The siderophore system is essential for viability of Aspergillus nidulans: functional analysis of two genes encoding l-ornithine N 5-monooxygenase (sidA) and a non-ribosomal peptide synthetase (sidC)

The filamentous ascomycete A. nidulans produces two major siderophores: it excretes triacetylfusarinine C to capture iron and contains ferricrocin intracellularly. In this study we report the characterization of two siderophore biosynthetic genes, sidA encoding l-ornithine N(5)-monooxygenase and sidC encoding a non-ribosomal peptide synthetase respectively. Disruption of sidC eliminated synthesis of ferricrocin and deletion of sidA completely blocked siderophore biosynthesis. Siderophore-deficient strains were unable to grow, unless the growth medium was supplemented with siderophores, suggesting that the siderophore system is the major iron assimilatory system of A. nidulans during both iron depleted and iron-replete conditions. Partial restoration of the growth of siderophore-deficient mutants by high concentrations of Fe(2+) (but not Fe(3+)) indicates the presence of an additional ferrous transport system and the absence of an efficient reductive iron assmilatory system. Uptake studies demonstrated that TAFC-bound iron is transferred to cellular ferricrocin whereas ferricrocin is stored after uptake. The siderophore-deficient mutant was able to synthesize ferricrocin from triacetylfusarinine C. Ferricrocin-deficiency caused an increased intracellular labile iron pool, upregulation of antioxidative enzymes and elevated sensitivity to the redox cycler paraquat. This indicates that the lack of this cellular iron storage compound causes oxidative stress. Moreover, ferricrocin biosynthesis was found to be crucial for efficient conidiation.     

Indications of neutralising anti-idiotypic antibodies and selective proteolytic fragmentation in polyclonal anti-D IgG preparations

Proteolytic fragmentation is the only suggested cause of potency losses during storage of liquid human polyclonal anti-D Ig. Besides the effect of fragmentation, we have investigated the potential contribution of neutralising anti-idiotypic antibodies (anti-Ids). Potency changes during storage and/or upon pH reduction in anti-D IgG batches with or without addition of plasminogen and urokinase were quantitatively analysed by the autoanalyser (AA) method or by a special procedure of flow cytometry (FC). Moreover, simultaneous changes of the molecular size distribution pattern have been determined by size exclusion chromatography. In contrast to the AA procedure, the particular FC methodology was found to be almost insensitive to proteolysis comprising up to 30% of total IgG. Data interpretation was based on the assumption that both assays cannot detect Ids with neutralised paratopes. In the absence of detectable neutralisation (functional absence of anti-Ids), it could be demonstrated that the anti-D IgG subpopulation is more sensitive to fragmentation by endogenous protease as compared to the unrelated bulk. However, both methods detected batch- and assay-dependently variable potency losses during storage. Moreover, the increase of potency induced by pH reduction correlated with the increase of monomeric IgG, essentially on the expense of dimers. This finding was interpreted to indirectly indicate the neutralising action of anti-Ids known to be the major driving force of dimer formation in polyclonal IgG. A more or less pronounced pH-dependent potency increase was also detectable in three arbitrarily selected batches of two other manufacturers. The data allows to assume that anti-Id-mediated neutralisation can significantly contribute to losses of anti-D potency. In addition, it turned out that anti-D plasma itself can be the source of anti-Ids.     

