Non–Culture-Based Methods for the Diagnosis of Invasive Candidiasis

Given the limitations of current fungal diagnostics, the use of non–culture-based methods for the diagnosis of invasive candidiasis (IC) is highly warranted. The implementation of molecular diagnostic strategies could permit the timely onset of appropriate therapy and may be expected to pave the way for improved clinical outcome of IC. Polymerase chain reaction (PCR) may have higher sensitivity for the diagnosis of IC than conventional blood cultures. The detection of fungal antigens generally requires a large fungal burden, and the presence of fungus-specific antibodies may not correlate with the underlying diseases. Therefore, the combined mannan and anti-mannan antibody testing is recommended. No single test has been shown convincingly to compensate for all the limitations of culture. Real-time PCR coupled with fungal culture and/or antigen detection will likely be required to significantly ameliorate the diagnostic problems in IC.     

Social Dimensions of the Human–Avian Bond: Parrots and Their Persons

Though birds are among the most popular companion animals in the United States, little scholarly research has focused on the human– companion parrot relationship. This study uses an ethnographic approach and qualitative analysis to examine the parrot–pet owner relationship. Two and onehalf weeks of ethnographic fieldwork were carried out in a veterinary clinic specializing in avian and exotic medicine. These observations complement the results of quantitative data and qualitative analysis of texts from questionnaires completed by 100 parrot owners outside the clinic. Both textual analysis and observations in the veterinary clinic revealed some interesting insights into the social dimensions of the human–companion parrot relationship, which was rated superior to that of cats and dogs by some bird owners. Various patterns of human–avian interactions emerged from the data, including childhood experience with birds, affection and physical contact with birds, birds as family members and the nature of the human–parrot bond, infantilization (delayed weaning and parrot as child surrogate), anthropomorphism (celebration of holidays, diet, death and spirituality, and misinterpretation of bird behavior), intersubjectivity and cognition, and anthropocentrism (bird as object). Misinterpretation of bird behavior and failure to recognize the unique physiological and social needs of their species may lead to impaired welfare. On the other hand, according the bird a social status as cherished family member, may enhance their welfare. In addition, other factors are considered that may enhance or detract from the welfare of companion parrots. In a discussion of hypotheses regarding the human–pet bond, it is concluded that the data presented by this study best support the social support hypothesis.     

A New Paradigm for Enzymatic Control of α-Cleavage and β-Cleavage of the Prion Protein

The cellular form of the prion protein (PrP^C) is found in both full-length and several different cleaved forms in vivo. Although the precise functions of the PrP^C proteolytic products are not known, cleavage between the unstructured N-terminal domain and the structured C-terminal domain at Lys-109↓His-110 (mouse sequence), termed α-cleavage, has been shown to produce the anti-apoptotic N1 and the scrapie-resistant C1 peptide fragments. β-Cleavage, residing adjacent to the octarepeat domain and N-terminal to the α-cleavage site, is thought to arise from the action of reactive oxygen species produced from redox cycling of coordinated copper. We sought to elucidate the role of key members of the ADAM (a disintegrin and metalloproteinase) enzyme family, as well as Cu²⁺ redox cycling, in recombinant mouse PrP (MoPrP) cleavage through LC/MS analysis. Our findings show that although Cu²⁺ redox-generated reactive oxygen species do produce fragmentation corresponding to β-cleavage, ADAM8 also cleaves MoPrP in the octarepeat domain in a Cu²⁺- and Zn²⁺-dependent manner. Additional cleavage by ADAM8 was observed at the previously proposed location of α-cleavage, Lys-109↓His-110 (MoPrP sequencing); however, upon addition of Cu²⁺, the location of α-cleavage shifted by several amino acids toward the C terminus. ADAM10 and ADAM17 have also been implicated in α-cleavage at Lys-109↓His-110; however, we observed that they instead cleaved MoPrP at a novel location, Ala-119↓Val-120, with additional cleavage by ADAM10 at Gly-227↓Arg-228 near the C terminus. Together, our results show that MoPrP cleavage is far more complex than previously thought and suggest a mechanism by which PrP^C fragmentation responds to Cu²⁺ and Zn²⁺.     

