DNA analysis and sorting of rat testis cells using two-parameter flow cytometry

By use of two-parameter flow cytometry of rat testis cell suspensions stained with mithramycin for DNA (the peak amplitude of the fluorescence signal versus total fluorescence intensity integrated over time), eight cell compartments could be distinguished without pre-enrichment of the samples. Cells in these compartments were identified by sorting and subsequent microscopic examination.     

Stereoselective Microbial Dehalorespiration with Vicinal Dichlorinated Alkanes

The suspected carcinogen 1,2-dichloroethane (1,2-DCA) is the most abundant chlorinated C₂ groundwater pollutant on earth. However, a reductive in situ detoxification technology for this compound does not exist. Although anaerobic dehalorespiring bacteria are known to catalyze several dechlorination steps in the reductive-degradation pathway of chlorinated ethenes and ethanes, no appropriate isolates that selectively and metabolically convert them into completely dechlorinated end products in defined growth media have been reported. Here we report on the isolation of Desulfitobacterium dichloroeliminans strain DCA1, a nutritionally defined anaerobic dehalorespiring bacterium that selectively converts 1,2-dichloroethane and all possible vicinal dichloropropanes and -butanes into completely dechlorinated end products. Menaquinone was identified as an essential cofactor for growth of strain DCA1 in pure culture. Strain DCA1 converts chiral chlorosubstrates, revealing the presence of a stereoselective dehalogenase that exclusively catalyzes an energy-conserving anti mechanistic dichloroelimination. Unlike any known dehalorespiring isolate, strain DCA1 does not carry out reductive hydrogenolysis reactions but rather exclusively dichloroeliminates its substrates. This unique dehalorespiratory biochemistry has shown promising application possibilities for bioremediation purposes and fine-chemical synthesis.     

A role for acetylcholine receptors in their own aggregation on muscle cells

Both neurotrophic factors and activity regulate synaptogenesis. At neuromuscular synapses, the neural factor agrin released from motor neuron terminals stimulates postsynaptic specialization by way of the muscle specific kinase MuSK. In addition, activity through acetylcholine receptors (AChRs) has been implicated in the stabilization of pre- and postsynaptic contacts on muscle at various stages of development. We show here that activation of AChRs with specific concentrations of nicotine is sufficient to induce AChR aggregation and that this induction requires the function of L-type calcium channels (L-CaChs). Furthermore, AChR function is required for agrin induced AChR aggregation in C2 muscle cells. The same concentrations of nicotine did not induce observable tyrosine phosphorylation on either MuSK or the AChR beta subunit, suggesting significant differences between the mechanisms of agrin and activity induced aggregation. The AChR/L-CaCh pathway provides a mechanism by which neuromuscular signal transmission can act in concert with the agrin-MuSK signaling cascade to regulate NMJ formation.     

A method for quantifying aggression in male Drosophila melanogaster

Aggressive behavior is a complex social behavior that is difficult to measure. Here, we describe a simple method for the quantitative analysis of aggression in male Drosophila melanogaster. Traditional measurements of aggressive behavior have relied on a territorial context with a food territory and a female as factors that induce or enhance aggression. The protocol described here is devoid of a food territory or a female, making it simpler than most existing methods used to measure aggressive behavior. Multiple pairs of males are tested simultaneously to obtain an average fighting score. Four parameters are used to quantify the behavior: frequency, index, latency and intensity of fighting based on unambiguous offensive fighting behaviors. The assay takes 15 min, during which time a frequency score is obtained for 20-35 pairs simultaneously. More in-depth analysis, including latency, index and intensity, can be performed on the videotaped record of the experiment. The assay is highly reproducible and requires limited resources in a simple setup.     

Crystal Structure of Yeast FAD Synthetase (Fad1) in Complex with FAD

Flavin adenine dinucleotide (FAD) synthetase is an essential enzyme responsible for the synthesis of FAD by adenylation of riboflavin monophosphate (FMN). We have solved the 1.9 Å resolution structure of Fad1, the yeast FAD synthetase, in complex with the FAD product in the active site. The structure of Fad1 shows it to be a member of the PP-ATPase superfamily. Important conformational differences in the two motifs involved in binding the phosphate moieties of FAD compared to the Candida glabrata FMNT ortholog suggests that this loop is dynamic and undergoes substantial conformational changes during its catalytic cycle.     

