No abstract

The source of energy for bacterial motility is the intermediate in In eukaryotes the transduction of chemical into mechanical energy (movement) involves ATP whenever it has been determined. Thus muscles contract and cilia and flagella beat by a sliding filament mechanism that uses ATP directly (1-3).In prokaryotes the immediate source of energy used in chemomechanical coupling is unknown.Cells can generate ATP either by oxidative phosphorylation, resulting from the oxidation of substrates via electron transport, or by substrate-level phosphorylation, even anaerobically as in glycolysis. The

A protein methylation reaction involved in chemotaxis of Escherichia coli has been identified. The involvement of this reaction in chemotaxis in indicated by four lines of evidence. (a) The methylation reaction is altered in several classes of generally nonchemotactic mutants and is coreverted with the chemotaxis defects. (b) The methylation level of the protein is affected by chemotactic stimuli. (c) The transferred methyl group is derived from methionine and is labile, in accord with the known fact that chemotaxis requires a continuous supply of methionine. (d) Methylation is abnormal in various mutants having defective or missing flagella.

Chronic alveolar hypoxia is the major cause of pulmonary hypertension. The cellular mechanisms involved in hypoxia- induced pulmonary arterial remodeling are still poorly understood. Mitogen-activated protein kinase (MAPK) is a key enzyme in the signaling pathway leading to cellular growth and proliferation. The purpose of this investigation was to determine the roles that MAPKs, specifically Jun-N-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), and p38 kinase, play in the hypoxia-induced pulmonary arterial remodeling. Rats were exposed to normobaric hypoxia (10% O(2)) for 1, 3, 7, or 14 d. Hypoxia caused significant remodeling in the pulmonary artery characterized by thickening of pulmonary arterial wall and increases in tissue mass and total RNA. JNK, ERK, and p38 kinase tyrosine phosphorylations and their activities were significantly increased by hypoxia. JNK activation peaked at Day 1 and ERK/p38 kinase activation peaked after 7 d of hypoxia. The results from immunohistochemistry show that hypoxia increased phospho-MAPK staining in both large and small intrapulmonary arteries. Hypoxia also upregulated vascular endothelial growth factor messenger RNA (mRNA) and platelet-derived growth factor receptor mRNA levels in pulmonary artery with a time course correlated to the activation of ERK and p38 kinase. The gene expressions of c-jun, c-fos, and egr-1, known as downstream effectors of MAPK, were also investigated. Hypoxia upregulated egr-1 mRNA but downregulated c-jun and c-fos mRNAs. These data suggest that hypoxia-induced activation of JNK is an early response to hypoxic stress and that activation of ERK and p38 kinase appears to be associated with hypoxia-induced pulmonary arterial remodeling.

Controversies abound concerning hematopoietic stem cell transduction by recombinant adeno-associated virus 2 (AAV) vectors. For human hematopoietic cells, we have shown that this problem is related to the extent of expression of the cellular receptor for AAV. At least a small subset of murine hematopoietic cells, on the other hand, does express both the AAV receptor and the coreceptor, yet is transduced poorly. In the present study, we have found that approximately 85% of AAV genomes were present in the cytoplasmic fraction of primary murine c-Kit(+)Lin- hematopoietic cells. However, when mice were injected intraperitoneally with hydroxyurea before isolation of these cells, the extent to which AAV genomes were detected in the cytoplasmic fraction was reduced to approximately 40%, with a corresponding increase to approximately 60% in the nuclear fraction, indicating that hydroxyurea facilitated nuclear transport of AAV. It was apparent, nonetheless, that a significant fraction of the AAV genomes present in the nuclear fraction from cells obtained from hydroxyurea-treated mice was single stranded. We next tested whether the single-stranded AAV genomes were derived from virions that failed to undergo uncoating in the nucleus. A substantial fraction of the signal in the nuclear fraction of hematopoietic cells obtained from hydroxyurea-treated mice was also resistant to DNase I. That AAV particles were intact and biologically active was determined by successful transduction of 293 cells by virions recovered from murine hematopoietic cells 48 hr postinfection. Although hydroxyurea facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second- strand DNA synthesis and transgene expression. A better understanding of the underlying mechanism of viral uncoating has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.