Protein interactions within the N-end rule ubiquitin ligation pathway

Rate studies have been employed as a reporter function to probe protein-protein interactions within a biochemically defined reconstituted N-end rule ubiquitin ligation pathway. The concentration dependence for E1-catalyzed HsUbc2b/E2(14kb) transthiolation is hyperbolic and yields K(m) values of 102 +/- 13 nm and 123 +/- 19 nm for high affinity binding to rabbit and human E1/Uba1 orthologs. Competitive inhibition by the inactive substrate and product analogs HsUbc2bC88A (K(i) = 104 +/- 15 nm) and HsUbc2bC88S-ubiquitin oxyester (K(i) = 169 +/- 17 nm), respectively, indicates that the ubiquitin moiety contributes little to E1 binding. Under conditions of rate-limiting E3alpha-catalyzed conjugation to human alpha-lactalbumin, HsUbc2b-ubiquitin thiolester exhibits a K(i) of 54 +/- 18 nm and is competitively inhibited by the substrate analog HsUbc2bC88S-ubiquitin oxyester (K(i) = 66 +/- 29 nm). In contrast, the ligase product analog HsUbc2bC88A exhibits a K(i) of 440 +/- 55 nm with respect to the wild type HsUbc2b-ubiquitin thiolester, demonstrating that ubiquitin binding contributes to the ability of E3alpha to discriminate between substrate and product E2. A survey of E1 and E2 isoform distribution in selected cell lines demonstrates that Ubc2 isoforms are the predominant intracellular ubiquitin carrier protein. Intracellular levels of E1 and Ubc2 are micromolar and approximately equal based on in vitro quantitation by stoichiometric (125)I-ubiquitin thiolester formation. Comparison of intracellular E1 and Ubc2 pools with the corresponding ubiquitin pools reveals that most of the free ubiquitin in cells is present as thiolesters to the components of the conjugation pathways. The present data represent the first comprehensive analysis of protein interactions within a ubiquitin ligation pathway.     

Inactivation of feline calicivirus and adenovirus type 40 by UV radiation

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm(2), respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm(2) in treated groundwater. A dose of 103 mJ/cm(2) was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.     

Transgenic expression of a soluble complement inhibitor protects against renal disease and promotes survival in MRL/lpr mice

To investigate the role of complement in lupus nephritis, we used MRL/lpr mice and a transgene overexpressing a soluble complement regulator, soluble CR1-related gene/protein y (sCrry), both systemically and in kidney. Production of sCrry in sera led to significant complement inhibition in Crry-transgenic mice relative to littermate transgene negative controls. This complement inhibition with sCrry conferred a survival advantage to MRL/lpr mice. In a total of 154 animals, 42.5% transgene-negative animals had impaired renal function (blood urea nitrogen > 50 mg/dl) compared with 16.4% mice with the sCrry-producing transgene (p < 0.001). In those animals that died spontaneously, MRL/lpr mice with the sCrry-producing transgene did not die of renal failure, while those without the transgene did (blood urea nitrogen values of 46.6 +/- 9 and 122 +/- 29 mg/dl in transgene-positive and transgene-negative animals, respectively; p < 0.001). Albuminuria was reduced in those transgenic animals in which sCrry expression was maximally stimulated (urinary albumin/creatinine = 12.4 +/- 4.3 and 36.9 +/- 7.7 in transgene-positive and transgene-negative animals, respectively; p < 0.001). As expected in the setting of chronic complement inhibition, there was less C3 deposition in glomeruli of sCrry-producing transgenic mice compared with transgene-negative animals. In contrast, there was no effect on glomerular IgG deposition, levels of anti-dsDNA Ab and rheumatoid factor, or spleen weights between the two groups. Thus, long-term complement inhibition reduces renal disease in MRL/lpr mice, which translates into improved survival. MRL/lpr mice in which complement is inhibited still have spontaneous mortality, yet this is not from renal disease.     

Fusaric acid-producing strains of Fusarium oxysporum alter 2,4-diacetylphloroglucinol biosynthetic gene expression in Pseudomonas fluorescens CHA0 in vitro and in the rhizosphere of wheat

The phytotoxic pathogenicity factor fusaric acid (FA) represses the production of 2,4-diacetylphloroglucinol (DAPG), a key factor in the antimicrobial activity of the biocontrol strain Pseudomonas fluorescens CHA0. FA production by 12 Fusarium oxysporum strains varied substantially. We measured the effect of FA production on expression of the phlACBDE biosynthetic operon of strain CHA0 in culture media and in the wheat rhizosphere by using a translational phlA'-'lacZ fusion. Only FA-producing F. oxysporum strains could suppress DAPG production in strain CHA0, and the FA concentration was strongly correlated with the degree of phlA repression. The repressing effect of FA on phlA'-'lacZ expression was abolished in a mutant that lacked the DAPG pathway-specific repressor PhlF. One FA-producing strain (798) and one nonproducing strain (242) of F. oxysporum were tested for their influence on phlA expression in CHA0 in the rhizosphere of wheat in a gnotobiotic system containing a sand and clay mineral-based artificial soil. F. oxysporum strain 798 (FA(+)) repressed phlA expression in CHA0 significantly, whereas strain 242 (FA(-)) did not. In the phlF mutant CHA638, phlA expression was not altered by the presence of either F. oxysporum strain 242 or 798. phlA expression levels were seven to eight times higher in strain CHA638 than in the wild-type CHA0, indicating that PhlF limits phlA expression in the wheat rhizosphere.     

