.12 mirom; 60 to 69 years-of-age was 545 +/- 40.22 microm; 70 to 79 years-of-age was 541 +/- 34.71 microm; 80 to 89 years-of-age was 541 +/- 34.05 microm; older than 90 years-of-age was 527 +/- 46.90 microm. CONCLUSIONS: Central corneal thickness of glaucoma patients in Puerto Rico was similar to that of Hispanics in the continental United States. However, the intraocular pressure and corrected intraocular pressure of glaucoma patients in Puerto Rico were statistically higher than that of Hispanics with glaucoma in the continental United States. In our study population, central corneal thickness and intraocular pressure were not affected by gender or age.

Early kinetics of amyloid fibril formation reveals conformational reorganisation of initial aggregates.

Understanding the initial steps of protein aggregation leading to the formation of amyloid fibrils remains a challenge. Here, the kinetics of such a process is determined for a misfolding protein model, ADA2h. The double nature of the very early kinetics suggests a step model of aggregation, where the denatured polypeptide folds into an aggregated beta-intermediate that subsequently reorganises into a more organised beta-sheet-richer structure that finally results in amyloid fibre formation. To determine the regions of the protein involved in amyloidosis, we have analysed a series of mutants previously made to study ADA2h folding. Using the algorithm TANGO, we have designed mutants that should enhance or decrease aggregation. Experimental analysis of the mutants shows that the C terminus of the molecule (comprising the last and edge beta-strand) is the major contributor to amyloid fibril formation, in good agreement with theoretical predictions. Comparison with proteins with similar topology reveals that family folds do not necessarily share the same principles of protein folding and/or aggregation.

Valoración del estado nutricional al ingreso de los pacientes internados en un hospital de niños / Evaluation of the nutritional status at admission in hospitalized patients in the pediatric hospital

Objetivos: conocer el estado nutricional de los pacientes que ingresan al hospital a través de un tamizaje nutricional. Determinar el día de inicio de la alimentación. Relacionar días medios de internación según estado nutricional y patología de base. Material y método: se estudiaron en forma prospectiva todos los pacientes ingresados en la salas de clínica médica (n:453) durante un período de tres meses. A través de una planilla se registraron los siguientes datos: fecha de ingreso y egreso; edad; sexo; diagnóstico de ingreso; peso; talla; día de inicio de la alimentación. Para evaluar el estado nutricional se tomó como patrón de referencia las tablas de NCHs/OMS y la clasificación del mismo se realizó en base a los criterios de Waterlow. Resultados: evaluaron 453 pacientes. El tamizaje nutricional mostró que el 47 por ciento de los pacientes al ingresar al hospital eran eutróficos y el 53 por ciento restante presentó algún grado de malnutrición: desnutrición aguda I° (15 por ciento), desnutrición aguda II° (8 por ciento), desnutrición aguda III° (3 por ciento); desnutrición crónica I° (9,5 por ciento)...

Valoración del estado nutricional al ingreso de los pacientes internados en un hospital de niños / Evaluation of the nutritional status at admission in hospitalized patients in the pediatric hospital

Objetivos: conocer el estado nutricional de los pacientes que ingresan al hospital a través de un tamizaje nutricional. Determinar el día de inicio de la alimentación. Relacionar días medios de internación según estado nutricional y patología de base. Material y método: se estudiaron en forma prospectiva todos los pacientes ingresados en la salas de clínica médica (n:453) durante un período de tres meses. A través de una planilla se registraron los siguientes datos: fecha de ingreso y egreso; edad; sexo; diagnóstico de ingreso; peso; talla; día de inicio de la alimentación. Para evaluar el estado nutricional se tomó como patrón de referencia las tablas de NCHs/OMS y la clasificación del mismo se realizó en base a los criterios de Waterlow. Resultados: evaluaron 453 pacientes. El tamizaje nutricional mostró que el 47 por ciento de los pacientes al ingresar al hospital eran eutróficos y el 53 por ciento restante presentó algún grado de malnutrición: desnutrición aguda Iº (15 por ciento), desnutrición aguda IIº (8 por ciento), desnutrición aguda IIIº (3 por ciento); desnutrición crónica Iº (9,5 por ciento)...(AU)

Protein engineering as a strategy to avoid formation of amyloid fibrils.

