Solid-State Microprocessor Controlled Detector for Doublet Peak Measurements in Flow Injection Analysis

A solid-state detector for time-based transmission measurements in flow-injection analysis (FIA) based on alight-emitting diode (LED) source and photodiode (PD) transducer with microprocessor controller is described. Theinstrument components cost less than US$ 500. A simple peak-finding algorithm was capable of accurate measurementsof the time interval between doublet peaks (.:1t). Comparison of results, obtained using the detector, withtheory for an acid-base reaction with bromothymol blue indicator is made. Linear relationships between themeasured lit and the logarithm of the injected concentration were obtained for two systems whose injection volumeswere 987 and 1650 μ.l. The correlation coefficients, based on 14 and 13 data points, were 0.999 and 0.992 ,respectively. The detector could measure transmittance signals corresponding to 0.00098 absorbance above thebaseline

Different Transport Pathways of Individual Precursor Proteins in Mitochondria

Transport of mitochondrial precursor proteins into mitochondria of Neurospora crassa was studied in a cellfree reconstituted system. Precursors were synthesized in a reticulocyte lysate programmed with Neurospora mRNA and transported into isolated mitochondria in the absence of protein synthesis. Uptake of the following precursors was investigated: apocytochrome c, ADP/ATP carrier and subunit 9 of the oligomycin-sensitive ATPase. Addition of high concentrations of unlabelled chemically prepared apocytochrome c (1–10 μM) inhibited the appearance in the mitochondrial of labelled cytochrome c synthesized in vitro because the unlabelled protein dilutes the labelled one and because the translocation system has a limited capacity [apparent V is 1–3 pmol × min−1× (mg mitochondrial protein)−1]. Concentrations of added apocytochrome c exceeding the concentrations of precursor proteins synthesized in vitro by a factor of about 104 did not inhibit the transfer of ADP/ATP carrier or ATPase subunit 9 into mitochondria. Carbonylcyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation, inhibited transfer in vitro of ADP/ATP carrier and of ATPase subunit 9, but not of cytochrome c. These findings suggest that cytochrome c and the other two proteins have different import pathways into mitochondria. It can be inferred from the data presented that different 'receptors' on the mitochondrial surface mediate the specific recognition of precursor proteins by mitochondria as a first step in the transport process

Large-Scale Agile Transformation: A Case Study of Transforming Business, Development and Operations

Today, product development organizations are adopting agile methods in units outside the software development unit, such as in sales, market, legal, operations working with the customer. This broader adoption of agile methods has been labeled large-scale agile transformation and is considered a particular type of organizational change, originating in the software development units. So far, there is little research-based advice on conducting such transformations. Aiming to contribute towards providing relevant research advice on large-scale agile transformation, we apply a research-based framework for evaluating organizational agility on a product development program in a maritime service provider organization. We found that doing a large-scale agile transformation involves many significant challenges, such as having a shared understanding of the problem, getting access to users, and getting commitment to change that needs to be done. In order to overcome such challenges, we discuss the need for a holistic and integrated approach to agile transformation involving all the units linked to software development.publishedVersio

Chronological changes of incidence and prognosis of children with asymptomatic congenital cytomegalovirus infection in Sapporo, Japan

BACKGROUND: Chronological changes of the incidence of congenital cytomegalovirus (CMV) infection and the longitudinal prognosis in children with asymptomatic congenital infection were investigated. METHODS: Congenital CMV infection, as demonstrated by isolation of the virus within the first week of life, was diagnosed in infants born in Sapporo, Japan, during the 26-year period between 1977 and 2002. RESULTS: Congenital infection was diagnosed in 37 (0.31%) of 11,938 infants. Thirty-two infants were (86.5%) asymptomatic and 5 (13.5%) were symptomatic at birth. CONCLUSIONS: Although a decrease in the total incidence of congenital CMV infection has been seen in recent years, screening of congenital infection at birth seems to be necessary to detect late-onset neurodevelopmental sequelae

Enterprise agility: A Balancing Act - a local government case study

Austerity and financial constraints have been threatening the public sector in the UK for a number of years. Foreseeing the threat of continued budget cuts, and addressing the situation many local councils face, requires internal transformations for financial stability without losing the key focus on public service. Agile transformations have been undertaken by organisations wanting to learn from the software development community and bringing agile principles into the wider organisation. This paper describes and analyses an ongoing behaviour-led transformation in a district council in the UK. It presents the results of the analysis of 19 interviews with internal stakeholders at the council, of observations of meetings among senior and middle management in a five-month period. The paper explores the successes and the challenges encountered towards the end of the transformation process and reflects on balancing acts to address the challenges, be-tween: disruption and business as usual, empowerment and goal setting, autonomy and processes and procedures, and behaviours and skills. Based on our findings, we suggest that behaviours on their own cannot guarantee a sustained agile culture, and that this is equally important for enterprise agility and for large-scale agile software development transformations

