bluuebunny/crossref_metadata_2025 huggingface dataset:
Heat Treatment Effects on Strawberry Plant Survival and Angular Leaf Spot, Caused by Xanthomonas fragariae, in Nursery Production
Authors: Turechek William W. , Peres Natalia A.
Published: February 2009

Angular leaf spot is an important disease in strawberry nursery production. The European and Mediterranean Plant Protection Organization (EPPO) lists Xanthomonas fragariae as an A2 quarantine pathogen. Therefore, nurseries wishing to export plants to European countries must maintain phytosanitary standards to exclude X. fragariae. To help nurseries achieve these standards, heat treatment for killing or reducing the number of viable bacterial cells in strawberry crown tissue was investigated. First, the sensitivity of bacteria to heat was determined by dispensing 1-ml aliquots of standardized cell suspensions in microcentrifuge tubes for each of four isolates of X. fragariae, including the type culture, and submerging the tubes in water at 36, 40, 44, 48, 52, and 56°C for 0, 1, 2.5, 5, 10, 15, 30, 60, 120, 240, 360, and 480 min. Bacteria were transferred to growth medium to determine the proportion surviving heat treatment. Two trials were conducted in a greenhouse to determine the sensitivity of bare-root plants to heat treatment. In the first trial, plants of cvs. Camarosa and Diamante from two different nurseries were heat treated as follows: (i) plants placed in metallic mesh cages and immersed directly into water (industry standard, direct dip); (ii) plants sealed in a plastic bag and the bag immersed in water (bagged dry); or (iii) plants wetted in warm water, sealed in a plastic bag, and then immersed in water (bagged wet). Plants were treated at 44 or 48°C for 0, 60, 120, 180, and 240 min. In the second trial, plants of cvs. Camarosa, Camino Real, Diamante, Oso Grande, Strawberry Festival, and Ventana from a single nursery were subjected to the same treatments. In both trials, plants were potted after treatment and rated for growth characteristics. Results showed that populations of bacteria exposed to 56 and 52°C were killed completely after 15 and 60 min of exposure, respectively; both treatments killed plants. Bacterial populations exposed to 44°C for 4 h or 48°C for 2 h were reduced by 105 or 106 CFU/ml. The same treatments minimally affected vegetative growth of plants bagged dry or wet, but flowering was adversely affected. These heat treatments were selected for testing of nursery stock of several cultivars in field trials established at two locations in successive years. The survival rate among cultivars was similar to that observed in greenhouse trials, and angular leaf spot developed appreciably only in non-heat-treated control plots. Heat treatment of strawberry nursery stock is feasible and can be used to supplement standard production practices for producing pathogen-free nursery stock.
First Report of Potato spindle tuber viroid in Cape Gooseberry (Physalis peruviana) from Turkey and Germany
Authors: Verhoeven J. Th. J. , Botermans M. , Roenhorst J. W. , Westerhof J. , Meekes E. T. M.
Published: February 2009

Since the recent identification of Potato spindle tuber viroid (PSTVd) in vegetatively propagated ornamental plant species (4), many growers have asked to have their mother plants tested for this viroid. In December of 2007, a grower from Turkey submitted cuttings of cape gooseberry (Physalis peruviana) to be tested for PSTVd. Initial testing by real-time reverse transcription (RT)-PCR according to Boonham et al. (1) indicated the presence of either Mexican papita viroid, PSTVd, or Tomato chlorotic dwarf viroid in four samples. To identify the viroid(s) present, isolated RNA from these samples was used for RT-PCR (2), and products of the expected full genome size for the three viroids were amplified from each sample. One of the PCR products was sequenced (GenBank Accession No. EU862230) and analysis of the 357 nt sequence indicated it was most related to PSTVd sequences belonging to the so-called ‘Oceanian’ strain of the viroid (3), with 99.7% identity to GenBank Accession No. AY962324. Therefore, the viroid was identified as PSTVd. Pathogenicity of this PSTVd genotype was demonstrated when 4 weeks after mechanical inoculation with sap extracts seedlings of tomato cv. Money-maker showed the expected viroid symptoms of chlorosis and stunting, and the presence of the viroid in these plants was confirmed by RT-PCR (2). In March of 2008, by use of RT-PCR (2) and sequencing of the PCR product (GenBank Accession No. EU862231), PSTVd was identified in young seedlings of P. peruviana from a German grower. The German isolate differed at only three nucleotide positions from the Turkish isolate. The identification of PSTVd in young seedlings indicates that seeds had been source of infection, whereas in the case of the PSTVd infected cuttings from Turkey, the infection originated from infected mother plants. To our knowledge, these are the first reports of PSTVd in P. peruviana. Although infected P. peruviana plants did not show symptoms, they might act as sources of inoculum for crops like potato and tomato, which may suffer serious damage.