Dynamic structural disorder of the FeO<Subscript>2</Subscript> moiety in oxymyoglobin studied by nuclear resonant forward scattering of synchrotron radiation

Oxy- as well as deoxymyoglobin exhibit a pronounced temperature dependence of the quadrupole splitting of the heme iron as detected by conventional M?ssbauer spectroscopy. With nuclear resonant forward scattering (NFS) of synchrotron radiation, which can be viewed as M?ssbauer spectroscopy in the time domain, it is shown that this spectroscopic behavior, although it is phenomenologically similar in the two cases, is based on completely different physical mechanisms. It is demonstrated that stochastic fluctuations of the iron electric field gradient in MbO(2), which are due to the dynamic structural disorder of the FeO(2) moiety, are the reason for the temperature-dependent alterations of the coherent quantum beat pattern in the NFS spectra of MbO(2), in contrast to deoxyMb where transitions between orbital states of iron take place. This subtle spectroscopic difference cannot be inferred from conventional M?ssbauer spectroscopy.     

Hippocampal ripple oscillations (200 Hz) in mechanisms of memory consolidation

A current status of knowledge about high-frequency (140-200 Hz) ripple oscillations in the CA1 hippocampal subfield is summarized and considered in the context of two-stage model of the hippocampal memory processing. A large body of evidence suggests highly-selective recruitment of pyramidal cells and interneurons in the generation of the oscillatory pattern after co-operative sharp-wave-related discharge of CA3 pyramidal neurons. We also discuss a role of transmission via gap junctions in the mechanisms of ripple oscillations as well as their adaptive aminergic (histaminergic) modulation. Patterns of neuronal firing in the hippocampus observed during ripple oscillations reproduce space-dependant neuronal activity from the previous waking period. Together with a data about efficacy of high-frequency stimulation for induction of synaptic modification it points out a role for ripples in the formation of long-term memory. Focal ultra fast ripples (up to 500 Hz) have been shown to participate in the development of temporal lobe epilepsy.     

Diplostomum spathaceum cercariae respond to a unique profile of cues during recognition of their fish host

During its normal life cycle, Diplostomum spathaceum cercariae attach to and invade fish intermediate hosts. They are also known to attach to various other aquatic animals in response to water currents, touch and carbon dioxide. The purpose of this study was to identify the specific stimuli used by D. spathaceum cercariae to recognise the appropriate fish host. We characterised the host cues which stimulate them to remain on the host (enduring contact) and to penetrate the skin. Cercariae were exposed to animal skin tissues and fish skin surface mucus, their extracts and chemical modifications integrated into agar or offered via membrane filters. Enduring contact was stimulated by hydrophilic extracts Mr<3kDa, which were sensitive to oxidation of carbohydrates. The stimulating cues are probably small molecular carbohydrates, as monosaccharides stimulated enduring contacts, but amino acids, urea, electrolytes and peptides did not. Penetration was stimulated by hydrophilic macromolecules, Mr>30kDa, and by lipids. The hydrophilic stimuli were protease resistant and precipitable with Alcian blue and they were sensitive to alkaline cleavage, to digestion with lysozyme and neuraminidase as well as to oxidation of sialic acids. They were considered to be glycoproteins with O-glycosidically linked carbohydrate chains and bound sialic acids as signal structures. The lipophilic penetration stimuli were contained exclusively in the fatty acid fractions, and the stimulating characteristics of these fatty acids resembled the stimulating penetrations in other cercarial species. Diplostomum spathaceum cercariae respond to a unique profile of cues in their sequence of host-recognition phases. These cues differ from those used in other fish parasites studied to date and underline the diversity of fish recognition strategies.