Generation of High-Affinity Chicken Single-Chain Fv Antibody Fragments for Measurement of the Pseudonitzschia pungens Toxin Domoic Acid

Antibody-based assay systems are now accepted by regulatory authorities for detection of the toxins produced by phytoplankton that accumulate in shellfish tissues. However, the generation of suitable antibodies for sensitive assay development remains a major challenge. We have examined the potential of using the chicken immune system to generate high-affinity, high-specificity recombinant antibody fragments against phytotoxins. Following immunization of the chicken with domoic acid-bovine serum albumin, a single-chain antibody variable region (scFv) gene library was generated from single V_H and V_L genes isolated from the immune cells in the spleen and bone marrow. scFvs reacting with domoic acid were isolated by phage display and affinity matured by light chain shuffling, resulting in an approximate 10-fold increase in sensitivity. The isolated scFvs were effectively expressed in Escherichia coli and readily purified by affinity chromatography. They were then used to develop a convenient and sensitive indirect competitive enzyme-linked immunosorbent assay for domoic acid, with a 50% effective dose of 156 ng/ml, which could be used reliably with shellfish extracts. This study demonstrates that chickens provide a valuable model system for the simplified, rapid generation of high-affinity recombinant antibody fragments with specificity for small toxin molecules.     

Secretion of cell-wall glycoproteins by yeast protoplasts. Effect of 2-deoxy-d-glucose and cycloheximide

The effect of 2-deoxy-d-glucose and cycloheximide on the synthesis and secretion of the cell-wall constituents protein and mannan in yeast protoplasts was examined in detail. Although the 2-deoxy-d-glucose hardly influenced protein synthesis, a significant parallel inhibition of carbohydrate and protein secretion into the medium was observed. The mechanism of this inhibition is considered as an interference of metabolites of 2-deoxy-d-glucose with the synthesis of yeast mannan. Cycloheximide, which is an effective inhibitor of protein synthesis in yeast (Kerridge, 1958), inhibited the secretion of non-diffusible carbohydrate in yeast protoplasts, but on the other hand had no effect on the activity of particulate yeast mannan synthetase. Our results clearly show that blocking the synthesis of either part of the mannan-protein complex prevents the extracellular appearance of the other component. The nature of this phenomenon is discussed.     

Disinfection of Aerosolized Pathogenic Fungi on Laboratory Surfaces: I. Tissue Phase

The effect of several fungicides on laboratory surfaces contaminated with the tissue phase of aerosolized Blastomyces dermatitidis, Coccidioides immitis, Cryptococcus neoformans, and Histoplasma capsulatum was ascertained. A statistical analysis of the data shows the correlation between fungi, surfaces, time, and concentration of disinfectant. All fungicides were effective at established times and concentrations, and the type of contaminated surface affected the fungicidal efficacy. By interpolating plotted graphs, laboratory personnel may determine, with a given fungicide, the concentration and time required to disinfect instruments, pipettes, gloves, bench tops, and floors contaminated with a tissue phase of pathogenic fungi.     

The Science of Vascular Contributions to Cognitive Impairment and Dementia (VCID): A Framework for Advancing Research Priorities in the Cerebrovascular Biology of Cognitive Decline

The World Health Organization reports that 47.5 million people are affected by dementia worldwide. With aging populations and 7.7 million new cases each year, the burden of illness due to dementia approaches crisis proportions. Despite significant advances in our understanding of the biology of Alzheimer’s disease (AD), the leading dementia diagnosis, the actual causes of dementia in affected individuals are unknown except for rare fully penetrant genetic forms. Evidence from epidemiology and pathology studies indicates that damage to the vascular system is associated with an increased risk of many types of dementia. Both Alzheimer’s pathology and cerebrovascular disease increase with age. How AD affects small blood vessel function and how vascular dysfunction contributes to the molecular pathology of Alzheimer’s are areas of intense research. The science of vascular contributions to cognitive impairment and dementia (VCID) integrates diverse aspects of biology and incorporates the roles of multiple cell types that support the function of neural tissue. Because of the proven ability to prevent and treat cardiovascular disease and hypertension with population benefits for heart and stroke outcomes, it is proposed that understanding and targeting the biological mechanisms of VCID can have a similarly positive impact on public health.     