References: (1) N. Boonham et al. J. Virol. Methods 116:139, 2004. (2) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) J. Th. J. Verhoeven et al. Plant Pathol. 57:399, 2008.
First Report of the Soybean Cyst Nematode, Heterodera glycines, on Soybean in Zhejiang, Eastern China
Authors: Zheng J. , Zhang Y. , Li X. , Zhao L. , Chen S.
Published: February 2009

The soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is a destructive pest of soybean. Damage to soybean by SCN was first reported from northeastern China in 1899 (1). SCN has been documented in Anhui, Beijing, Hebei, Heilongjiang, Henan, Jiangsu, Jilin, Liaoning, Neimenggu, Shaanxi, Shandong, and Shanxi provinces in mainland China (1). These provinces are situated in the Heilongjiang and Songhuajing valleys in northeastern China and the eastern region of the Yangtze and Yellow rivers in northern China and have cold to temperate climates. In June of 2008, cyst-forming nematodes were detected in two soybean-growing areas of Hangzhou and Xiaoshan in Zhejiang Province, in subtropical eastern China. The soybean plants at the Hangzhou site showed symptoms of stunting and chlorosis, whereas no aboveground or root symptoms were observed on soybean plants at the Xiaoshan site, except for the presence of SCN females on the roots. The two populations had the same morphological and molecular characters. The cysts were lemon shaped with posterior protuberance, ambifenestrate, underbridge and bullae strongly developed, and lateral field of second-stage juveniles consisted of four incisures. The key morphometrics of cysts were fenestra length (41 to 52 μm) and width (33 to 48 μm), vulval silt (47 to 55 μm), and underbridge length (79 to 94 μm), all of which were coincident with that of SCN (2). Amplification of rDNA-internal transcribed spacer (ITS) region using primers TW81 (5′-GTT TCC GTA GGT GAA CCT GC-3′) and AB28 (5′-ATA TGC TTA AGT TCA GCG GGT-3′) yielded a PCR fragment of approximately 1,030 bp. The digestion patterns of the PCR fragments of the ITS region with AluI, AvaI, CfoI, MvaI, and RsaI showed identical restriction profiles to H. glycines (3), and the sequences exhibited 100% similarity with those of H. glycines isolates, Accession No. AY667456 from GenBank. Morphological and molecular identification confirmed that the two populations of cyst-forming nematodes from Zhejiang are SCN. To our knowledge, this is the first report of SCN in Zhejiang, now the most southern location in mainland China with confirmed infestation of SCN.

References: (1) Z. X. Liu et al. Int. J. Nematol. 7:18, 1997. (2) R. H. Mulvey. Can. J. Zool. 50:1277, 1972. (3) J. Zheng et al. Russ. J. Nematol. 8:109, 2000.
Transgenic Suppression of Cell Death Limits Penetration Success of the Soybean Rust Fungus Phakopsora pachyrhizi into Epidermal Cells of Barley
Authors: Hoefle Caroline , Loehrer Marco , Schaffrath Ulrich , Frank Markus , Schultheiss Holger , Hückelhoven Ralph
Published: February 2009

The basidiomycete Phakopsora pachyrhizi (P. pachyrhizi) causes Asian soybean rust, one of the most devastating plant diseases on soybean. When inoculated on the nonhost barley P. pachyrhizi caused only very small necrotic spots, typical for an incompatible interaction, which involves a hypersensitive cell death reaction. A microscopic inspection of the interaction of barley with P. pachyrhizi revealed that the fungus germinated on barley and formed functional appressoria on epidermal cells. The fungus attempted to directly penetrate through periclinal cell walls but often failed, arrested in plant cell wall appositions that stained positively for callose. Penetration resistance depends on intact ROR1(REQUIRED FOR mlo-SPECIFIED RESISTANCE 1) and ROR2 genes of barley. If the fungus succeeded in penetration, epidermal cell death took place. Dead epidermal cells did not generally restrict fungal development but allowed for mesophyll invasion, which was followed by mesophyll cell death and fungal arrest. Transient or stable over expression of the barley cell death suppressor BAX inhibitor-1 reduced both epidermal cell death and fungal penetration success. Data suggest that P. pachyrhizi provokes a programmed cell death facilitating fungal entry into epidermal cells of barley.
ThePi40Gene for Durable Resistance to Rice Blast and Molecular Analysis ofPi40-Advanced Backcross Breeding Lines
Authors: Suh J. P. , Roh J. H. , Cho Y. C. , Han S. S. , Kim Y. G. , Jena K. K.
Published: February 2009

Rice blast severely reduces production in both irrigated and water-stressed upland ecosystems of tropical and temperate countries. Nearly 50 blast resistance genes have been identified and some of those are incorporated into several rice cultivars. However, most of the resistance genes break down in a few years because of their race specificity and the rapid change in pathogenicity of the blast fungus (Magnaporthe grisea). The objective of this study was to analyze advanced backcross breeding lines (ABL) possessing the gene Pi40 for durable rice blast resistance. In all, 4 resistant genotypes, 4 japonica cultivars, and 10 monogenic differential rice genotypes with some known resistance genes were bioassayed in the greenhouse using seven sequential plantings and 29 virulent M. grisea isolates of Korea. The genotypes with the Pi40 gene had <3% diseased leaf area, which was significantly below the disease threshold level of 40% considered for durable blast resistance. Moreover, the genotypes with the Pi40 gene expressed compatibility with only two to three virulent M. grisea isolates supporting durability of resistance, in contrast to susceptible cultivars with >50% diseased leaf area and 10 compatible isolates. Of the 10 known resistance genes tested, Piz-t, Piz-5, and Pi9 showed differential reactions to the pathogen isolates in seven plantings. Genotyping of the ABL with 260 simple sequence repeat (SSR) markers revealed rapid conversion toward recurrent parent genotypes with fewer donor chromosomal segments (5.3 to 14.5%). Our study based on a sequential testing and background selection of breeding lines with the resistance gene Pi40 provided valuable information for durable blast resistance breeding in rice.
Laparoscopic Transdiaphragmatic Access to the Apex of the Left Ventricle of the Heart
Authors: Thompson Jess L. , Suri Rakesh M. , McKellar Stephen H. , Schaff Hartzell V.
Published: February 2009

Objective

Delivery of a collapsable aortic valve prosthesis via the left ventricular (LV) apex has proven problematic clinically. We describe our experience with a novel technique for a laparoscopic transdiaphragmatic approach to the LV outflow tract (LVOT).

