Iron stress responses in the cyanobacterium Synechococcus sp. PCC7942

In the present study, we describe the sequential events by which the cyanobacterium Synechococcus sp. PCC 7942 adapts to iron deficiency. In doing so, we have tried to elucidate both short and long-term acclimation to low iron stress in order to understand how the photosynthetic apparatus adjusts to low iron conditions. Our results show that after an initial step, where CP43' is induced and where ferredoxin is partly replaced by flavodoxin, the photosynthetic unit starts to undergo major rearrangements. All measured components of Photosystem I (PSI), PSII and cytochrome (Cyt) ƒ decrease relative to chlorophyll (Chl) a . The photochemical efficiencies of the two photosystems also decline during this phase of acclimation. The well-known drop in phycobilisome content measured as phycocyanin (PC)/Chl was not due to an increased degradation, but rather to a decreased rate of synthesis. The largest effects of iron deficiency were observed on PSI, the most iron-rich structure of the photosynthetic apparatus. In the light of the recent discovery of an iron deficiency induced CP43' ring around PSI a possible dual function of this protein as both an antenna and a quencher is discussed. We also describe the time course of a blue shift in the low temperature Chl emission peak around 715 nm, which originates in PSI. The shift might reflect the disassembly and/or degradation of PSI during iron deficiency and, as a consequence, PSI might under these conditions be found predominantly in a monomeric form. We suggest that the observed functional and compositional alterations represent cellular acclimation enabling growth and development under iron deficiency, and that growth ceases when the acclimation capacity is exhausted. However, the cells remain viable even after growth has ceased, since they resumed growth once iron was added back to the culture.     

Identification of residues important for NAD+ binding by the Thermotoga maritima alpha-glucosidase AglA,a member of glycoside hydrolase family 4

The NAD+-requiring enzymes of glycoside hydrolase family 4 (GHF4) contain a region with a conserved Gly-XXX-Gly-Ser (GXGS) motif near their N-termini that is reminiscent of the fingerprint region of the Rossmann fold, a conserved structural motif of classical nicotinamide nucleotide-binding proteins. The function of this putative NAD+-binding motif in the alpha-glucosidase AglA of Thermotoga maritima was probed by directed mutagenesis. The K(d) for NAD+ of the AglA mutants G10A, G12A and S13A was increased by about 300-, 5-, and 9-fold, respectively, while their K(m) for p-nitrophenyl-alpha-glucopyranoside was not seriously affected. The results indicate that the GXGS motif is indeed important for NAD+ binding by the glycosidases of GHF4.     

Mesothelial cells in suspension expose an enriched integrin repertoire capable of capturing soluble fibronectin and laminin

Pleural cavities are lined by a polarized monolayer of mesothelial cells (MC). During pleuritis, MC are shed into effusions, and pleural obstruction may occur. Integrins are cell surface receptors mediating interactions with extracellular matrix (ECM) proteins. The distribution of beta 1-, beta 3-, beta 4-integrins and fibronectin and laminin in normal and chronically inflamed pleura and in/on MC from pleural effusions was examined by immunomorphology and flow cytometry. Adhesion assays of MC to fibronectin and laminin were performed. In situ, resting MC expressed beta 1-, beta 3-, and beta 4-, and alpha v-subunits. Activated MC were beta 1- and alpha v-positive and also expressed alpha 3 and alpha 6; beta 4 was confined to the basal surface of MC; beta 3 was absent. Floating MC from effusions neoexpressed alpha 5 and reexpressed beta 3. In vitro, MC surface expressed beta 1, beta 3, alpha 3, alpha 5, alpha 6, alpha v, and also alpha 1 and alpha 2. In normal pleura, fibronectin and laminin were components of the basement membrane. In pleuritis, the basement membrane was desintegrated. Instead, newly formed fibronectin/laminin containing fibrils extended into the submesothelial connective tissue. Floating MC freshly isolated from effusions carried fibronectin and laminin on their surface and showed specific binding to these ECM proteins. Binding was blocked by anti-beta 1 or anti-alpha 5 and anti-alpha 6 antibodies, respectively. MC incubated with fibronectin showed a clear shift to the S phase, while laminin had no effect. In conclusion, activated and detached MC progressively enrich their integrin repertoire. By capturing soluble fibronectin and laminin and by matrix-mediated bridging, readhering MC may contribute to pleural obstruction. Further, soluble fibronectin bound to alpha 5 beta 1 might be life-sustaining for floating MC by driving cells into cell cycle.     

