Transformation of Aspergillus oryzae using the A. niger pyrG gene

Summary A transformation system for Aspergillus oryzae based on the orotidine-5-phosphate decarboxylase gene (pyrG) was developed. Transformation frequencies of up to 16 transformants per g of DNA were obtained with the vector pAB4-1, which carries the pyrG gene of A. niger. Southern blotting analysis showed that vector DNA sequences were integrated into the chromosomal DNA, in various copy numbers and presumably at different sites. Efficient cotransformation of an unselectable gene was also shown. Under the conditions used no transformants were obtained with the equivalent pyr4 gene of Neurospora crassa.     

99mTc-CD19 monoclonal antibody is not useful for imaging of B cell non-Hodgkin’s lymphoma

 In this study we investigated the applicability of ^99mTc-labeled CD19 monoclonal antibody (mAb) for tumor imaging in patients with B cell non-Hodgkin’s lymphoma. A 1-mg sample of murine CD19 mAb was labeled with approximately 550 MBq [^99mTc]pertechnetate. The labeled mAb was administered i. v. to seven patients, four without and three with pretreatment with 10 mg unlabeled CD19 mAb. The number of circulating B cells was decreased by 44±5% 1 h after injection of the radiolabeled mAb. Peripheral B cells were coated with CD19, resulting in partial modulation of CD19, most pronounced in the three pretreated patients. Whole-body images were obtained with a gamma camera and compared with results obtained by conventional imaging techniques. Initially, blood-pool activity dominated, whereas 24 h after injection the radioactivity was mainly located in the spleen, kidneys and liver. In two patients, a lesion in the spleen appeared as an unlabeled spot. In one patient, a lesion in the femur, which was detected by computed tomography (CT) and gallium-67 scans, was also seen on the CD19 scan from 1 h after administration of the radioimmunoconjugate onwards. Good imaging of bone marrow infiltration was observed in one of three patients. Lymph node involvement was not observed in any of the patients in whom affected lymph nodes were detected by CT or gallium-67 scan. In conclusion, in the present study radioimmunodetection with ^99mTc-labeled CD19 mAb was found to be inferior to CT and gallium-67 scanning in the diagnosis of patients with B cell non-Hodgkin’s lymphoma. Received: 14 March 1996 / Accepted: 14 May 1996     

Versatility of the Endoplasmic Reticulum Protein Folding Factory

ABSTRACT

The endoplasmic reticulum (ER) is dedicated to import, folding and assembly of all proteins that travel along or reside in the secretory pathway of eukaryotic cells. Folding in the ER is special. For instance, newly synthesized proteins are N-glycosylated and by default form disulfide bonds in the ER, but not elsewhere in the cell. In this review, we discuss which features distinguish the ER as an efficient folding factory, how the ER monitors its output and how it disposes of folding failures.     

BODY SIZE DECLINES DESPITE POSITIVE DIRECTIONAL SELECTION ON HERITABLE SIZE TRAITS IN A BARNACLE GOOSE POPULATION

Analyses of more than 2000 marked barnacle geese (Branta leucopsis) in the largest Baltic colony, Sweden, showed that structurally large females generally produced larger clutches and larger eggs, hatched their broods earlier in the season, and produced more and heavier young than smaller females. In males, the corresponding relationships between reproductive parameters and structural body size were weaker or nonsignificant. Because structural body size traits have previously been found to be significantly heritable and positively genetically correlated, an increase in mean structural body size of individuals as a response to selection might have been expected. By contrast, we found that the mean adult head length and mean adult tarsus length decreased significantly in the largest colony by approximately 0.7 and 0.5 standard deviations, respectively, in both males and females during the 13-year study period. Environmental factors, such as the amount of rain in different years, were found to affect the availability of high-quality food for growing geese. As a consequence of this temporal variability in the availability of high-quality food, the mean adult structural body size of different cohorts differed by up to 1.3 standard deviations. Comparisons of mean body size of cohorts born in different colonies suggest that the most likely explanation for the body-size decline in the main study colony is that a density-dependent process, which mainly was in effect during the very early phase of colony growth, negatively affected juvenile growth and final size. We conclude that large environmental effects on growth and final structural body size easily can mask microevolutionary responses to selection. Analyses of environmental causes underlying temporal and spatial body size variation should always be considered in the reconstruction and prediction of evolutionary changes in natural populations.     