Methods

Laparoscopic transabdominal access to the left ventricle was achieved in four live swine and one human cadaver using three abdominal laparoscopic trocars. Pericardioscopy and cardioscopy were performed. A hemostatic purse-string suture was placed laparoscopically at the apex of the left ventricle in one pig, and an instrument passed through the purse-string and into the LVOT. After the procedure, a thoracotomy was performed and the heart explanted for gross examination.

Results

Transdiaphragmatic exposure of the LV apex was accomplished at a mean of 23 minutes. Hemodynamic stability was maintained in all animals and the pleural cavities were not violated. Pericardioscopy allowed visualization of both right and left pulmonary veins along with the left atrial appendage. In situ cardioscopy confirmed that access to the LVOT and the aortic annulus was possible from the LV apex entry site.

Conclusions

Laparoscopic transdiaphragmatic approach to the LVOT is a possible route for minimally invasive beating heart aortic valve implantation. This strategy permits a straight transapical line of access to the aortic valve; avoiding the potential for cardiac torsion via a thoracotomy.
p53 isoform Δ113p53 is a p53 target gene that antagonizes p53 apoptotic activity via BclxL activation in zebrafish
Authors: Chen Jun , Ng Sok Meng , Chang Changqing , Zhang Zhenhai , Bourdon Jean-Christophe , Lane David P. , Peng Jinrong
Published: February 2009

p53 is a well-known tumor suppressor and is also involved in processes of organismal aging and developmental control. A recent exciting development in the p53 field is the discovery of various p53 isoforms. One p53 isoform is human Δ133p53 and its zebrafish counterpart Δ113p53. These N-terminal-truncated p53 isoforms are initiated from an alternative p53 promoter, but their expression regulation and physiological significance at the organismal level are not well understood. We show here that zebrafish Δ113p53 is directly transactivated by full-length p53 in response to developmental and DNA-damaging signals. More importantly, we show that Δ113p53 functions to antagonize p53-induced apoptosis via activating bcl2L (closest to human Bcl-xL), and knockdown of Δ113p53 enhances p53-mediated apoptosis under stress conditions. Thus, we demonstrate that the p53 genetic locus contains a new p53 response gene and that Δ113p53 does not act in a dominant-negative manner toward p53 but differentially modulates p53 target gene expression to antagonize p53 apoptotic activity at the physiological level in zebrafish. Our results establish a novel feedback pathway that modulates the p53 response and suggest that modulation of the p53 pathway by p53 isoforms might have an impact on p53 tumor suppressor activity.
Inhibition of SNF1-Related Protein Kinase1 Activity and Regulation of Metabolic Pathways by Trehalose-6-Phosphate
Authors: Zhang Yuhua , Primavesi Lucia F. , Jhurreea Deveraj , Andralojc P. John , Mitchell Rowan A.C. , Powers Stephen J. , Schluepmann Henriette , Delatte Thierry , Wingler Astrid , Paul Matthew J.
Published: February 2009

Trehalose-6-phosphate (T6P) is a proposed signaling molecule in plants, yet how it signals was not clear. Here, we provide evidence that T6P functions as an inhibitor of SNF1-related protein kinase1 (SnRK1; AKIN10/AKIN11) of the SNF1-related group of protein kinases. T6P, but not other sugars and sugar phosphates, inhibited SnRK1 in Arabidopsis (Arabidopsis thaliana) seedling extracts strongly (50%) at low concentrations (1–20 μ  m). Inhibition was noncompetitive with respect to ATP. In immunoprecipitation studies using antibodies to AKIN10 and AKIN11, SnRK1 catalytic activity and T6P inhibition were physically separable, with T6P inhibition of SnRK1 dependent on an intermediary factor. In subsequent analysis, T6P inhibited SnRK1 in extracts of all tissues analyzed except those of mature leaves, which did not contain the intermediary factor. To assess the impact of T6P inhibition of SnRK1 in vivo, gene expression was determined in seedlings expressing Escherichia coli  otsA encoding T6P synthase to elevate T6P or otsB encoding T6P phosphatase to decrease T6P. SnRK1 target genes showed opposite regulation, consistent with the regulation of SnRK1 by T6P in vivo. Analysis of microarray data showed up-regulation by T6P of genes involved in biosynthetic reactions, such as genes for amino acid, protein, and nucleotide synthesis, the tricarboxylic acid cycle, and mitochondrial electron transport, which are normally down-regulated by SnRK1. In contrast, genes involved in photosynthesis and degradation processes, which are normally up-regulated by SnRK1, were down-regulated by T6P. These experiments provide strong evidence that T6P inhibits SnRK1 to activate biosynthetic processes in growing tissues.
Rituximab‐based immunosuppression for autoimmune haemolytic anaemia in infants
Authors: Svahn Johanna , Fioredda Francesca , Calvillo Michaela , Molinari Angelo C. , Micalizzi Concetta , Banov Laura , Schmidt Madalina , Caprino Daniela , Marinelli Doretta , Gallisai Domenico , Dufour Carlo
Published: February 2009

