SummaryThe minimization of a genome is necessary to identify experimentally the minimal gene set that contains only those genes that are essential and sufficient to sustain a functioning cell. Recent developments in genetic techniques have made it possible to generate bacteria with a markedly reduced genome. We developed a simple system for formation of markerless chromosomal deletions, and constructed and characterized a series of large-scale chromosomal deletion mutants of Escherichia coli that lack between 2.4 and 29.7% of the parental chromosome. Combining deletion mutations changes cell length and width, and the mutant cells with larger deletions were even longer and wider than the parental cells. The nucleoid organization of the mutants is also changed: the nucleoids occur as multiple small nucleoids and are localized peripherally near the envelope. Inhibition of translation causes them to condense into one or two packed nucleoids, suggesting that the coupling of transcription and translation of membrane proteins peripherally localizes chromosomes. Because these phenotypes are similar to those of spherical cells, those may be a consequence of the morphological change. Based on the nucleoid localization observed with these mutants, we discuss the cellular nucleoid dynamics.
The pathogenesis of idiopathic Parkinson's disease is unknown, but nigral degeneration and depigmentation are associated with microglial inflammation and anti-inflammatory medications appear to protect against the disease. The possibility that humoral immunity may play a role in initiating or regulating the inflammation has been suggested by experimental studies triggering dopamine cell death using a variety of transfer strategies and the observation of CD8+ T lymphocytes and complement in the nigra in Parkinson's disease. We analysed the association between degeneration and humoral immune markers in brain tissue of patients with idiopathic (n = 13) or genetic (n = 2 with alpha-synuclein and n = 1 with parkin mutations) Parkinson's disease and controls without neurological disease (n = 12) to determine the humoral immune involvement in Parkinson's disease. Formalin-fixed tissue samples from the substantia nigra and primary visual cortex for comparison were stained for alpha-synuclein, major histocompatibility complex II (HLA), immunoglobulin M (IgM), immunoglobulin G (IgG), IgG subclasses 1-4 and IgG receptors FcgammaR I-III. Antigen retrieval and both single immunoperoxidase and double immunofluorescence procedures were employed to determine the cell types involved and their pattern and semiquantitative densities. Significant dopamine neuron loss occurred in all patients with Parkinson's disease, negatively correlating with disease duration (r = -0.76, P = 0.002). Although all patients had increased inflammatory HLA immunopositive microglia, the degree of inflammation was similar throughout the disease (r = 0.08, P = 0.82). All patients with Parkinson's disease had IgG binding on dopamine neurons but not IgM binding. Lewy bodies were strongly immunolabelled with IgG. A mean 30 +/- 12% of dopamine nigral neurons were immunoreactive for IgG in Parkinson's disease with the proportion of IgG immunopositive neurons negatively correlating with the degree of cell loss in the substantia nigra (r = -0.67, P < 0.0001) and positively correlating with the number of HLA immunopositive microglia (r = 0.51, P = 0.01). Most neuronal IgG was the IgG1 subclass with some IgG3 and less IgG2 also found in the damaged substantia nigra. The high affinity activating IgG receptor, FcgammaRI, was expressed on nearby activated microglia. The low affinity activating IgG receptor, FcgammaRIII was expressed on cells morphologically resembling lymphocytes, whereas immunoreactivity for the inhibitory IgG receptor FcgammaRII was absent in all cases. This pattern of humoral immune reactivity is consistent with an immune activation of microglia leading to the targeting of dopamine nigral neurons for destruction in both idiopathic and genetic cases of Parkinson's disease.
Pulmonary nosocomial infection.
In Parkinson disease (PD), alpha-synuclein aggregates called Lewy bodies often involve and sequester Septin4 (Sept4), a polymerizing scaffold protein. However, the pathophysiological significance of this phenomenon is unclear. Here, we show the physiological association of Sept4 with alpha-synuclein, the dopamine transporter, and other presynaptic proteins in dopaminergic neurons; mice lacking Sept4 exhibit diminished dopaminergic neurotransmission due to scarcity of these presynaptic proteins. These data demonstrate an important role for septin scaffolds in the brain. In transgenic mice that express human alpha-synuclein(A53T) (a mutant protein responsible for familial PD), loss of Sept4 significantly enhances neuropathology and locomotor deterioration. In this PD model, insoluble deposits of Ser129-phosphorylated alpha-synuclein(A53T) are negatively correlated with the dosage of Sept4. In vitro, direct association with Sept4 protects alpha-synuclein against self-aggregation and Ser129 phosphorylation. Taken together, these data show that Sept4 may be involved in PD as a dual susceptibility factor, as its insufficiency can diminish dopaminergic neurotransmission and enhance alpha-synuclein neurotoxicity.
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