Biochemical and spectroscopic properties of Brucella microti glutamate decarboxylase, a key component of the glutamate-dependent acid resistance system

In orally acquired bacteria, the ability to counteract extreme acid stress (pH < 2.5) ensures survival during transit through the animal host stomach. In several neutralophilic bacteria, the glutamate-dependent acid resistance system (GDAR) is the most efficient molecular system in conferring protection from acid stress. In Escherichia coli its structural components are either of the two glutamate decarboxylase isoforms (GadA, GadB) and the antiporter, GadC, which imports glutamate and exports γ-aminobutyrate, the decarboxylation product. The system works by consuming protons intracellularly, as part of the decarboxylation reaction, and exporting positive charges via the antiporter. Herein, biochemical and spectroscopic properties of GadB from Brucella microti (BmGadB), a Brucella species which possesses GDAR, are described. B. microti belongs to a group of lately described and atypical brucellae that possess functional gadB and gadC genes, unlike the most well-known "classical" Brucella species, which include important human pathogens. BmGadB is hexameric at acidic pH. The pH-dependent spectroscopic properties and activity profile, combined with in silico sequence comparison with E. coli GadB (EcGadB), suggest that BmGadB has the necessary structural requirements for the binding of activating chloride ions at acidic pH and for the closure of its active site at neutral pH. On the contrary, cellular localization analysis, corroborated by sequence inspection, suggests that BmGadB does not undergo membrane recruitment at acidic pH, which was observed in EcGadB. The comparison of GadB from evolutionary distant microorganisms suggests that for this enzyme to be functional in GDAR some structural features must be preserved

The glutaminase-dependent acid resistance system. Qualitative and quantitative assays and analysis of its distribution in enteric bacteria

Neutralophilic bacteria have developed several strategies to overcome the deleterious effects of acid stress. In particular, the amino acid-dependent systems are widespread, with their activities overlapping, covering a rather large pH range, from 6 to <2. Recent reports showed that an acid resistance (AR) system relying on the amino acid glutamine (AR2_Q), the most readily available amino acid in the free form, is operative in Escherichia coli, Lactobacillus reuteri and some Brucella species. This system requires a glutaminase active at acidic pH and the antiporter GadC to import L-glutamine and export either glutamate (the glutamine deamination product) or GABA. The latter occurs when the deamination of glutamine to glutamate, via acid-glutaminase (YbaS/GlsA), is coupled to the decarboxylation of glutamate to GABA, via glutamate decarboxylase (GadB), a structural component of the glutamate-dependent AR (AR2) system, together with GadC. Taking into account that AR2_Q could be widespread in bacteria and that until now assays based on ammonium ion detection were typically employed, this work was undertaken with the aim to develop assays that allow a straightforward identification of the acid-glutaminase activity in permeabilised bacterial cells (qualitative assay) as well as a sensitive method (quantitative assay) to monitor in the pH range 2.5-4.0 the transport of the relevant amino acids in vivo. The qualitative assay is colorimetric, rapid and reliable and provides several additional information, such as co-occurrence of AR2 and AR2_Q in the same bacterial species and assessment of the growth conditions that support maximal expression of glutaminase at acidic pH. The quantitative assay is HPLC-based and allows to concomitantly measure the uptake of glutamine and the export of glutamate and/or GABA via GadC in vivo and depending on the external pH. Finally, an extensive bioinformatic genome analysis shows that the gene encoding the glutaminase involved in AR2_Q is often nearby or in operon arrangement with the genes coding for GadC and GadB. Overall, our results indicate that AR2_Q is likely to be of prominent importance in the AR of enteric bacteria and that it modulates the enzymatic as well as antiport activities depending on the imposed acidic stress

How Overly Cautious Risk Assessment Methods Overstate Risk from PCBs in Indoor Air