We report a case series of four infants with severe autoimmune haemolytic anaemia (AIHA) who responded to treatment with rituximab and cyclosporine after having failed first line therapy with high‐dose steroid (prednisolone 4–8 mg/kg/d). Rituximab was started at 11–90 d from onset due to continued haemolysis; three infants also received cyclosporine A. Three of four infants reached complete response, defined as normal haemoglobin, reticulocytes and negative indices of haemolysis, at 7–21 months from diagnosis. In long‐term follow‐up two infants remained disease‐free with normal immunology, one had undefined immunodeficiency and one had autoimmune lymphoproliferative syndrome.
Composition of the immunoglobulin classic antigen‐binding site regulates allergic airway inflammation in a murine model of experimental asthma
Authors: Kerzel S. , Wagner J. , Rogosch T. , Yildirim A.‐Ö. , Sikula L. , Fehrenbach H. , Garn H. , Maier R. F. , Schroeder Jr H. W. , Zemlin M.
Published: February 2009

Background
When bound to mast cell FcɛRI, IgE serves as antigen receptor for allergic reactions, permitting specific identification of the allergen. Although the core of the classic antigen‐binding site is heavy chain complementarity determining region 3 (CDR‐H3), recent studies suggest that allergens might also bind IgE in a superantigen‐like fashion outside the classic antigen‐binding site.

Objective
We sought to evaluate the contribution of the classic CDR‐H3‐centric antigen‐binding site to the development of an allergic phenotype.

Methods
Using a murine model of experimental asthma, we characterized a gene‐targeted mouse strain expressing an altered range of CDR‐H3s (ΔD‐iD mice) in response to the hydrophobic allergen ovalbumin (OVA). Mutant and wild‐type (wt) mice were sensitized intraperitoneally with OVA; non‐sensitized mice served as controls.

Results
We found the composition of the classic CDR‐H3‐centric antigen‐binding site to be critical for the development of characteristic aspects of allergic asthma. (i) Compared with wt animals, ΔD‐iD mice showed a significantly less pronounced OVA ‐induced rise in allergen‐specific IgE levels and hence in total serum IgE levels. (ii) In addition, ΔD‐iD mice demonstrated a significant reduction in eosinophilic airway inflammation, as well as in interleukin‐4 (IL‐4), IL‐5 and IL‐13 levels in BAL fluids.

Conclusion
Allergic sensitization and airway inflammation depend on the composition of the predominant CDR‐H3 repertoire, suggesting that the classic CDR‐H3‐centric antigen‐binding site plays a crucial role in creating the immunological interface between allergen and IgE. Our results further emphasize a central role of IgE, not only in mediating but also in regulating the allergic immune response.
T‐cell seeding: neonatal transfer of anti‐myelin basic protein T‐cell lines renders Fischer rats susceptible later in life to the active induction of experimental autoimmune encephalitis
Authors: Volovitz Ilan , Mor Felix , Machlenkin Athur , Goldberger Ofir , Marmor Yotvat , Eisenbach Lea , Cohen Irun
Published: February 2009

Fischer strain rats resist active induction of experimental autoimmune encephalomyelitis (EAE) following immunization with guinea‐pig myelin basic protein (MBP) in complete Freund’s adjuvant (CFA). Nevertheless, we now report that an encephalitogenic CD4+ anti‐MBP T‐cell line could be developed from actively immunized Fischer rats. Adoptive transfer of the activated line mediated acute EAE in adult Fischer rats, but not in 1‐day‐old rats. Moreover, we found that both resting and activated anti‐MBP T cells injected 1 day post‐natally rendered these rats susceptible later in life to the active induction of EAE by immunization with MBP/CFA. The actively induced EAE manifested the accelerated onset of a secondary, memory‐type response. Resting anti‐MBP T cells injected even up to 2 weeks post‐natally produced no clinical signs but seeded 50–100% of the recipients for an active encephalitogenic immune response to MBP. An earlier T‐cell injection (1–2 days) produced a higher incidence and stronger response. The transferred resting T cells entered the neonatal spleen and thymus and proliferated there but did not change the total anti‐MBP precursor number in adults. Splenocytes harvested from rats that were injected neonatally but not exposed to MBP in vivo proliferated strongly and produced significant amounts of interferon‐γ to MBP in vitro. Similar results were observed in rats injected with resting T‐cell lines reactive to ovalbumin, suggesting that the neonatal injection of resting T cells specific for a self or for a foreign antigen can seed the immune system with the potential for an enhanced effector response to that antigen later in life.
Adenosine regulates CD8 T‐cell priming by inhibition of membrane‐proximal T‐cell receptor signalling
Authors: Linnemann Carsten , Schildberg Frank A. , Schurich Anna , Diehl Linda , Hegenbarth Silke I. , Endl Elmar , Lacher Svenja , Müller Christa E. , Frey Jürgen , Simeoni Luca , Schraven Burkhart , Stabenow Dirk , Knolle Percy A.
Published: February 2009

Adenosine is a well‐described anti‐inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naïve CD8 T cells by professional and semi‐professional antigen‐presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naïve CD8 T cells after αCD3/CD28‐mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naïve CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T‐cell priming by professional antigen‐presenting cells (dendritic cells) and semi‐professional antigen‐presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP‐70 as well as Akt and ERK1/2 in naïve αCD3/CD28‐stimulated CD8 cells. Consequently, αCD3/CD28‐induced calcium‐influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane‐proximal T‐cell receptor signalling and thereby also differentiation of naïve CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T‐cell priming and differentiation in peripheral organs.
Serological evidence of infection with hepatitis E virus among wild Yezo‐deer, Cervus nippon yesoensis, in Hokkaido, Japan
Authors: Tomiyama D. , Inoue E. , Osawa Y. , Okazaki K.
Published: February 2009