Bacillus sporothermodurans and other highly heat-resistant spore formers in milk

A recent example of a micro-organism causing undesired growth in consumer milk is Bacillus sporothermodurans producing highly heat-resistant spores (HRS) which may survive ultra-high temperature (UHT) treatment or industrial sterilization. Molecular typing showed a heterogeneous group of farm isolates (non-HRS strains), but a clonal group of UHT isolates from diverse European countries and other continents (HRS-clone) suggesting a common source. During a survey of Belgian dairy farms for the presence of potentially highly heat-resistant spore formers, high numbers of these spores were detected in filter cloth, green crop and fodder samples. The strain collection showed a high taxonomic diversity with 18 potentially new species and with Bacillus licheniformis and Geobacillus pallidus as predominating species overall. Seventeen B. sporothermodurans isolates were identified, mainly originating from feed concentrate. Heat resistance studies showed the UHT resistance of B. sporothermodurans spores present in industrially contaminated UHT milk, but a lower heat resistance of laboratory-grown strains (HRS and non-HRS). Hydrogen peroxide, used as sanitizer in the dairy industry, was found to induce higher heat resistance of laboratory-grown B. sporothermodurans strains to a certain level. This indicates that sublethal stress conditions may affect the heat resistance. By transmission electron microscopy, structural differences at the spore level were found between HRS and non-HRS strains. The data indicate that the attainment of extreme heat resistance is rather multifactorial.     

Reduction of lamin B receptor levels by miR-340-5p disrupts chromatin,promotes cell senescence and enhances senolysis

A major stress response influenced by microRNAs (miRNAs) is senescence, a state of indefinite growth arrest triggered by sublethal cell damage. Here, through bioinformatic analysis and experimental validation, we identified miR-340-5p as a novel miRNA that foments cellular senescence. miR-340-5p was highly abundant in diverse senescence models, and miR-340-5p overexpression in proliferating cells rendered them senescent. Among the target mRNAs, miR-340-5p prominently reduced the levels of LBR mRNA, encoding lamin B receptor (LBR). Loss of LBR by ectopic overexpression of miR-340-5p derepressed heterochromatin in lamina-associated domains, promoting the expression of DNA repetitive elements characteristic of senescence. Importantly, overexpressing miR-340-5p enhanced cellular sensitivity to senolytic compounds, while antagonization of miR-340-5p reduced senescent cell markers and engendered resistance to senolytic-induced cell death. We propose that miR-340-5p can be exploited for removing senescent cells to restore tissue homeostasis and mitigate damage by senescent cells in pathologies of human aging.     

Dependence of acetylcholine and ADP dilation of pial arterioles on heme oxygenase after transfusion of cell-free polymeric hemoglobin

Polymers of cell-free hemoglobin have been designed for clinical use as oxygen carriers, but limited information is available regarding their effects on vascular regulation. We tested the hypothesis that the contribution of heme oxygenase (HO) to acetylcholine-evoked dilation of pial arterioles is upregulated 2 days after polymeric hemoglobin transfusion. Dilator responses to acetylcholine measured by intravital microscopy in anesthetized cats were blocked by superfusion of the HO inhibitor tin protoporphyrin-IX (SnPPIX) in a group that had undergone exchange transfusion with hemoglobin 2 days earlier but not in surgical sham and albumin-transfused groups. However, immunoblots from cortical brain homogenates did not reveal changes in expression of the inducible isoform HO1 or the constitutive isoform HO2 in the hemoglobin-transfused group. To test whether the inhibitory effect of SnPPIX was present acutely after hemoglobin transfusion, responses were measured within an hour of completion of the exchange transfusion. In control and albumin-transfused groups, acetylcholine responses were unaffected by SnPPIX but were blocked by addition of the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine (l-NNA) to the superfusate. In hemoglobin-transfused groups, the acetylcholine response was blocked by either SnPPIX or l-NNA alone. The effect of another HO inhibitor, chromium mesoporphyrin (CrMP), was tested on ADP, another endothelial-dependent dilator, in anesthetized rats. Pial arteriolar dilation to ADP was unaffected by CrMP in controls but was attenuated 62% by CrMP in rats transfused with hemoglobin. It is concluded that 1) polymeric hemoglobin transfusion acutely upregulates the contribution of HO to acetylcholine-induced dilation of pial arterioles in cats, 2) this upregulation persists 2 days after transfusion when 95% of the hemoglobin is cleared from the circulation, and 3) this acute upregulation of HO signaling is ubiquitous in that similar effects were observed with a different endothelial-dependent agonist (i.e., ADP) in a another species (rat).