Spatial and Developmental Differentiation of Mannitol Dehydrogenase and Mannitol-1-Phosphate Dehydrogenase in Aspergillus niger

The presence of a mannitol cycle in fungi has been subject to discussion for many years. Recent studies have found no evidence for the presence of this cycle and its putative role in regenerating NADPH. However, all enzymes of the cycle could be measured in cultures of Aspergillus niger. In this study we have analyzed the localization of two enzymes from the pathway, mannitol dehydrogenase and mannitol-1-phosphate dehydrogenase, and the expression of their encoding genes in nonsporulating and sporulating cultures of A. niger. Northern analysis demonstrated that mpdA was expressed in both sporulating and nonsporulating mycelia, while expression of mtdA was expressed only in sporulating mycelium. More detailed studies using green fluorescent protein and dTomato fused to the promoters of mtdA and mpdA, respectively, demonstrated that expression of mpdA occurs in vegetative hyphae while mtdA expression occurs in conidiospores. Activity assays for MtdA and MpdA confirmed the expression data, indicating that streaming of these proteins is not likely to occur. These results confirm the absence of the putative mannitol cycle in A. niger as two of the enzymes of the cycle are not present in the same part of A. niger colonies. The results also demonstrate the existence of spore-specific genes and enzymes in A. niger.Mannitol has been described as one of the main compatible solutes in fungi (20) and may play a role as a storage carbon source (3) or a protectant against a variety of stresses (10, 16, 20, 22). Mannitol metabolism in fungi has been the subject of study for decades. It was proposed to exist in the form of a cyclic pathway, the mannitol cycle (9). This cycle consists of four steps enabling the conversion of fructose into mannitol and back to fructose (Fig. 1). The main role proposed for this cycle was regenerating NADPH (9, 10). Subsequently, many studies have questioned the existence of a mannitol cycle (reviewed in reference 20), and it has been shown that a mannitol cycle is not involved in NADPH regeneration in Stagonospora nodorum (19), Aspergillus niger (16), and Alternaria alternata (21). However, all enzymes of the cycle were detected in both sporulating and nonsporulating mycelia in A. niger (16), suggesting that a cycle could operate in this fungus. Fungi are able to use mannitol as a sole carbon source but do so in various ways (7).Open in a separate windowFig. 1.Putative mannitol cycle in fungi as proposed by Hult and Gatenbeck (9). HXK, hexokinase (EC 2.7.1.1); MTD, mannitol dehydrogenase (EC 1.1.1.138); MPD, mannitol-1-phosphate dehydrogenase (EC 1.1.1.17); MPP, mannitol-1-phosphate phosphatase (EC 3.1.3.22).d-Mannitol plays an important role in germination of Aspergillus conidia. In A. niger (23) and Aspergillus oryzae (8), mannitol accumulates in conidiospores and is utilized during the initial stages of germination. Production of mannitol appears to be largely dependent on mannitol-1-phosphate dehydrogenase (MPD) while mannitol dehydrogenase (MTD) contributes to a lesser extent (16, 19, 20).In this study we demonstrate that MTD and MPD as well as the expression of the corresponding genes (mtdA and mpdA) are spatially separated in colonies of A. niger. This demonstrates that a mannitol cycle does not exist in this fungus and shows that spores express specific genes that are involved in germination.     

The tumor-promoting effect of TNF-alpha involves the induction of secretory leukocyte protease inhibitor

According to the cancer immunoediting concept, inflammatory mediators play not only a critical role in promoting host protection against cancer but also contribute to cancer cell growth and survival. TNF-alpha is a critical factor in this network. However, the mechanisms underlying the tumor-promoting effect of TNF-alpha have not been fully elucidated yet. We previously reported that in vitro culture of Lewis lung carcinoma 3LL cells with TNF-alpha-producing macrophages resulted in enhanced resistance toward TNF-alpha-mediated lysis and increased malignancy of the 3LL cells. In this study, we analyzed the effects of endogenous TNF-alpha on TNF-alpha resistance and malignant behavior in vivo of low-malignant/TNF-alpha-sensitive 3LL-S cells and cancer cells derived from 3LL-S tumors that developed in wild-type or TNF-alpha(-/-) mice. Interestingly, 3LL-S cells acquired a malignant phenotype in vivo depending on the presence of host TNF-alpha, whereas acquisition of TNF-alpha resistance was TNF-alpha-independent. This result suggested that malignancy-promoting characteristics of 3LL-S cells other than TNF-alpha resistance are influenced in vivo by TNF-alpha. We previously identified the malignancy-promoting genes, secretory leukocyte protease inhibitor (SLPI) and S100A4, as being up-regulated in 3LL-S cells upon their s.c. growth in wild-type mice. In this study, we show that SLPI, but not S100A4, was induced in 3LL-S cells both in vitro and in vivo by TNF-alpha, and that silencing of in vivo induced 3LL-S SLPI expression using RNA interference abrogated in vivo progression but did not influence TNF-alpha resistance. These data indicate that SLPI induction may be one mechanism whereby TNF-alpha acts as an endogenous tumor promoter.     