Summary.  In this study, 520 serum samples from Yezo‐deer in the Hidaka district, Hokkaido, Japan were examined by enzyme‐linked immunosorbent assay to investigate whether the animals were infected with hepatitis E virus (HEV). The distribution of optical density values showed a bimodal pattern and 181 samples (34.8%) were deemed to be antibody‐positive against HEV. At least five (2.8%) of the positive sera gave specific bands by Western blot analysis. An age‐dependent increase in prevalence of the antibodies was found among the animals. These findings indicate that Yezo‐deer are a possible host for HEV infection. To avoid the risk of becoming HEV infected, the consumption of raw Yezo‐deer meat must be prohibited.
GLP‐1 regulates gastroduodenal motility involving cholinergic pathways
Authors: Schirra J. , Nicolaus M. , Woerle H. J. , Struckmeier C. , Katschinski M. , Göke B.
Published: February 2009

Abstract  The gut‐born incretin hormone glucagon‐like peptide‐1 (GLP‐1) delays gastric emptying. To elucidate the mechanisms by which GLP‐1 affects gastroduodenal motility and glycaemia, we studied the effects of exendin(9–39), a potent GLP‐1 receptor antagonist, on gastroduodenal motility and pancreatic hormones. In this randomized, double‐blind, placebo‐controlled, four‐arm, cross‐over trial, 10 healthy volunteers were studied during the interdigestive period followed by duodenal perfusion of a mixed liquid meal (250 kcal). On four separate days, exendin(9–39), atropine, exendin(9–39) + atropine or saline were infused intravenously. Antro‐pyloro‐duodenal and fundic motility were assessed. The compliance of the proximal stomach was determined by isobaric distensions. During fasting, exendin(9–39) did not influence proximal gastric volume, pyloric tone, and duodenal contractility. Exendin(9–39) significantly increased antral waves only in the absence of atropine. During duodenal meal perfusion, exendin(9–39) significantly reduced proximal gastric volume accommodation, abbreviated postprandial antral inhibition, reduced the postprandial increase in pyloric tone, and reduced gastric compliance. Atropine abolished the effects of exendin(9–39) on gastric volume accommodation but did not affect its effects on postprandial antroduodenal motility and on gastric compliance. Exendin(9–39) increased fasting and postprandial glycaemia and plasma glucagon but not insulin concentrations. Atropine did not affect GLP‐1 secretion. Cholinergic mechanisms mediate the effects of GLP‐1 on postprandial gastric accommodation but not on antro‐pyloro‐duodenal motility. GLP‐1 reduces fasting and postprandial glycaemia, in part by reducing glucagon secretion.
Comparison of Antigens: the Reaction of “Identity”
Authors: Axelsen N. H. , Bock Elisabeth , Krøll J.
Published: February 2009

Summary. The many possibilities for comparison of two antigen samples by means of quantitative immunoelectrophoresis methods are reviewed in diagrams.
II. Comparison of Antigens: the Reaction of Partial Identity
Authors: BOCK ELISABETH , AXELSEN N. H.
Published: February 2009

Summary. The patterns of partial identity obtained by various quantitative immunoelectrophoretic methods are reviewed by means of diagrams. The methods are sensitive and the reaction of partial identity is characteristic and easily recognized when it appears, even in complicated systems.
16. Red Cell Carbonic Anhydrases B and C (CA B and CA C)
Authors: NøRGAARD‐PEDERSEN BENT
Published: February 2009

Summary. After carbamylation of unpurified haemolysates the human carbonic anhydrases B and C are determined by rocket immunoelectrophoresis. Quantitation of CA B is of diagnostic value in thyroid disorders since a definite low concentration is observed in hyperthyroidism and a less constant elevation is observed in hypothyroidism. In cord blood from newborn infants a close positive correlation is found between both CA B and CA C and gestational age. Without carbamylation it is possible to quantitate only the CA B enzyme.
18. Determination of Insulin Binding to Human IgG by Quantitative Radioimmunoelectrophoresis
Authors: CHRISTIANSEN AA. HEIN , Krøll J.
Published: February 2009

Summary. The quantitative radioimmunoelectrophoresis for the determination of insulin binding to IgG is described. In 95% of 267 Danish diabetics treated with insulin, insulin‐binding IgG has been demonstrated. The method has the advantage of being specific, sensitive, and simple. A number of precautionary measures to be taken when using isotopes in electrophoresis are mentioned.
20. Carbamylated Antibodies Used for Quantitation of Human IgG. A Routine Method
Authors: BJERRUM O. J. , INGILD A. , LØWENSTEIN H. , WEEKE B.
Published: February 2009

Summary. In the present paper it is demonstrated that the pH at which rocket immunoelectrophoresis can be performed is not restricted to the conventional value of 8.6. By carbamylation the pI of rabbit antibodies was lowered until their mean electrophoretic migration in an agarose gel at pH 5.0 was zero. Using such carbamylated antibodies, IgG from normal human sera were quantitated by rocket immunoelectrophoresis at pH 5.0. The results obtained were shown to be concordant with the IgG concentrations determined at pH 8.6 using carbamylated antigen as described by Weeke (134). The advantage of utilizing carbamylated antibodies for quantitation of IgG in routine laboratory work is pointed out, and additional applications are stated.
Antisera against Purified Asialo‐Orosomucoid and Asialo‐Cholinesterase
Authors: BROGREN C.‐H. , BøG‐HANSEN T. C. , SVENDSEN P. JUST , BJERRUM O. J.
Published: February 2009