Mating tactics in the sub-Antarctic deep-sea squid <Emphasis Type="Italic">Onykia ingens</Emphasis> (Cephalopoda: Onychoteuthidae)

The behavior of polar deep-sea nekton is very poorly known. To obtain insight into mating behavior of the abundant and ecologically important sub-Antarctic deep-sea squid Onykia ingens, our goals were to (1) quantify spermatophore production; (2) determine the preferred location for spermatangia deposition; (3) investigate whether male and female O. ingens mate with multiple mates; and (4) discuss the location of implanted spermatangia in light of mating behavior and egg fertilization. Toward this end, we examined male and female O. ingens specimens from Falkland Island and New Zealand waters. Male O. ingens store up to 198 spermatophores (mean 103 ± 61; n = 12) in their reproductive system, which are produced over a period of considerable somatic growth (200–400 mm ML), and which may have a considerable size range. Males insert their long extendible terminal organ in the mantle cavity of the female, potentially through the funnel, to deposit spermatophores in one or more of four regions on the female’s body. Most implanted spermatangia (52.5 %) were found in the funnel region, but many were also found inside the mantle cavity closer to the oviducts. Males with longer terminal organs therefore may be able to position closer to the oviducts where fertilization chances are higher than for spermatangia located in the funnel region. The number of implanted spermatangia per individual female (4–60, mean 29 ± 20; n = 24), the multiregional spermatangia deposition, and the different outer appearance of spermatangia, suggested that females have multiple mating events. Since males produce more spermatophores (up to 200) than the number of spermatangia in one region (<60), it is likely that males too mate with more than one female. We show how quantitative assessment of reproductive characteristics can provide insight into the reproductive behavior of deep-sea species for which in situ observations are currently lacking.     

Wheat plant selection for high yields entailed improvement of leaf anatomical and biochemical traits including tolerance to non-optimal temperature conditions

Assessment of photosynthetic traits and temperature tolerance was performed on field-grown modern genotype (MG), and the local landrace (LR) of wheat (Triticum aestivum L.) as well as the wild relative species (Aegilops cylindrica Host.). The comparison was based on measurements of the gas exchange (A/c_i, light and temperature response curves), slow and fast chlorophyll fluorescence kinetics, and some growth and leaf parameters. In MG, we observed the highest CO₂ assimilation rate \(\left( {{A_{{\text{C}}{{\text{O}}_2}}}} \right),\) electron transport rate (J_max) and maximum carboxylation rate \(\left( {{V_{{{\text{C}}_{\hbox{max} }}}}} \right)\). The Aegilops leaves had substantially lower values of all photosynthetic parameters; this fact correlated with its lower biomass production. The mesophyll conductance was almost the same in Aegilops and MG, despite the significant differences in leaf phenotype. In contrary, in LR with a higher dry mass per leaf area, the half mesophyll conductance (g_m) values indicated more limited CO₂ diffusion. In Aegilops, we found much lower carboxylation capacity; this can be attributed mainly to thin leaves and lower Rubisco activity. The difference in CO₂ assimilation rate between MG and others was diminished because of its higher mitochondrial respiration activity indicating more intense metabolism. Assessment of temperature response showed lower temperature optimum and a narrow ecological valence (i.e., the range determining the tolerance limits of a species to an environmental factor) in Aegilops. In addition, analysis of photosynthetic thermostability identified the LR as the most sensitive. Our results support the idea that the selection for high yields was accompanied by the increase of photosynthetic productivity through unintentional improvement of leaf anatomical and biochemical traits including tolerance to non-optimal temperature conditions.     

Mutations Causing Constitutive Invertase Synthesis in Yeast: Genetic Interactions with snf Mutations

We have selected 210 mutants able to grow on sucrose in the presence of 2-deoxyglucose. We identified recessive mutations in three major complementation groups that cause constitutive (glucose-insensitive) secreted invertase synthesis. Two groups comprise alleles of the previously identified HXK2 and REG1 genes, and the third group was designated cid1 (constitutive invertase derepression). The effect of cid1 on SUC2 expression is mediated by the SUC2 upstream regulatory region, as judged by the constitutive expression of a SUC2-LEU2-lacZ fusion in which the LEU2 promoter is under control of SUC2 upstream sequences. A cid1 mutation also causes glucose-insensitive expression of maltase. The previously isolated constitutive mutation ssn6 is epistatic to cid1, reg1 and hxk2 for very high level constitutive invertase expression. Mutations in SNF genes that prevent derepression of invertase are epistatic to cid1, reg1 and hxk2; we have previously shown that ssn6 has different epistasis relationships with snf mutations. The constitutive mutation tup1 was found to resemble ssn6 in its genetic interactions with snf mutations. These findings suggest that CID1, REG1 and HXK2 are functionally distinct from SSN6 and TUP1.