PLCβ isoforms differ in their subcellular location and their CT-domain dependent interaction with Gαq

Phospholipase C (PLC) β isoforms are implicated in various physiological processes and pathologies. However, mechanistic insight into the localization and activation of each of the isoforms is limited. Therefore, it is crucial to gain more in-depth knowledge as to the regulation of the different isoforms. Here we describe the subcellular location of full-length PLCβ isozymes and their C-terminal (CT) domains. Strikingly, we found isoforms PLCβ1 and PLCβ4 to be enriched at the plasma membrane, contrary to isoforms PLCβ2 and PLCβ3. We determined that the CT domain is an inhibitor of Gq-mediated increases in intracellular calcium, the potency of its effect being dependent upon the CT domain isoform used. Furthermore, ratiometric fluorescence resonance energy transfer (FRET) imaging was used to study the kinetics of the Gαq–CTβx interactions. By the use of recently developed tools, which enable the on-demand activation of Gαq, we could show that the interaction between constitutively active Gαq and PLCβ3 prolongs the residence time of PLCβ3 at the plasma membrane. These findings suggest that under physiological circumstances, PLCβ3 and Gαq interact in a kiss-and-run fashion, likely due to the GTPase-activating activity of PLCβ towards Gαq.     

Low predictive value of seroprevalence of Toxoplasma gondii in cattle for detection of parasite DNA

The role of beef in human infections with Toxoplasma gondii is not clear. To get a better understanding of the value of seroprevalence as an indication of the role of beef in human infections with T. gondii we studied the seroprevalence of T. gondii in Dutch cattle and analysed the correlation between detection of antibodies and parasitic DNA. An indirect ELISA was developed and used to test a sample of the Dutch cattle population. Since validation of the ELISA was hampered by a lack of sufficient bovine reference sera, the results were analysed in two different ways: using a cut-off value that was based on the course of the OD in 27 calves followed from birth until 16 months of age, and by fitting a mixture of two normal distributions (binormal mixture model) to the log-transformed ODs observed for the different groups of cattle in the study population. Using the cut-off value, the seroprevalence was estimated at 0.5% for white veal, 6.4% for rosé veal and 25.0% for cattle. However, using the frequency distributions the prevalences were higher: 1.9% for white veal, 15.6% for rosé veal and 54.5% for cattle. Next, for 100 cattle the results with two different serological assays (ELISA and Toxo-Screen DA) were compared with detection of parasites by our recently developed sensitive magnetic capture PCR. Toxoplasma gondii DNA was detected in only two seronegative cattle. This discordance demonstrates that seroprevalence cannot be used as an indicator of the number of cattle carrying infectious parasites. Demonstrating parasitic DNA in seronegative cattle and not in seropositive cattle suggests that only recent infections are detectable. Whether beef from these PCR-positive cattle is infectious to humans remains to be studied.     

A search filter for increasing the retrieval of animal studies in Embase

Collecting and analysing all available literature before starting a new animal experiment is important and it is indispensable when writing systematic reviews of animal research. In practice, finding all animal studies relevant to a specific research question turns out to be anything but simple. In order to facilitate this search process, we previously developed a search filter for retrieving animal studies in the most often used biomedical database, PubMed. It is a general requirement for systematic reviews, however, that at least two databases are searched. In this report, we therefore present a similar search filter for a second important database, namely Embase. We show that our filter retrieves more animal studies than (a combination of) the options currently available in Embase. Our search filters for PubMed and Embase therefore represent valuable tools for improving the quality of (systematic) reviews and thereby of new animal experiments.     

Assessing the search for information on Three Rs methods, and their subsequent implementation: a national survey among scientists in the Netherlands

A local survey conducted among scientists into the current practice of searching for information on Three Rs (i.e. Replacement, Reduction and Refinement) methods has highlighted the gap between the statutory requirement to apply Three Rs methods and the lack of criteria to search for them. To verify these findings on a national level, we conducted a survey among scientists throughout The Netherlands. Due to the low response rate, the results give an impression of opinions, rather than being representative of The Netherlands as a whole. The findings of both surveys complement each other, and indicate that there is room for improvement. Scientists perceive searching the literature for information on Three Rs methods to be a difficult task, and specific Three Rs search skills and knowledge of Three Rs databases are limited. Rather than using a literature search, many researchers obtain information on these methods through personal communication, which means that published information on possible Three Rs methods often remains unfound and unused. A solution might be to move beyond the direct search for information on Three Rs methods and choose another approach. One approach that seems rather appropriate is that of systematic review. This provides insight into the necessity for any new animal studies, as well as optimal implementation of available data and the prevention of unnecessary animal use in the future.