Rabbits were immunized with highly purified desialylated human serum cholinesterase and orosomucoid. In immunoelectrophoresis and immunodiffusion the resulting antibodies had precipitation properties which were identical to those of antibodies against the native proteins. Spurring between native and asialo‐proteins was not seen. A linear correlation was observed between the rocket height obtained with the antiserum raised against the asialo‐protein and the rocket height with a normal antiserum. A monospecific antiserum against cholinesterase was obtained without absorption. The use of desialylated glycoproteins as immunogens for the production of monospecific antisera is discussed.
An Artefact in Quantitative Immunoelectrophoresis of Spectrin Caused by Proteolytic Activity in Antibody Preparations
Authors: BJERRUM O. J. , RAMLAU J. , CLEMMESEN I. , INGILD AGNETE , BøG‐HANSEN T. C.
Published: February 2009

Many antibody preparations exhibit proteolytic activity due to the presence of plasmin. In crossed immunoelectrophoresis at pH 8.6 this enzyme can degrade certain proteins during electrophoresis in the antibody‐containing gel, resulting in artefacts in the form of extra precipitation arcs of congruent shape. The degradation behaviour of spectrin, a major protein of human erythrocyte membranes, was investigated. The artefact could be completely abolished by the addition of protease inhibitors, e.g. aprotinin and soya bean trypsin inhibitor, to the antibody preparations.
Analysis of Partially Degraded Proteins by Quantitative Immunoelectrophoresis
Authors: BJERRUM O. J. , BøG‐HANSEN T. C.
Published: February 2009

For the recognition of partially degraded proteins in quantitative immunoelectrophoresis, controlled proteolytic degradation of two model systems was investigated: human serum proteins and solubilized human erythrocyte membrane proteins. The following characteristic precipitates were found: 1) unending ('flying') precipitates, 2) precipitates splitting into several lines, 3) precipitation arcs showing shoulders or double peaks ('camels'), and 4) skew precipitation arcs. The same types of precipitates were also found for solubilized erythrocyte membrane proteins containing contaminating proteolytic enzymes and normal concentrated urine proteins.
Thin‐Layer Gel Filtration Immunoelectrophoresis
Authors: NIELSEN C. SCHäFER
Published: February 2009

A method that directly combines separation of proteins by thin‐layer gel filtration (TLG) with quantitative immunoelectrophoresis is described. All the operations are performed in monolayer gels on a single glass plate. The resolving power of the method is comparable to that of an ordinary column technique monitored by fused rocket immunoelectrophoresis.
The Adsorption of Serum Proteins to Aluminium Hydroxide Gel Examined by Means of Quantitative Immunoelectrophoresis
Authors: WEEKE B. , WEEKE E. , LøWENSTEIN H.
Published: February 2009

By means of crossed immunoelectrophoresis with intermediate gel containing a known amount of aluminium hydroxide the adsorption of the individual human serum proteins to the aluminium hydroxide was determined. The extent of adsorption varies considerably with the particular protein and is independent of the net charge, the molecular weight and the carbohydrate content of the proteins. The possible use of the method in connection with diagnosis and immunotherapy in human allergology is outlined.
Identification of Bovine Serum Proteins by Quantitative Immunoelectrophoretic Methods
Authors: ANDERSEN METTE MARIE , KRøLL JENS
Published: February 2009

Line immunoelectrophoresis of bovine serum revealed a minimum of 37 bovine serum proteins. In line‐absorption immunoelectrophoresis, 13 of these proteins were identified by their cross‐reactivity with monospecific antisera against human serum proteins. One additional protein was identified by monospecific antisera against bovine immunoglobulins. Crossed immunoelectrophoresis revealed a minimum of 40 bovine proteins and demonstrated their electrophoretic mobility. In crossed‐line immunoelectrophoresis all 37 line precipitates were related to their corresponding peak precipitates and the electrophoretic mobility of the 14 proteins identificd was determined.
Immunological Removal of Contaminants: a Useful Principle in Protein Purification Applied to Human Secretory Component and J Chain
Authors: BRANDTZAEG P.
Published: February 2009

Summary.An outline is given of the purification of free secretory component (SC) from human colostrum and of J chain released from polymeric IgA by mild reduction. Immunological removal of impurities is a crucial purification step, but one that has to be combined with conventional biochemical methods. The yield of highly purified material was 10% for SC and 30% for J chain.
Sepharose‐C3
Authors: PEPYS M. B. , BELL A. J. , ROWE I. F.
Published: February 2009

Summary. Sepharose beads induced complement activation in normal human, murine and equine sera. C3 became fixed to the Sepharose, and the Sepharose‐C3 complex was used as a solid phase immunosorbent for isolation of anti‐C3 antibodies and immunoconglutinin.
Sepharose‐C3
Authors: PEPYS M. B. , BELL A. J. , ROWE I. F.
Published: February 2009

Summary. Effective conditions were devised for the specific depletion from lymphocyte populations of complement receptor lymphocytes (CRL) on columns of Sepharose‐C3. CRL among both human and murine lymphocytes were found by this method to be a subpopulation of B cells.
Purification, Fractionation and Assay of Antibody‐Dependent Lymphocytic Effector Cells (K Cells) in Human Blood
Authors: PERLMANN H. , PERLMANN P. , PAPE G. R. , HALLDEN G.
Published: February 2009

