Further improvement of broad specificity hapten recognition with protein engineering

Sulfa-antibiotics (sulfonamides) are widely used in veterinary medicine. Meat and milk from treated animals can be contaminated with sulfa residues. Current sulfonamide assays are unfit for screening of food, because they are either too laborious, insensitive or specific for a few sulfa compounds only. An immunoassay for detection of all sulfas in a single reaction would be useful for screening. Previously we have improved the broad specificity sulfa binding of antibody 27G3 with random mutagenesis and phage display. In order to improve the properties of this antibody further, mutants from the previous study were recombined and more mutations introduced. These new libraries were enriched with phage display and several different mutant antibodies were isolated. The cross-reaction profile of the best mutant was better than that of the wild-type antibody and the mutants of the previous study: it was capable of binding 10 of the tested 13 sulfonamides within a narrow concentration range and also bound the rest of the sulfas 5- to 11-fold better than the mutants of the previous study.     

Methodologies for the characterization of microbes in industrial environments: a review

There is growing interest in research and development to develop novel tools to study, detect, and characterize microbes and their communities in industrial environments. However, knowledge about their validity in practical industrial use is still scarce. This review describes the advantages and limitations of traditional and molecular methods used for biofilm and/or planktonic cell studies, especially those performed with Listeria monocytogenes, Bacillus cereus, and/or Clostridium perfringens. In addition, the review addresses the importance of isolating the microorganisms from the industrial environment and the possibilities and future prospects for exploiting the described methods in the industrial environment.     

Phosphorylation of a peptide related to subunit c of the F0F1-ATPase/ATP synthase and relationship to permeability transition pore opening in mitochondria

A phosphorylated polypeptide (ScIRP) from the inner membrane of rat liver mitochondria with an apparent molecular mass of 3.5 kDa was found to be immunoreactive with specific antibodies against subunit c of F₀F₁-ATPase/ATP synthase (Azarashvily, T. S., Tyynelä, J., Baumann, M., Evtodienko, Yu. V., and Saris, N.-E. L. (2000). Biochem. Biophys. Res. Commun. 270, 741–744. In the present paper we show that the dephosphorylation of ScIRP was promoted by the Ca²⁺-induced mitochondrial permeability transition (MPT) and prevented by cyclosporin A. Preincubation of ScIRP isolated in its dephosphorylated form with the mitochondrial suspension decreased the membrane potential (_M) and the Ca²⁺-uptake capacity by promoting MPT. Incorporation of ScIRP into black-lipid membranes increased the membrane conductivity by inducing channel formation that was also suppressed by antibodies to subunit c. These data indicate that the phosphorylation level of ScIRP is influenced by the MPT pore state, presumably by stimulation of calcineurin phosphatase by the Ca²⁺ used to induce MPT. The possibility of ScIRP being part of the MPT pore assembly is discussed in view of its capability to induced channel activity.     

Interactions of the Males and Females of Three Sympatric Drosophila virilis-Group Species, D. montana, D. littoralis, and D. lummei, (Diptera: Drosophilidae) in Intra- and Interspecific Courtships in the Wild and in the Laboratory

Four species of the Drosophila virilis group, D. montana, D. littoralis, D. lummei, and D. ezoana, occur sympatrically in several locations in northern Europe. Courtship interactions between the flies of the three first-mentioned species were observed at malt baits in Kemi, northern Finland, to find out how the flies of different species recognize conspecific individuals and how interspecific courtships differ from intraspecific ones in the wild. Intraspecific courtships (including females of different reproductive stages) and interspecific courtships were also videotaped and analyzed in laboratory. In the wild the males courted both conspecific and allospecific females, even though the species varied in how much the males were attracted to females of different species. Interspecific courtships usually broke off when the male touched the female or when the male and/or the female vibrated his/her wings, producing acoustic cues. In the laboratory males courted conspecific females irrespective of the reproductive stage of the female, even though the courtships directed toward immature and fertilized females usually included only orienting and touching (no licking and singing). D. littoralis, and very rarely D. montana and D. lummei, males courted also allospecific females. In the few interspecific courtships between these three species, where the male proceeded to singing, females responded to male singing by vibrating their wings. This ended the courtship. It is suggested that both the chemical cues affecting female attractivity and the acoustic signals of males and females, which are produced by wing vibration, function in maintaining sexual isolation between these three species.     