Synthesis of 2-Deoxy-β-D-ribose 1-Phosphate,NMR Comparison and Its Enzymatic Activity for Structural Elucidation of Synthetic α-Isomer

Abstract

2-Deoxy-β-D-ribose 1-phosphate (1) was synthesized in a stereoselective manner and isolated with no detectable contamination by its α-isomer (4). Explicit configuration of 4 was first determined by NMR comparison with 1 judging from NOE results and their coupling constants. Natural purine nucleoside phosphorylase (PNPase) did not recognize 1 and gave no products such as α- or β-deoxynucleosides.     

Regioselectivity in β-Galactosidase-catalyzed Transglycosylation for the Enzymatic Assembly of D-Galactosyl-D-mannose

The regioselectivity of β-galactosidase derived from Bacillus circulans ATCC 31382 (β-1,3-galactosidase) in transgalactosylation reactions using D-mannose as an acceptor was investigated. This D-mannose associated regioselectivity was found to be different from reactions using either GlcNAc or GalNAc as acceptors, not only for β-1,3-galactosidase but also for β-galactosidases of different origins. The relative hydrolysis rate of Galβ-pNP and D-galactosyl-D-mannoses, of various linkages, was also measured in the presence of β-1,3-galactosidase and was found to correlate well with the ratio of disaccharides formed by transglycosylation. The unexpected regioselectivity using D-mannose can therefore be explained by an anomalous specificity in the hydrolysis reaction. By utilizing the identified characteristics of both regioselectivity and hydrolysis specificity using D-mannose, an efficient method for enzymatic synthesis of β-1,3-, β-1,4- and β-1,6-linked D-galactosyl-D-mannose was subsequently established.     

Perturbation Theory in the Catalytic Rate Constant of the Henri–Michaelis–Menten Enzymatic Reaction

The Henry–Michaelis–Menten (HMM) mechanism of enzymatic reaction is studied by means of perturbation theory in the reaction rate constant k ₂ of product formation. We present analytical solutions that provide the concentrations of the enzyme (E), the substrate (S), as well as those of the enzyme-substrate complex (C), and the product (P) as functions of time. For k ₂ small compared to k _?1, we properly describe the entire enzymatic activity from the beginning of the reaction up to longer times without imposing extra conditions on the initial concentrations E _o and?S _o , which can be comparable or much different.     

Enzymatic Resolution of (±)-Epoxy-β-cyclogeraniol,a Synthetic Precursor for Abscisic Acid Analogs

Lipase-catalyzed optical resolution of (±)-epoxy-β-cyclogeraniol (1), a key synthetic intermediate for epoxy-β-ionylideneacetic acid, was achieved in high enantiomeric purity. Transesterification with vinyl acetate by using lipase P (Nagase) made enriched (-)-1, while hydrolysis of the corresponding acetate by using lipase P (Amano) afforded (+)-1 with a high E value (E=1600).     

Biotechnology education and the training of engineer-technologists in the St. Petersburg State Chemical–Pharmaceutical Academy and the State University of Technology and Design

Some facts about biotechnological education in St. Petersburg (Russia) are presented by authors at two different higher schools: the State Chemical–Pharmaceutical Academy and the State University of Technology and Design.     