Abstract. In this article we present methods for the purification and fractionation of human blood lymphocytes, which have been used in our laboratory to characterize antibody‐dependent cytotoxic effector cells (K cells). The assay system consists of highly purified lymphocytes, 51Cr‐labelled chicken erythrocytes (Ec) and IgG rabbit anti‐Ec in high dilutions. Various ways of comparing K‐cell potentials of different lymphocyte preparations in this system are discussed. When purified lymphocytes are partially depleted (60‐85% depletion) of cells forming rosettes with sheep erythrocytes (E+ cells), the K‐cell activity of the depleted fraction is increased, indicating that the majority of the E+ cells are inactive in this assay. Depletion of EAC‐rosette‐forming cells shows that most or all K cells have complement receptors. For depletion of B cells, the lymphocytes may be passed through glass bead columns, charged with F(ab')2 fragments of human IgG and F(ab')2 fragments of rabbit antibodies to the F(ab')2 part of human IgG. These columns give high yields of B‐cell depleted fractions. These preparations are rich in E+ cells and contain ˜80% of the Fc‐receptor lymphocytes which form rosettes with bovine erythrocytes, coated with IgG antibodies. Their K‐cell activity is unchanged or slightly elevated, indicating that mature B cells, i.e. SIg+ cells, have little or no K‐cell activity. In contrast, passage of the lymphocytes through immune complex columns (ovalbumin/anti‐ovalbumin) leads to ˜ 70% depletion of Fc receptor‐bearing cells, while most of the B cells (SIg+ cells) pass through the columns. The relative frequency of E+ cells in the passed fraction frequently shows a slight reduction. These preparations have a very low K‐cell activity, indicating that K cells are lymphocytes with Fc receptors of relatively strong avidity.
Lymphocyte Surface Membrane Immunoglobulin
Authors: WINCHESTER R. J. , FU S. M.
Published: February 2009

Abstract. Demonstration of surface Ig on lymphocytes is subject to interference by the formation of immune complexes between the fluorochrome tagged antibodies and minute amounts of residual serum Ig present in the fluid phase or loosely bound to the Fc receptor. These newly formed immune complexes accordingly bind to Fc receptors, including those of the Fc‐positive Ig‐negative third population, and result in falsely elevated percentages of Ig‐bearing cells. In principle, this immune complex formation can occur with any soluble antigens, but in practice it is most commonly encountered with IgG and to a lesser extent with IgM or IgA determinations. The use of fluorochrome tagged F(ab')2 fragments of antibodies in fluorescence results in an immune complex without an exposed Fc region that does not bind to Fc receptors. Methods for preparation of the F(ab')2 fragments are described. The use of latex ingestion as an aid for the identification of monocytes has the additional effect of allowing autoreactive anti‐lymphocyte antibodies to elute from the cell surface and thereby diminishes this additional source of false positive surface fluorescence.
Assay for the Two Different Types of Lymphocyte Complement Receptors
Authors: ROSS G. D. , POLLEY M. J.
Published: February 2009

Abstract. This paper describes the assay system for two different types of lymphocyte complement receptors, the immune adherence receptor (C3b receptor) and the C3d receptor.
The Generation, Detection and Characterization of Cytotoxic T Lymphocyte Activity in Vitro
Authors: ENGERS HOWARD D. , MACDONALD H. ROBSON
Published: February 2009

Abstract. This paper describes the generation, detection and characterization of cytotoxic T lymphocyte activity in vitro and the application of this assay system to various immunological problems.
Human Lymphocyte Subpopulations Involved in MLC and CML
Authors: ZEIJLEMAKER W. P. , VAN OERS M. H. J. , EIJSVOOGEL V. P.
Published: February 2009

Abstract. Lymphocyte subpopulations from human peripheral blood were tested for their capacity to stimulate allogeneic cells in MLC and for their ability to develop into cytotoxic cells in cell‐mediated lympholysis (CML). By various cell separation experiments lymphocyte suspensions were depleted or enriched with respect to Fc receptor‐bearing cells (detected as EA‐RFC) and Ig‐bearing cells. It was concluded that Fc receptor‐bearing cells and Ig‐bearing cells belong to different subpopulations. Furthermore, about 30% of the Fc receptor‐bearing cells had T cell characteristics. The MLC stimulatory capacity was mainly confined to the Ig‐bearing lymphocytes, and the Fc receptor‐bearing cells and T cells exerted very little stimulation. Removal of Fc receptor‐bearing cells strongly decreased the development of cytotoxic lymphocytes in CML. However, removal of Fc receptor‐bearing cells after MLC activation no longer affected cytotoxicity in CML. The activity in antibody‐dependent lymphocytotoxicity was strongly decreased when Fc receptor‐bearing cells were removed either before or after MLC activation. It was concluded that the cytotoxic precursor cells in CML bear an Fc receptor, which is either lost or blocked during MLC activation.
Typing for MLC Determinants
Authors: THOMSEN M. , RYDER L. P. , SVEJGAARD A.
Published: February 2009

Abstract.MLC typing of random individuals can be performed using a panel of inactivated HLA‐D homozygous cells. Eight different HLA‐D specificities are now internationally accepted. The evaluation of the results must take into account both the general responding capacity of the cells to be typed and the general stimulating capacity of the typing cells. An evaluation based on the 75th percentile is discussed in detail and some pitfalls are mentioned. Furthermore a description is given of primed lymphocyte typing (PLT), where cells primed in ordinary MLC cultures to one HLA‐D determinant have the ability to respond in an accelerated way to similar HLA‐D antigens when re‐exposed to such cells in secondary cultures. In our experiments, an excellent correlation is found between these two ways of MLC typing provided that the cells used for priming are well characterized (i.e. HLA‐D homozygous cells). Finally, some clinical applications of MLC typing are described, especially in connection with transplantation and association between HLA and various diseases.
Human Leucocyte Migration Inhibition
Authors: BENDIXEN G. , BENDTZEN K. , CLAUSEN J. E. , KJÉR M. , SØBORG M.
Published: February 2009