The past use of building materials that contained polychlorinated biphenyls (PCBs) is prompting public concern and expensive PCB removal projects. Building materials that may contain PCBs include paint, caulk, floor finishes and many other interior and exterior construction components manufactured before 1972. While initial concern about PCBs in buildings has focused on schools, it is likely that PCB-containing materials will also be found in many residential, commercial, and industrial structures. In buildings, the primary route of human exposure is the inhalation of PCBs that volatilize out of building materials. The USEPA has indicated that inhalation of airborne PCBs may pose a significant human exposure pathway in schools. EPA’s approach to PCBs in schools is evolving quickly. Initial estimates of health risk from indoor PCBs were calculated using risk assessment methods and EPA published toxicity factors. Recently, EPA established guidance titled “Public Health Levels for PCBs in Indoor School Air” to assist school systems in remediation efforts. These criteria are intended to be “prudent public health levels that maintain PCB exposures below the \u27reference dose\u27 – the amount of PCB exposure that EPA does not believe will cause harm. This article considers two factors central to the accurate assessment of PCB indoor air risk: 1) the difference in the chemical make up of PCBs in air compared to the chemical make up of the PCBs in the building materials they originated from; and 2) how the variability in certain commercial lots of PCBs (Aroclors) can result in different degrees of toxicity. Each of these factors may act to significantly modify the level of risk associated with the inhalation of PCBs in buildings. At the present time, EPA has not incorporated these factors into its calculation of its Public Health Levels

The Glutaminase-dependent system confers extreme acid resistance to new species and atypical strains of Brucella

Neutralophilic bacteria have developed specific mechanisms to cope with the acid stress encountered in environments such as soil, fermented foods, and host compartments. In Escherichia coli, the glutamate decarboxylase (Gad)-dependent system is extremely efficient: it requires the concerted action of glutamate decarboxylase (GadA/GadB) and of the glutamate (Glu)/γ-aminobutyrate antiporter, GadC. Notably, this system is operative also in new strains/species of Brucella, among which Brucella microti, but not in the "classical" species, with the exception of marine mammals strains. Recently, the glutaminase-dependent system (named AR2_Q), relying on the deamination of glutamine (Gln) into Glu and on GadC activity, was described in E. coli. In Brucella genomes, a putative glutaminase (glsA)-coding gene is located downstream of the gadBC genes. We found that in B. microti these genes are expressed as a polycistronic transcript. Moreover, using a panel of Brucella genus-representative strains, we show that the AR2_Q system protects from extreme acid stress (pH =2.5), in the sole presence of Gln, only the Brucella species/strains predicted to have functional glsA and gadC. Indeed, mutagenesis approaches confirmed the involvement of glsA and gadC of B. microti in AR2_Q and that the acid-sensitive phenotype of B. abortus can be ascribed to a Ser248Leu substitution in GlsA, leading to loss of glutaminase activity. Furthermore, we found that the gene BMI_II339, of unknown function and downstream of the gadBC-glsA operon, positively affects Gad- and GlsA-dependent AR. Thus, we identified novel determinants that allow newly discovered and marine mammals Brucella strains to be better adapted to face hostile acidic environments. As for significance, this work may contribute to the understanding of the host preferences of Brucella species and opens the way to alternative diagnostic targets in epidemiological surveillance of brucellosis

Speculazioni Nostalgiche. L'eredità della New Hollywood nel cinema americano contemporaneo