Electron-paramagnetic-resonance measurements of the electron-transfer components of the reaction centre of Rhodopseudomonas viridis. Oxidation–reduction potentials and interactions of the electron acceptors

Oxidation-reduction potentiometry was carried out on Rhodopseudomonas viridis chromatophores. Measurements of e.p.r. signals of the semiquinone-iron type at g=1.82 have revealed a more complex situation than previously reported. The presence of three different components is indicated. The midpoint potential (E(m)) of the primary acceptor quinone/semiquinone couple was found to be approx. -165mV at pH10, with a pK being reached at around pH7.5. The primary acceptor also accepts a second electron with an E(m) of -525mV, but this redox transition exhibits a hysteresis effect. Interaction effects indicate the presence of another component with E(m) values at pH10 of approx. -165mV (pK reached at around pH7.5) for single reduction and -350mV (pK at pH10 or greater) for double reduction. It is suggested that this component is the secondary acceptor. Another semiquinone-iron-type component which gives a g=1.82 signal is also present. This component is distinguishable from the primary acceptor by its e.p.r. spectrum, which shows a double peak at g=1.82 and a g(x) line at g=1.76. This component has E(m) values at pH10 for single and double reduction of -15mV and approx. -150mV respectively. Both of these E(m) values are pH-dependent. The presence of an interaction between this component and the photoreduced primary acceptor indicates the close proximity of these components. However, the midpoint potential of this component indicates a function as a secondary electron-transport component rather than an electron acceptor in the reaction centre. The dependence of the bacteriopheophytin intermediate (I) doublet e.p.r. signal on the presence of the semiquinone-iron form of the primary acceptor is demonstrated. The midpoint potential of the I/I(-) couple is estimated to be lower than -600mV.     

Contribution of charged and polar residues for the formation of the E1–E2 heterodimer from Hepatitis C Virus

The transmembrane domains of the envelope glycoprotein E1 and E2 have crucial multifunctional roles in the biogenesis of hepatitis C virus. We have performed molecular dynamics simulations to investigate a structural model of the transmembrane segments of the E1–E2 heterodimer. The simulations support the key role of the Lys370–Asp728 ion pair for mediating the E1–E2 heterodimerization. In comparison to these two residues, the simulation results also reveal the differential effect of the conserved Arg730 residue that has been observed in experimental studies. Furthermore, we discovered the formation of inter-helical hydrogen bonds via Asn367 that stabilize dimer formation. Simulations of single and double mutants further demonstrate the importance of the ion-pair and polar interactions between the interacting helix monomers. The conformation of the E1 fragment in the simulation of the E1–E2 heterodimer is in close agreement with an NMR structure of the E1 transmembrane segment. The proposed model of the E1–E2 heterodimer supports the postulated cooperative insertion of both helices by the translocon complex into the bilayer.     

Dimensions of the Human–Animal Bond and Evacuation Decisions among Pet Owners during Hurricane Ike

ABSTRACT

Flagship species play an important role in promoting nature conservation. However, although the significance of invertebrates in biodiversity and ecosystem services is undisputed, they are rarely used as flagship species. A focused approach to better understand the drivers of differences in attitudes toward invertebrates, and insects in particular, would be helpful for selecting and establishing insects as flagship species, especially in a local context for local conservation purposes. Using a standard questionnaire, a total of 363 children, predominantly aged 10 to 12 years, were asked about their attitudes toward 18 invertebrate species indigenous to Switzerland. The species, 14 insect species and four other invertebrates, were individually presented in a color photograph without any background information. Based on ordinal regression models, the survey revealed substantial affinity rating differences across the invertebrates selected. Gender, species knowledge, preferred leisure activities, and family membership of a nature protection organization proved to be significant predictors for children's attitudes in general, and for some specific species in particular. Additionally, existing species knowledge was analyzed and was found to neither depend on school location (urban/rural) nor on gender. The authors propose the inclusion of local invertebrates and species knowledge in the curriculum in early years at primary school while applying teaching methods that allow for real-life experience.     