Subunit composition and functional properties of G-protein heterotrimers on rat chromaffin granules

Heterotrimeric G-proteins at the plasma membrane serve as switches between heptahelical receptors and intracellular signal cascades. Likewise endomembrane associated G-proteins may transduce signals from intracellular compartments provided they consist of a functional trimer. Using quantitative immunoelectron microscopy we found heterotrimeric G-protein subunits Galpha2, Galpha(q/11), Gbeta2 and Gbeta5 to reside on secretory granules in chromaffin cells of rat adrenal glands.Thus rat chromaffin granules are equipped with functional G-proteins that consist of a specific alpha-, beta- and probably gamma-subunit combination. Serotonin uptake into a crude rat chromaffin granule preparation was inhibited by activated Galphao2 (10 nM) to nearly the same extent as by GMppNp (50 microM) whereas GDPbetaS was ineffective. The data support the idea that vesicular G-proteins directly regulate the transmitter content of secretory vesicles. In this respect Galphao2 appears to be the main regulator of vesicular momoamine transporter activity.     

Marshall Barber and the century of microinjection: from cloning of bacteria to cloning of everything

A hundred years ago, Dr. Marshall A. Barber proposed a new technique - the microinjection technique. He developed this method initially to clone bacteria and to confirm the germ theory of Koch and Pasteur. Later on, he refined his approach and was able to manipulate nuclei in protozoa and to implant bacteria into plant cells. Continuous improvement and adaptation of this method to new applications dramatically changed experimental embryology and cytology and led to the formation of several new scientific disciplines including animal cloning as one of its latest applications. Interestingly, microinjection originated as a method at the crossroad of bacteriology and plant biology, demonstrating once again the unforeseen impact that basic research in an unrelated field can have on the development of entirely different disciplines.     

Subnetwork hierarchies of biochemical pathways

MOTIVATION: The vastness and complexity of the biochemical networks that have been mapped out by modern genomics calls for decomposition into subnetworks. Such networks can have inherent non-local features that require the global structure to be taken into account in the decomposition procedure. Furthermore, basic questions such as to what extent the network (graph theoretically) can be said to be built by distinct subnetworks are little studied. RESULTS: We present a method to decompose biochemical networks into subnetworks based on the global geometry of the network. This method enables us to analyze the full hierarchical organization of biochemical networks and is applied to 43 organisms from the WIT database. Two types of biochemical networks are considered: metabolic networks and whole-cellular networks (also including for example information processes). Conceptual and quantitative ways of describing the hierarchical ordering are discussed. The general picture of the metabolic networks arising from our study is that of a few core-clusters centred around the most highly connected substances enclosed by other substances in outer shells, and a few other well-defined subnetworks. AVAILABILITY: An implementation of our algorithm and other programs for analyzing the data is available from http://www.tp.umu.se/forskning/networks/meta/ SUPPLEMENTARY INFORMATION: Supplementary material is available at http://www.tp.umu.se/forskning/networks/meta/     

Broad and complex antifungal activity among environmental isolates of lactic acid bacteria

More than 1200 isolates of lactic acid bacteria isolated from different environments were screened for antifungal activity in a dual-culture agar plate assay. Approximately 10% of the isolates showed inhibitory activity and 4% showed strong activity against the indicator mould Aspergillus fumigatus. The antifungal spectra for 37 isolates with strong activity and five isolates with low or no activity were determined. Several of the strains showed strong inhibitory activity against the moulds A. fumigatus, Aspergillus nidulans, Penicillium commune and Fusarium sporotrichioides, and also against the yeast Rhodotorula mucilaginosa. Penicillium roqueforti and the yeasts Pichia anomala and Kluyveromyces marxianus were not inhibited. Several isolates showed reduced antifungal activity after storage and handling. The majority of the fungal inhibitory isolates were identified by 16S rDNA sequencing as Lactobacillus coryniformis. Lactobacillus plantarum and Pediococcus pentosaceus were also frequently identified among the active isolates. The degree of fungal inhibition was not only related to production of lactic or acetic acid. In addition, antifungal cyclic dipeptides were identified after HPLC separation and several other active fractions were found suggesting a highly complex nature of the antifungal activity.     