The activation domain of human procarboxypeptidase A2 (ADA2h) aggregates following thermal or chemical denaturation at acidic pH. The aggregated material contains well-defined ordered structures with all the characteristics of the fibrils associated with amyloidotic diseases. Variants of ADA2h containing a series of mutations designed to increase the local stability of each of the two helical regions of the protein have been found to have a substantially reduced propensity to form fibrils. This arises from a reduced tendency of the denatured species to aggregate rather than from a change in the overall stability of the native state. The reduction in aggregation propensity may result from an increase in the stability of local relative to longer range interactions within the polypeptide chain. These findings show that the intrinsic ability of a protein to form amyloid can be altered substantially by protein engineering methods without perturbing significantly its overall stability or activity. This suggests new strategies for combating diseases associated with the formation of aggregated proteins and for the design of novel protein or peptide therapeutics.

Thermodynamic analysis of helix-engineered forms of the activation domain of human procarboxypeptidase A2.

Thermodynamic characterization of the activation domain of human procarboxypeptidase A2, ADA2h, and its helix-engineered mutants was carried out by differential scanning calorimetry. The mutants were engineered by changing residues in the exposed face of the two alpha helices in order to increase their stability. At neutral and alkaline pH the three mutants, alpha-helix 1 (M1), alpha-helix 2 (M2) and alpha-helix 1 and alpha-helix 2 (DM), were more stable than the wild-type domain, in the order DM, M2, M1 and wild-type. Under these conditions the CD and NMR spectra of all the variants are very similar, indicating that this increase in stability is not the result of gross structural changes. Calorimetric analysis shows that the stabilizing effect of mutating the water-exposed surfaces of the helices seems to be mainly entropic, because the mutations do not change the enthalpy or the increase in heat capacity of denaturation. The unfolding behavior of all variants changes under acidic conditions: whereas wild-type and M1 have a strong tendency to aggregate, giving rise to a beta conformation upon unfolding, M2 and DM unfold reversibly, M2 being more stable than DM. CD and NMR experiments at pH 3.0 suggest that a region involving residues of the second and third beta strands as well as part of alpha-helix 1 changes its conformation. It seems that the enhanced stability of the altered conformation of M2 and DM reduces the aggregation tendency of ADA2h at acidic pH.

Evidencias de la participación del ácido glutámico en la neurotransmisión de la vía genículo-cortical en la rata / Evidence for glutamatergic neurotransmission in the geniculo-cortical pathway in the rat

Objetivo: Investigar la participación del ácido glutámico en la neurotransmisión de la vía genículo-cortical en la rata. Método: En una primera serie de experimentos, se han estudiado los efectos de la administración iontoforética del ácido kinurénico, un antagonista de los receptores de glutamato, en las neuronas de la corteza visual activadas mediante la estimulación eléctrica del núcleo geniculado lateral dorsal y/o del tracto óptico. La actividad neuronal se analizó mediante registros unitarios extracelulares. En una segunda serie de experimentos, se han estudiado los efectos de la estimulación eléctrica del núcleo geniculado lateral dorsal y del tracto óptico sobre la liberación de glutámico en la corteza visual de la rata in vivo. Para ello, y mediante el método de perfusión intracerebral push-pull, se realizaron perfundidos en pequeñas áreas del córtex visual con líquido cefalorraquídeo artificial. El contenido en glutámico y aspártico de los perfundido obtenidos se analizó mediante cromatografía líquida de alta resolución. Resultados: La microiontoforesis de kinurénico en el área 17 inhibió selectivamente la respuesta excitatoria de las neuronas producida por la estimulación del núcleo geniculado lateral dorsal o del tracto óptico. La estimulación del núcleo geniculado lateral dorsal o del tracto óptico incrementó significativamente la liberación de ácido glutámico, pero no de ácido aspártico en el área 17.Conclusiones: Estos resultados demuestran que el ácido glutámico es el neurotransmisor de la vía genículo-cortical en la rata (AU)

Hydrogen exchange monitored by MALDI-TOF mass spectrometry for rapid characterization of the stability and conformation of proteins.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to monitor hydrogen exchange on entire proteins. Two alternative methods have been used to carry out the hydrogen exchange studies, exchanging deuteron (H to D experiments) or proton (D to H experiments). In the former case, the use of a deuterated matrix has made possible to overcome back-exchange problems and attain reproducible results. The methods presented have been used to determine the slow exchange core of the potato carboxypeptidase inhibitor in different folding states, and to differentially compare the activation domain of human procarboxypeptidase A2 versus three site-directed mutants of different conformational stability. In this work, we show that by using MALDI-TOF MS to monitor hydrogen exchange in entire proteins, it is possible to rapidly check the folding state of a protein and characterize mutational effects on protein conformation and stability, while requiring minimal amounts of sample.