Effect of nutrient enrichment on bacterioplankton biomass and community composition in mesocosms in the Archipelago Sea, northern Baltic

Effects of nutrient enrichment on the biomass and communitycomposition of heterotrophic bacteria and picocyanobacteriawere studied in large (42 m³) mesocosms in the brackish-waterArchipelago Sea (Baltic Sea) in late summer 2000 using cellcounts and denaturing gradient gel electrophoresis (DGGE) ofpolymerase chain reaction (PCR)-amplified 16S rRNA gene fragments.The identity of the major DNA bands was determined by sequencing.The obtained sequences were related to - and -proteobacteria,actinobacteria, verrucomicrobia and cyanobacteria. Nitrogenand phosphorus additions increased the biomasses of heterotrophicbacteria and picocyanobacteria and caused significant changesin their community composition judging from the DGGE bandingpatterns. Most verrucomicrobial bands had their highest relativeintensity in the control treatment and their lowest in the highernutrient addition treatment, whereas most Synechococcus-relatedbands had their lowest relative intensity in the lower nutrientaddition treatment. The responses of proteobacteria and actinobacteriawere more variable. The presence of both freshwater and marinesequences among the closest relatives to our sequences highlightsthe intermediate character of the Archipelago Sea between afreshwater and truly marine environment.     

Cryopreservation of in vitro-grown shoot tips of raspberry (Rubus idaeus L.) by encapsulation–vitrification and encapsulation–dehydration

The first efficient cryopreservation procedure for in vitro-grown shoot tips of raspberry (Rubus idaeus L.) has been developed based on encapsulation–vitrification (EnVi) and encapsulation–dehydration (EnDe). EnVi resulted in higher survival (85%) and regrowth (75%) of cryopreserved shoot tips than EnDe (65 and 50%, respectively). In both cryogenic procedures, shoots regenerated from cryopreserved shoot tips without intermediary callus formation. Histological studies showed that a much larger number of meristematic cells survived following EnVi than EnDe. The EnVi procedure was applied to seven raspberry genotypes with an average survival and regrowth of 71 and 68%, respectively. Regenerated plants showed normal morphology. Results here indicate EnVi as a simple and efficient method for long-term preservation of R. idaeus germplasm.     

Frequent HIN-1 promoter methylation and lack of expression in multiple human tumor types

HIN-1 (high in normal-1) is a candidate tumor suppressor identified as a gene silenced by methylation in the majority of breast carcinomas. HIN-1 is highly expressed in the mammary gland, trachea, lung, prostate, pancreas, and salivary gland, and in the lung, its expression is primarily restricted to bronchial epithelial cells. In this report, we show that, correlating with the secretory nature of HIN-1, high levels of HIN-1 protein are detected in bronchial lavage, saliva, plasma, and serum. To determine if, similar to breast carcinomas, HIN-1 is also silenced in tumors originating from other organs with high HIN-1 expression, we analyzed its expression and promoter methylation status in lung, prostate, and pancreatic carcinomas. Nearly all prostate and a significant fraction of lung and pancreatic carcinomas showed HIN-1 hypermethylation, and the majority of lung and prostate tumors lacked HIN-1 expression. In lung carcinomas, the degree of HIN-1 methylation differed among tumor subtypes (P = 0.02), with the highest level of HIN-1 methylation observed in squamous cell carcinomas and the lowest in small cell lung cancer. In lung adenocarcinomas, the expression of HIN-1 correlated with cellular differentiation status. Hypermethylation of the HIN-1 promoter was also frequently observed in normal tissue adjacent to tumors but not in normal tissue from noncancer patients, implying that HIN-1 promoter methylation may be a marker of premalignant changes. Thus, silencing of HIN-1 expression and methylation of its promoter occurs in multiple human cancer types, suggesting that elimination of HIN-1 function may contribute to several forms of epithelial tumorigenesis.     

Income and Physical Activity among Adults: Evidence from Self-Reported and Pedometer-Based Physical Activity Measurements

This study examined the relationship between income and physical activity by using three measures to illustrate daily physical activity: the self-reported physical activity index for leisure-time physical activity, pedometer-based total steps for overall daily physical activity, and pedometer-based aerobic steps that reflect continuous steps for more than 10 min at a time. The study population consisted of 753 adults from Finland (mean age 41.7 years; 64% women) who participated in 2011 in the follow-up of the ongoing Young Finns study. Ordinary least squares models were used to evaluate the associations between income and physical activity. The consistency of the results was explored by using register-based income information from Statistics Finland, employing the instrumental variable approach, and dividing the pedometer-based physical activity according to weekdays and weekend days. The results indicated that higher income was associated with higher self-reported physical activity for both genders. The results were robust to the inclusion of the control variables and the use of register-based income information. However, the pedometer-based results were gender-specific and depended on the measurement day (weekday vs. weekend day). In more detail, the association was positive for women and negative or non-existing for men. According to the measurement day, among women, income was positively associated with aerobic steps despite the measurement day and with totals steps measured on the weekend. Among men, income was negatively associated with aerobic steps measured on weekdays. The results indicate that there is an association between income and physical activity, but the association is gender-specific and depends on the measurement type of physical activity.