Evaluation of exudates by solid phase microextraction-gas chromatography

Head-space solid phase microextraction combined with gas chromatography (SPME-GC) was used for the determination of bacterial volatile fatty acid (VFA) patterns. The method was validated with cultures of reference bacterial strains. It was confirmed that VFA production depends on the composition of the cultivation medium, which limits accurate characterisation of particular bacterial species. A set of 195 clinical exudates of various origin and consistence was analysed using SPME-GC and compared with 73 samples extracted using tert-butyl methyl ether. Approximate agreement of VFA profiles with cultivation findings was found in most cases. However, 20.5% of clinical exudates with distinct VFA profiles appeared to be false-negative by cultivation. Using SPME-GC of exudates, the frequency of false-negative cultivations was higher than that of solvent extraction of exudates or blood cultures found previously. The described method is suitable for preliminary detection of bacteria, particularly non-sporulating anaerobes, in clinical samples. It can reveal false-negative findings due to cultivation. Analysis can be performed in 30 min without the need for cultivation.     

Histological process and dynamics of germ cell degeneration in pejerrey Odontesthes bonariensis larvae and juveniles during exposure to warm water

Elevated temperature causes degeneration and disappearance of the germ cells in the males of scrotal mammals. It was recently shown that heat-induced germ cell degeneration occurs also in fish but, unlike in mammals, it occurs not only in males but also in females. The purpose of this study was to clarify the histological process and dynamics of heat-induced germ cell disappearance in pejerrey Odontesthes bonariensis larvae and juveniles. Monosex and mixed-sex fish produced by thermal manipulation of sex (temperature-dependent sex determination) were subjected to 29 degrees C for periods between 1 and 12 weeks, and used to analyze, by histological methods, the changes in gonadal size and the number of normal and degenerating germ cells. Groups exposed to 29 degrees C for 8-12 weeks were subsequently transferred to 24 degrees C to verify if any gonadal damage would be permanent. Germ cell degeneration, histologically characterized by nuclear pyknosis or eosinophilia and cytoplasmic eosinophilia, was observed with increasing frequency at higher temperatures (29>24> 17 degrees C) and more in males than in females. Clear degenerative changes in the germinal epithelium usually began within one week of exposure to 29 degrees C and appeared clearer in females than in males. Complete loss of germ cells was observed only in individuals exposed for periods of 8-12 weeks to 29 degrees C but no treatment produced 100% sterile fish. Germ cells that remained in the gonads after exposure to 29 degrees C retained the capacity to rapidly recolonize germ cell-depleted areas, suggesting that the associated somatic cells in the gonads are little or not affected by this temperature.     

Evidence of cytochrome P450-catalyzed cleavage of the ether bond of phenoxybutyrate herbicides in Rhodococcus erythropolis K2-3

Bacterial strain Rhodococcus erythropolis K2-3 can cleave theether bond of the phenoxybutyrate herbicides, i.e., 4-(2,4-dichlorophenoxy)butyrate(2,4-DB) and 4-(4-chloro-2-methylphenoxy)butyrate (MCPB), by anenzyme system that is constitutively expressed. The enzyme(s) involved were investigated in this study. The rate ofdisappearance of 2,4-DB determined in a whole cell assay amounted to0.6 mmol/h ¶ g_{dry mass}.Carbon monoxide difference spectra of dithionite-reduced wholecells and crude cell extracts suggested that strain K2-3 contains a soluble cytochrome P450(P450), named P450_PB-1. The addition of various phenoxybutyrate substrates to crude cell extracts resulted in typical difference spectra following the type I pattern ofsubstrate binding with P450. The rate of 2,4-DB cleavage was reduced by inhibitors of P450: 5 mM metyrapone and carbon monoxide at a CO/O₂ ratio of 10 reduced the activity by about 20%, and 70%, respectively. The ether cleaving activity completely disappearedafter disruption of the cells and could not be detected in crude extracts. To elucidate theenzymatic basis of this reaction, P450 was partially purified. With the resulting enzyme preparation,2,4-DB cleavage activity was re-established, becoming measurable after the addition of eitherphenazine methosulfate or ferredoxin and ferredoxin/NADP oxidoreductase from spinach. We detected no activities attributable to -ketoglutarate-dependent dioxygenase orNAD(P)H-dependent monooxygenase. These results collectively indicatethat cleavage of the ether bond of phenoxybutyrate herbicides is catalyzed by P450-mediated activityin this strain. One of the products derived from this reaction is dichlorophenol, and comparativechromatographic analyses suggest that the other product is a C4-carbonicacid, most likely succinic semialdehyde/succinate.