Diversity and Habitat Specificity of Free-Living Protozoa in Commercial Poultry Houses

Despite stringent biosecurity measures, infections by bacterial food pathogens such as Campylobacter are a recurrent problem in industrial poultry houses. As the main transmission route remains unclear, persistence of these infections has been linked to bacterial survival and possibly multiplication within protozoan vectors. To date, however, virtually no information is available on the diversity and occurrence of free-living protozoa in these environments. Using a combination of microscopic analyses of enrichment cultures and molecular methods (denaturing gradient gel electrophoresis [DGGE]) on natural samples, we show that, despite strict hygiene management, free-living protozoa are common and widespread throughout a 6-week rearing period in both water and dry samples from commercial poultry houses. Protozoan communities were highly diverse (over 90 morphotaxa and 22 unique phylotypes from sequenced bands) and included several facultative pathogens and known bacterial vectors. Water samples were consistently more diverse than dry ones and harbored different communities, mainly dominated by flagellates. The morphology-based and molecular methods yielded markedly different results: amoebic and, to a lesser degree, ciliate diversity was seriously underestimated in the DGGE analyses, while some flagellate groups were not found in the microscopic analyses. Some recommendations for improving biosecurity measures in commercial poultry houses are suggested.The microaerobic bacterial genus Campylobacter (Campylobacteraceae) is recognized as the primary cause of food-borne diseases in industrialized countries, with poultry products as one of the main infection sources (3). To prevent contamination of the flocks, stringent biosecurity and control measures are recommended in industrial broiler houses in Europe (26). These include the application of a down period of at least 2 weeks between rearing cycles, during which the pathogen hosts (chickens) are absent and the broiler houses are cleansed and disinfected (23). However, despite these measures, recurrent contamination of flocks is common. Numerous studies have been carried out to identify the main infection sources and transmission routes during the rearing process, but to date these remain unclear (32). Campylobacter strains are not detected in the empty broiler houses after the cleansing and disinfection process (47). Epidemiological investigations have shown that most flocks become infected only 2 to 3 weeks after the introduction of new chicks, ruling out vertical transmission via contaminated eggs as the primary infection source (32). It has also been shown that the presence of animal vectors (e.g., rodents, insects, birds, pets, and livestock) plays only a minor role in the transmission of the pathogens (32). It thus seems that other, as yet not identified, vehicles may be present that allow the bacteria to persist in the environment and/or rapidly colonize the chickens.There is increasing evidence, mainly derived from in vitro cocultivation studies, that bacteria, including Campylobacter (5, 30, 41), can survive and probably even multiply within protozoa (reviewed, e.g., in reference 43). The bacteria are able to survive protozoan grazing using pre- and postingestion adaptations (31). These interactions may protect the bacteria from unfavorable conditions such as desiccation and disinfection and in this way can have a major impact on their epidemiology, survival, and transmission (30). Some pathogens, such as Legionella pneumophila and Mycobacterium, have been shown to become more virulent after coculture with protozoa (see references 8 and 9).The present study forms part of a larger investigation aimed at evaluating the role of protozoa in the transmission of Campylobacter jejuni and Campylobacter coli in industrial broiler houses and reports on the results of a comprehensive study on the occurrence and diversity of protozoa in various habitats inside commercial poultry farms. Since to date very little information is available on this topic and on the occurrence of protozoa in anthropogenic environments in general, the main aims of our study were the following: (i) to assess the biodiversity of the protozoan communities present in commercial broiler houses throughout a rearing period; (ii) to evaluate to what degree the spatial and temporal dynamics of these communities can be related to farm-specific environmental conditions and management strategies (including cleansing and disinfection) and within-farm microhabitat; and (iii) to translate these findings into a number of management and biosecurity recommendations. Protozoa are difficult to survey in any locality as many of them are present in a cryptic state; i.e., they are too rare to detect or are present as resting cysts (12). In order to get an inventory of their true diversity, we combined an incubation approach (aimed at enriching the pool of cryptic taxa) and microscopical observations with molecular-genetic fingerprints of the original samples.     

Priority effects in experimental populations of the cyanobacterium Microcystis

The arrival order of colonists in developing populations can have a lasting influence on community and population structure, a phenomenon referred to as priority effects. To explore whether such priority effects are important in determining strain composition of populations of the cyanobacterium Microcystis , four Microcystis strains, isolated from a single lake and differing in functional traits, were grown during 4 weeks in the laboratory in all possible pairwise combinations, with the two strains either inoculated at the same time or with a time lag of 1 week, in the presence or absence of grazing Daphnia magna . The relative abundance of strains in the mixtures was assessed using denaturing gradient gel electrophoresis, and the growth rate of each strain in the mixtures was determined for the last 2 weeks of the experiment. We observed strong effects of inoculation order on the final population structure, and these effects were influenced by grazing Daphnia . The priority effects were strain-specific and occurred in two directions: some of the strains grew slower while others grew faster when inoculated second compared with when inoculated first. Our results indicate that priority effects may have a profound impact on strain composition of Microcystis populations.     