[Evidence for glutamatergic neurotransmission in the geniculo-cortical pathway in the rat]. / Evidencias de la participación del ácido glutámico en la neurotransmisión de la vía genículo-cortical en la rata.

PURPOSE: To investigate the possible participation of glutamate in the neurotransmission of the rat geniculo-cortical pathway. METHODS: In a first set of experiments, we studied the effects of microiontophoresis of kynurenate, a broad spectrum antagonist of glutamate receptors, on the excitation evoked in visual cortex cells by electrical stimulation of the dorsal lateral geniculate nucleus and the optic tract. Cell activity was determined by extracellular unit recordings. In a second set of experiments, we studied the effects of electrical stimulation of the dorsal lateral geniculate nucleus and the optic tract on the release of glutamate and aspartate in the visual cortex in vivo. Push pull perfusions were made in the visual cortex and the amino acids contents of the perfusates were analysed by high performance liquid chromatography. RESULTS: Microiontophoresis of kynureate in area 17 cells selectively supressed the excitatory response to dorsal geniculate nucleus and optic tract stimulation. On the other hand, stimulation of these structures significantly increased glutamate, but not aspartate, release in area 17. CONCLUSIONS: These results demonstrate that glutamate is the neurotransmitter of the geniculo-cortical pathway in the rat.

Mapping the pro-region of carboxypeptidase B by protein engineering. Cloning, overexpression, and mutagenesis of the porcine proenzyme.

The proteolytic processing of pancreatic procarboxypeptidase B to a mature and functional enzyme is much faster than that of procarboxypeptidase A1. This different behavior has been proposed to depend on specific conformational features at the region that connects the globular domain of the pro-segment to the enzyme and at the contacting surfaces on both moieties. A cDNA coding for porcine procarboxypeptidase B was cloned, sequenced, and expressed at high yield (250 mg/liter) in the methylotrophic yeast Pichia pastoris. To test the previous hypothesis, different mutants of the pro-segment at the putative tryptic targets in its connecting region and at some of the residues contacting the active enzyme were obtained. Moreover, the complete connecting region was replaced by the homologous sequence in procarboxypeptidase A1. The detailed study of the tryptic processing of the mutants shows that limited proteolysis of procarboxypeptidase B is a very specific process, as Arg-95 is the only residue accessible to tryptic attack in the proenzyme. A fast destabilization of the connecting region after the first tryptic cut allows subsequent proteolytic processing and the expression of carboxypeptidase B activity. Although all pancreatic procarboxypeptidases have a preformed active site, only the A forms show intrinsic activity. Mutational substitution of Asp-41 in the globular activation domain, located at the interface with the enzyme moiety, as well as removal of the adjacent 310 helix allow the appearance of residual activity in the mutated procarboxypeptidase B, indicating that the interaction of both structural elements with the enzyme moiety prevents the binding of substrates and promotes enzyme inhibition. In addition, the poor heterologous expression of such mutants indicates that the mutated region is important for the folding of the whole proenzyme.

Structure of the transition state in the folding process of human procarboxypeptidase A2 activation domain.

The transition state for the folding pathway of the activation domain of human procarboxypeptidase A2 (ADA2h) has been analyzed by the protein engineering approach. Recombinant ADA2h is an 81-residue globular domain with no disulfide bridges or cis-prolyl bonds, which follows a two-state folding transition. Its native fold is arranged in two alpha-helices packing against a four-stranded beta-sheet. Application of the protein engineering analysis for 20 single-point mutants spread throughout the whole sequence indicates that the transition state for this molecule is quite compact, possessing some secondary structure and a hydrophobic core in the process of being consolidated. The core (folding nucleus) is made by the packing of alpha-helix 2 and the two central beta-strands. The other two strands, at the edges of the beta-sheet, and alpha-helix 1 seem to be completely unfolded. These results, together with previous analysis of ADA2h with either of its two alpha-helices stabilized through improved local interactions, suggest that alpha-helix 1 does not contribute to the folding nucleus, even though it is partially folded in the denatured state under native conditions. On the other hand, alpha-helix 2 folds partly in the transition state and is part of the folding nucleus. It is suggested that a good strategy to improve folding speed in proteins would be to stabilize the helices that are not folded in the denatured state but are partly present in the transition state. Comparison with other proteins shows that there is no clear relationship between fold and/or size with folding speed and level of structure in the transition state of proteins.