Requirement of a Membrane Potential for the Posttranslational Transfer of Proteins into Mitochondsria

Posttranslational transfer of most precursor proteins into mitochondria is dependent on energization of the mitochondria. Experiments were carried out to determine whether the membrane potential or the intramitochondrial ATP is the immediate energy source. Transfer in vitro of precursors to the ADP/ATP carrier and to ATPase subunit 9 into isolated Neurospora mitochondria was investigated. Under conditions where the level of intramitochondrial ATP was high and the membrane potential was dissipated, import and processing of these precursor proteins did not take place. On the other hand, precursors were taken up and processed when the intramitochondrial ATP level was low, but the membrane potential was not dissipated. We conclude that a membrane potential is involved in the import of those mitochondrial precursor proteins which require energy for intracellular translocatio

Specific sequences within arginine–glycine-rich domains affect mRNA-binding protein function

The discovery of roles for arginine methylation in intracellular transport and mRNA splicing has focused attention on the methylated arginine–glycine (RG)-rich domains found in many eukaryotic RNA-binding proteins. Sequence similarity among these highly repetitive RG domains, combined with interactions between RG-rich proteins, raises the question of whether these regions are general interaction motifs or whether there is specificity within these domains. Using the essential Saccharomyces cerevisiae mRNA-binding protein Npl3 (ScNpl3) as a model system, we first tested the importance of the RG domain for protein function. While Npl3 lacking the RG domain could not support growth of cells lacking Npl3, surprisingly, expression of the RG domain alone supported partial growth of these cells. To address the specificity of this domain, we created chimeric forms of ScNpl3 with RG-rich domains of S. cerevisiae nucleolar proteins, Gar1 and Nop1 (ScGar1, ScNop1), or of the Candida albicans Npl3 ortholog (CaNpl3). Whereas the CaNpl3 RG chimeric protein retained nearly wild-type function in S. cerevisiae, the ScGar1 and ScNop1 RG domains significantly reduced Npl3 function and self-association, indicating RG domain specificity. Nuclear localization of Npl3 also requires specific RG sequences, yet heterologous RG domains allow similar modulation of Npl3 transport by arginine methylation

Biosynthesis of Mitochondrial Porin and Insertion into the Outer Mitochondrial Membrane of Neuruspora crassa

Mitochondrial porin, the major protein of the outer mitochondrial membrane is synthesized by free cytoplasmic polysomes. The apparent molecular weight of the porin synthesized in homologous or heterologous cell-free systems is the same as that of the mature porin. Transfer in vitro of mitochondrial porin from the cytosolic fraction into the outer membrane of mitochondria could be demonstrated. Before membrane insertion, mitochondrial porin is highly sensitive to added proteinase; afterwards it is strongly protected. Binding of the precursor form to mitochondria occurs at 4°C and appears to precede insertion into the membrane. Unlike transfer of many precursor proteins into or across the inner mitochondrial membrane, assembly of the porin is not dependent on an electrical potential across the inner membrane

Transport of Proteins into Mitochondria

The mitochondrial ADP/ATP carrier is an integral transmembrane protein of the inner membrane. It is synthesized on cytoplasmic ribosomes. Kinetic data suggested that this protein is transferred into mitochondria in a posttranslational manner. The following results provide further evidence for such a mechanism and provide information on its details. 1. In homologous and heterologous translation systems the newly synthesized ADP/ATP carrier protein is present in the postribosomal supernatant. 2. Analysis by density gradient centrifugation and gel filtration shows, that the ADP/ATP carrier molecules in the postribosomal fraction are present as soluble complexes with apparent molecular weights of about 120000 and 500000 or larger. The carrier binds detergents such as Triton X-100 and deoxycholate forming mixed micelles with molecular weights of about 200000–400000. 3. Incubation of a postribosomal supernatant of a reticulocyte lysate containing newly synthesized ADP/ATP carrier with mitochondria isolated from Neurospora spheroplasts results in efficient transfer of the carrier into mitochondria. About 20–30% of the transferred carrier are resistant to proteinase in whole mitochondria. The authentic mature protein is also largely resistant to proteinase in whole mitochondria and sensitive after lysis of mitochondria with detergent. Integrity of mitochondria is a prerequisite for translocation into proteinase resistant position. 4. The transfer in vitro into a proteinase-resistant form is inhibited by the uncoupler carbonyl-cyanide m-chlorophenylhydrazone but not the proteinase-sensitive binding. These observations suggest that the posttranslational transfer of ADP/ATP carrier occurs via the cytosolic space through a soluble oligomeric precursor form. This precursor is taken up by intact mitochondria into an integral position in the membrane. These findings are considered to be of general importance for the intracellular transfer of insoluble membrane proteins. They support the view that such proteins can exist in a water-soluble form its precursors and upon integration into the membrane undergo a conformational change. Uptake into the membrane may involve the cleavage of an additional sequence in some proteins, but this appears not to be a prerequisite as demonstrated by the ADP/ATP carrier protein