Characterization of the GbpD-activated Rap1 pathway regulating adhesion and cell polarity in Dictyostelium discoideum

The regulation of cell polarity plays an important role in chemotaxis. GbpD, a putative nucleotide exchange factor for small G-proteins of the Ras family, has been implicated in adhesion, cell polarity, and chemotaxis in Dictyostelium. Cells overexpressing GbpD are flat, exhibit strongly increased cell-substrate attachment, and extend many bifurcated and lateral pseudopodia. These cells overexpressing GbpD are severely impaired in chemotaxis, most likely due to the induction of many protrusions rather than an enhanced adhesion. The GbpD-overexpression phenotype is similar to that of cells overexpressing Rap1. Here we demonstrate that GbpD activates Rap1 both in vivo and in vitro but not any of the five other characterized Ras proteins. In a screen for Rap1 effectors, we overexpressed GbpD in several mutants defective in adhesion or cell polarity and identified Phg2 as Rap1 effector necessary for adhesion, but not cell polarity. Phg2, a serine/threonine-specific kinase, directly interacts with Rap1 via its Ras association domain.     

Distinct roles of PI(3,4,5)P3 during chemoattractant signaling in Dictyostelium: a quantitative in vivo analysis by inhibition of PI3-kinase

The role of PI(3,4,5)P(3) in Dictyostelium signal transduction and chemotaxis was investigated using the PI3-kinase inhibitor LY294002 and pi3k-null cells. The increase of PI(3,4,5)P(3) levels after stimulation with the chemoattractant cAMP was blocked >95% by 60 microM LY294002 with half-maximal effect at 5 microM. This correlated well with the inhibition of the membrane translocation of the PH-domain protein, PHcracGFP. LY294002 did not reduce cAMP-mediated cGMP production, but significantly reduced the cAMP response up to 75% in wild type and completely in pi3k-null cells. LY294002-treated cells were round, not elongated as control cells. Interestingly, cAMP induced a time and dose-dependent recovery of cell elongation. These elongated LY294002-treated wild-type and pi3k-null cells exhibited chemotactic orientation toward cAMP that is statistically identical to chemotactic orientation of control cells. In control cells, PHcrac-GFP and F-actin colocalize upon cAMP stimulation. However, inhibition of PI3-kinases does not affect the first phase of the actin polymerization at a wide range of chemoattractant concentrations. Our data show that severe inhibition of cAMP-mediated PI(3,4,5)P(3) accumulation leads to inhibition of cAMP relay, cell elongation and cell aggregation, but has no detectable effect on chemotactic orientation, provided that cAMP had sufficient time to induce cell elongation.     

CD103 is a marker for alloantigen-induced regulatory CD8+ T cells

The alphaEbeta7 integrin CD103 may direct lymphocytes to its ligand E-cadherin. CD103 is expressed on T cells in lung and gut and on allograft-infiltrating T cells. Moreover, recent studies have documented expression of CD103 on CD4+ regulatory T cells. Approximately 4% of circulating CD8+ T cells bear the CD103 molecule. In this study, we show that the absence or presence of CD103 was a stable trait when purified CD103- and CD103+ CD8+ T cell subsets were stimulated with a combination of CD3 and CD28 mAbs. In contrast, allostimulation induced CD103 expression on approximately 25% of purified CD103- CD8+ T cells. Expression of CD103 on alloreactive cells was found to be augmented by IL-4, IL-10, or TGF-beta and decreased by addition of IL-12 to MLCs. The alloantigen-induced CD103+ CD8+ T cell population appeared to be polyclonal and retained CD103 expression after restimulation. Markedly, in vitro-expanded CD103+ CD8+ T cells had low proliferative and cytotoxic capacity, yet produced considerable amounts of IL-10. Strikingly, they potently suppressed T cell proliferation in MLC via a cell-cell contact-dependent mechanism. Thus, human alloantigen-induced CD103+ CD8+ T cells possess functional features of regulatory T cells.     

Multiple regulatory mechanisms for the Dictyostelium Roco protein GbpC

GbpC is a multidomain Roco protein in Dictyostelium, involved in transduction of intracellular cGMP that is produced by chemotactic signals. We have shown previously that cGMP binding to GbpC induces an intramolecular signaling cascade by activating subsequently the GEF, Ras, and kinase domains. In this study, we report on the cellular localization of GbpC. In resting cells, the protein is present in the cytoplasm, but GbpC rapidly translocates to the cell boundary upon stimulation with the chemoattractant cAMP. Also, during the formation of cell-cell streams and osmotic shock, the protein localizes toward the plasma membrane and actin cytoskeleton. The translocation upon cAMP stimulation occurs downstream of heterotrimeric G proteins but is independent of guanylyl cyclases and the previously identified cGMP-induced intramolecular signaling cascade in GbpC. Mutations in the GRAM domain of GbpC lead to disturbed membrane association and inactivation of GbpC function during chemotaxis in vivo. Furthermore, we show that the GRAM domain itself associates with cellular membranes and binds various phospholipids in vitro. Together, the results show that GbpC receives multiple input signals that are both required for functional activity in vivo. cAMP-stimulation induces a cGMP-dependent signaling cascade, leading to activation of kinase activity, and, independently, cAMP induces a GRAM-dependent translocation of GbpC toward the plasma membrane and cell cortex, where it may locally phosphorylate effector proteins, which are needed for proper biological activity.