Il periodo storico che intercorre dai primi anni 60 fino alla prima metà degli anni 70 è tornato ad essere un argomento rilevante per il cinema americano contemporaneo. Registi quali Martin Scorsese, Steven Spielberg, Quentin Tarantino e Paul Thomas Anderson, hanno rimesso in scena quel preciso contesto storico e culturale, sfruttando la macchina produttiva del cinema odierno per rimetterla in scena dopo mezzo secolo, se non proprio riesumandola per interpellare il presente. L’indagine su questa tendenza anima l’obiettivo di questo studio, indirizzato ad interpretare le rispettive eredità storiche, letterarie e culturali nel cinema contemporaneo. Il primo capitolo è dedicato alle ultime opere di Tarantino e Anderson nella loro comune rappresentazione di un particolare momento storico dellla California e Los Angeles, di conseguenza l’attenzione è focalizzata sul rapporto tra la nostalgia e lo spazio californiano, con tutto le istanze cuturali che questa comporta nel rievocare il passato urbano e territoriale di Hollywood in quelle che definisco geografie dell’infanzia. Un secondo capitolo apre il discorso sulle ultime opere di due registi come Scorsese e Eastwood, in particolare al modo in cui queste opere “testamentarie” siano i punti di arrivo di carriere longeve che intrattengono un legame culturale con la memoria cinematografica dei registi della New Holywood. Considerando alcune opere si vuole tuttavia approfondire il legame della vecchiaia iconografica di registi e attori che ritornani ai loro generi cinematografici per riflettere su di esso. Infine la terza parte si concentra sulla materia storica presa in esame da questi film. Tenendo ben chiari in mente i concetti di memoria, Storia e trauma si vogliono ricapitolare le forme di racconto e indagine proposte da alcuni titoli, nel loro rifare luci su punti nascosti o già ampiamente noti della storia americana di quegli anni

Le Condizioni di Esistenza dell' Arco fra Carboni

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Diagnóstico da pesca de isca-viva empregada pela frota atuneira no sudeste e sul do Brasil

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias, Programa de Pós-Graduação em Aquicultura, Florianópolis, 2013.A sardinha-verdadeira (Sardinella brasiliensis), espécie de peixe pelágico, nativa do litoral Sudeste e Sul do Brasil, classificada como sobreexplotada pela legislação brasileira, sustenta as duas maiores cadeias de processamento industrial de pescados nacional, os enlatados de atum e de sardinha. A pesca do atum, bonito-listrado (Katsuwonus pelamis), utiliza juvenis de sardinha como fonte de isca-viva. Esta pesquisa realizou o diagnóstico da pesca de isca-viva empregada pela frota atuneira que opera no sudeste e sul do Brasil, por meio da avaliação das características da frota atuante; da caracterização da captura e estimativa da demanda e mortalidade das iscas-vivas; e identificou as principais causas da mortalidade das iscas-vivas, mantidas sob confinamento nas tinas das embarcações atuneiras para contribuir com a sustentabilidade do recurso pesqueiro sardinha-verdadeira. Os resultados obtidos neste diagnóstico mostraram que: (i) apesar da frota atuneira ter se mantido constante em número de barcos, aumentou o poder de pesca; (ii) a disponibilidade de isca-viva, em especial juvenis de sardinha, é fator limitante à produção de atum pela frota de atuneiros, que as espécies utilizadas como isca (sardinha e manjubas) tem preferências e padrões distintos de ocorrência, que a frota atuneira tem aumentado o esforço, a demanda e consumo de isca-viva no decorrer dos anos, capturando anualmente 760 milhões de indivíduo e, portanto, promovem impacto sobre a população selvagem de sardinha, que existem elevadas perdas de iscas por mortalidade, atingindo cerca de 49% do total capturado; e (iii) que o manejo de transbordo com uso de sarico, a elevada densidade de estocagem, a reduzida concentração de oxigênio dissolvido, grande amplitude de temperatura em curto intervalo de tempo, excessiva variação do pH, são as principais causas de mortalidade das iscas, além de ser determinada por fatores como a espécie e o tamanho dos indivíduos utilizados. A redução da mortalidade de isca-viva, a partir da adoção de boas práticas de manejo, constitui a solução para maximizar os rendimentos e reduzir os impactos negativos da atividade sobre o ecossistema marinho.Abstract : The true sardine (Sardinella braziliensis), species of pelagic fish, native from Southeast coast and South of Brazil, classified by the legislation as an overexploited fish, it is sustaining two larger chains of national industry processing of fish, the tuna and sardine canned foods. The first one, fishing of skipjack tuna (Katsuwonus pelamis), involves the use of juvenile sardines as source of bait-alive for capture of tuna. This research has made the diagnosis of bait-alive fishing employed by the tuna fleet operating in southeastern and southern Brazil, through the evaluation of the characteristics of the tuna fleet, characterized the capture and estimated demand and mortality of bait-alive, and identified the main causes of mortality of bait-alive, kept under confinement in tubs of tuna fleet to contribute to the sustainability of the fisheries resource sardine. The results of this diagnosis showed that: (i) although the tuna fleet has remained constant in number of boats increased fishing power, (ii) the availability of bait-alive, especially juvenile sardines, is a limiting factor for tuna production by the tuna fleet, the species used as bait (sardines and anchovies) have distinct preferences and patterns of occurrence, the tuna fleet has increased the effort, the demand and consumption of bait alive through the years, catching annually 760 million the individual and thus promote impact on the wild population of sardines, which are high losses of lures for mortality, reaching about 49% of the total catch, and (iii) the management, especially related to the transfer of live baits the use of dip nets, the high density of stockpiling, low dissolved oxygen concentration, wide temperature range in a short period of time, excessive pH changes, as major causes of mortality of bait, and is determined by factors such as the species and size of fish used. The decrease of mortality of live-bait, from the adoption of good management practices, is the solution to maximize yields and reduce the negative impacts of the activity on the marine ecosystem