Abstract. Within the last decade a variety of techniques have been developed and used for the detection of cell‐mediated immunity in man by means of leucocyte migration inhibition in vitro. A detailed description of the leucocyte migration capillary tube technique (LMCT) and the leucocyte migration agarose technique (LMAT) is given. The procedure for selecting and using the proper antigen concentration is described. A description is also given of the indirect LMAT and the technique for determination of concanavalin A‐induced lymphocyte release of leucocyte migration inhibition factor. Applications of these techniques are mainly intended for the exploration of the immunobiology of lymphocytes and cellular interactions associated with the immune response and the investigation of clinical conditions in man, i.e. infectious diseases, autoimmune diseases, transplantation states, tumour diseases, contact hypersensitivity and immunological deficiency states. Selection and adaptation to suit the experimental aim is necessary to obtain optimal results with these techniques. Their usefulness may be increased through more extensive use of purified antigens and indirect assays.
Serum Alpha‐fetoprotein (AFP) Levels as an Index of Response to Chemotherapy in Endodermal Sinus Tumours (EST) in Children
Authors: RATCLIFFE J. G. , BURT R. W. , BAIRD G. M. , CAMPBELL A. M. , BARTER D. A. C. , WILLOUGHBY M. L. N.
Published: February 2009

The value of a sensitive radioimmunoassay for serum AFP was assessed in three children with gonadal EST. In each patient, serial AFP levels predicted accurately the clinical response to surgery and/or chemotherapy. It is suggested that frequent (at least weekly) AFP determination should be made after a course of chemotherapy to compare the observed disappearance rate with that predicted theoretically assuming complete tumour cell kill. Incomplete cell kill is indicated by a slower rate of decay than predicted. This method should help to identify optimal chemotherapy in the individual patient with EST.
Alpha‐1 Antitrypsin (AAT) and Alpha‐fetoprotein (AFP) in Sera of Patients with Germ Cell Neoplasm
Authors: HAIJE W. G. , TALERMAN A. , BAGGERMAN L.
Published: February 2009

Serum alpha‐fetoprotein (AFP) and serum alpha‐1 antitrypsin (AAT) was determined in 24 patients with germ cell neoplasms of the gonads and extra‐gonadal sites.

Although when serial determinations are performed a correlation between AAT and AFP can be seen, 71 determinations of AAT as well as AFP in the serum of the patients with germ cell tumours gave the following results: when the AFP‐concentration was below 20 ng/ml, the AAT values were always below the upper limit of the normal range (4.0 g/l); with AFP‐values well above 20 ng/ml, the AAT concentration was elevated (>4.0 g/l) in only 33% of the cases; in all the sera with an elevated AAT‐level, the AFP‐concentration was—mostly considerably—enhanced; and in 45% of the sera with normal (or lowered) AAT, there was a diagnostically meaningful elevation of the AFP‐concentration. In addition data from 9 patients with tumours not of germ cell origin undergoing therapy showed considerable variations of AAT‐values at normal AFP concentrations.

It is therefore considered that although there is evidence that AAT is produced by EST, serum AAT is not a useful monitor of disease activity in these patients, especially when compared with serum AFP.
Protein Synthesis in Yolk Sac Tumor: Histochemical Studies of Human and Rat Yolk Sac Tumor
Authors: ENDO Y. , URANO Y. , TSUCHIDA Y. , ASAKA T. , KANEKO Y. , KANEKO M. , SAKASHITA S. , TSUKADA Y. , WATABE H. , HIRAI H. , ODA T.
Published: February 2009

Synthesis of various serum proteins, such as albumin, prealbumin, α1‐antitrypsin, transferrin and α‐fetoprotein, by human and rat yolk sac tumors was studied by indirect immunofluorescence and immunoperoxidase techniques. These serum proteins were found to be present in the cytoplasm of tumor cells forming the lining of vitelline cysts, as well as Schiller‐Duval bodies and vacuolated meshwork on histological sections. Two of these tumors were cultured. Immunopathological studies revealed that albumin, prealbumin, α1‐antitrypsin, transferrin and α‐fetoprotein were found in the cytoplasm of these cultured tumor cells. The immunofluorescence of these proteins was also found on the PAS‐positive granules, which existed intra‐ and extra‐cellularly.

The ability to synthesize these serum proteins of these tumor cells was not affected by tissue culture and was maintained during subcloning.

It was demonstrated by electron microscope ferritin antibody technique that α‐fetoprotein was mainly synthesized on the ribosomes of the rough endoplasmic reticula of rat yolk sac tumor cells.
Isolation of α‐fetoprotein Messenger RNA
Authors: MIURA K. , LAW S. W. , TAMAOKI T.
Published: February 2009

Messenger RNA for α‐fetoprotein (AFP mRNA) was isolated from mouse yolk sac by a combination of indirect immunoprecipitation of AFP‐synthesizing polysomes and poly(U)‐Sepharose affinity chromatography of poly(A)‐containing mRNA. Over 200‐fold purification was achieved. Purified AFP mRNA migrated as a single band at about 21S on agarose gels in the presence of 6 M urea. Upon translation in wheat germ extracts, 98% of the protein synthesized reacted with specific antibodies to AFP. The total translation product was characterized by means of gel electrophoresis under two different conditions and by tryptic peptide analysis. It was concluded that the AFP preparation was essentially free of other yolk sac mRNA and was able to direct the synthesis of authentic AFP.