Loop C and the mechanism of acetylcholine receptor–channel gating

Agonist molecules at the two neuromuscular acetylcholine (ACh) receptor (AChR) transmitter-binding sites increase the probability of channel opening. In one hypothesis for AChR activation (“priming”), the capping of loop C at each binding site transfers energy independently to the distant gate over a discrete structural pathway. We used single-channel analyses to examine the experimental support for this proposal with regard to brief unliganded openings, the effects of loop-C modifications, the effects of mutations to residues either on or off the putative pathway, and state models for describing currents at low [ACh]. The results show that (a) diliganded and brief unliganded openings are generated by the same essential, global transition; (b) the radical manipulation of loop C does not prevent channel opening but impairs agonist binding; (c) both on- and off-pathway mutations alter gating by changing the relative stability of the open-channel conformation by local interactions rather than by perturbing a specific site–gate communication link; and (d) it is possible to estimate directly the rate constants for agonist dissociation from and association to both the low and high affinity forms of the AChR-binding site by using a cyclic kinetic model. We conclude that the mechanism of energy transfer between the binding sites and the gate remains an open question.     

Chemical and microbiological characteristics of rice husk bedding having distinct depths and used for growing–finishing swine

This study compared the effects of different bedding depths on the chemical and microbiological characteristics of the bedding material used to raise pigs during growing and finishing. The experiment was conducted in two pens housing 5 pigs from 60 to 145 days of age, with rice husk beddings 0.50 or 0.25 m deep. Four lots of pigs (replicates) were raised over time in each bedding depth: each bedding was used by two consecutive lots. Bedding samples were collected quarterly to determine the most probable number (MPN) of thermophilic and mesophilic bacteria, fungi and actinomycetes. Contents of N, P, K, C, organic, mineral and dry matter, C:N ratio and pH were also determined. The MPN of thermophilic bacteria was higher for the 0.50 m than for the 0.25 m bedding (p < 0.05). The compost of 0.25 m deep bedding had a higher N, P and K content than that from the 0.50 m bedding (p < 0.05). Thus, the use of the 0.25 m deep bedding would be recommended due to its greater agronomical value in comparison with the deeper bedding.     

Systems for the detection and analysis of protein–protein interactions

The analysis of protein–protein interactions is important for developing a better understanding of the functional annotations of proteins that are involved in various biochemical reactions in vivo. The discovery that a protein with an unknown function binds to a protein with a known function could provide a significant clue to the cellular pathway concerning the unknown protein. Therefore, information on protein–protein interactions obtained by the comprehensive analysis of all gene products is available for the construction of interactive networks consisting of individual protein–protein interactions, which, in turn, permit elaborate biological phenomena to be understood. Systems for detecting protein–protein interactions in vitro and in vivo have been developed, and have been modified to compensate for limitations. Using these novel approaches, comprehensive and reliable information on protein–protein interactions can be determined. Systems that permit this to be achieved are described in this review.K. Kuroda, M. Kato and J. Mima contributed equally to this work.     

Systematic Comparative Evaluation of Methods for Investigating the TCRβ Repertoire

High-throughput sequencing has recently been applied to profile the high diversity of antibodyome/B cell receptors (BCRs) and T cell receptors (TCRs) among immune cells. To date, Multiplex PCR (MPCR) and 5’RACE are predominately used to enrich rearranged BCRs and TCRs. Both approaches have advantages and disadvantages; however, a systematic evaluation and direct comparison of them would benefit researchers in the selection of the most suitable method. In this study, we used both pooled control plasmids and spiked-in cells to benchmark the MPCR bias. RNA from three healthy donors was subsequently processed with the two methods to perform a comparative evaluation of the TCR β chain sequences. Both approaches demonstrated high reproducibility (R² = 0.9958 and 0.9878, respectively). No differences in gene usage were identified for most V/J genes (>60%), and an average of 52.03% of the CDR3 amino acid sequences overlapped. MPCR exhibited a certain degree of bias, in which the usage of several genes deviated from 5’RACE, and some V-J pairings were lost. In contrast, there was a smaller rate of effective data from 5’RACE (11.25% less compared with MPCR). Nevertheless, the methodological variability was smaller compared with the biological variability. Through direct comparison, these findings provide novel insights into the two experimental methods, which will prove to be valuable in immune repertoire research and its interpretation.