An Activity Monitor for Diabetic Individuals

An activity monitor that diabetic individuals can wear continuously will provide important information on how these individuals should make adjustments to their exercise, diet, and insulin dosage in order to maintain a healthy lifestyle. The device is composed of both heart rate sensing components and components to measure the magnitude of physical movement. The energy expenditure is calculated using an algorithm that continuously adjusts depending on the type of activity. The system display provides the carbohydrates burned in order to be adjunctive to carbohydrate counting, a common technique used for glucose management

DESCRIPTION AND ANALYSIS OF THE TEMPORAL VARIATION OF THE PURSE-SEINE FLEET OPERATION IN ITAJAÍ HARBOR, BRAZIL, BETWEEN 1997 AND 1999.

A principal frota que desembarca no porto pesqueiro de Itajaí, SC, é a frota de traineiras, a qual representa 30% do peso dos desembarques. Essa frota tem como espécie-alvo a sardinhaverdadeira (Sardinella brasiliensis). O objetivo deste trabalho é descrever e analisar as variações temporais da operação de pesca da frota de traineiras entre 1997 e 1999. Os aspectos analisados foram: (1) descrição da frota através do comprimento dos barcos, potência do motor, número de tripulantes, tipo de casco e conservação, capacidade do porão e comprimento e altura da rede de cerco; e (2) variação temporal da operação de pesca através do número de lances por viagem, tempo de duração dos lances, número de dias no mar por viagem, número de dias de pesca por viagem e tempo de procura. No período analisado foram obtidos um total de 797 registros. De forma geral, a descrição da frota indica que houve um aumento do poder de pesca nos últimos 10 anos, apesar da redução do número de barcos. Por outro lado, as características da operação de pesca foram relacionadas com as flutuações na disponibilidade da espécie-alvo.xThe main fishing fleet that lands in Itajaí harbor, Brazil, is the purse-seine one, which represents 30% of the landed weight. This fleet has as target species the Brazilian sardine (Sardinella brasiliensis). The objective of this work is to describe and to analyze the temporal variations of the fishing operation of the purse-seiners between 1997 and 1999. The analyzed aspects were: (1) description of the fleet using the ships length, motor power, crew size, skull and conservation type, hold capacity, and net’s length and height; and (2) temporal variation of the fishing operation using number of tows per trip, time and duration of tows, number of days at sea per trip, number of fishing days per trip and searching time of schools. In the analyzed period, a total of 797 records were obtained. In general, the fleet description indicated that there was an increase of the fishing power in the last 10 years, in spite of the reduction of the number of ships. On the other hand, the results suggest that the characteristics of the fishing operation are related with the oscillations of the target species availability