Function of the oxidative burst in hypersensitive disease resistance.

Microbial elicitors or attempted infection with an avirulent pathogen strain causes the rapid production of reactive oxygen intermediates. Recent findings indicate that H2O2 from this oxidative burst plays a central role in the orchestration of the hypersensitive response: (i) as the substrate driving the cross-linking of cell wall structural proteins to slow microbial ingress prior to the deployment of transcription-dependent defenses and to trap pathogens in cells destined to undergo hypersensitive cell death, (ii) as a local threshold trigger of this programmed death in challenged cells, and (iii) as a diffusible signal for the induction in adjacent cells of genes encoding cellular protectants such as glutathione S-transferase and glutathione peroxidase. These findings provide the basis for an integrated model for the orchestration of the localized hypersensitive resistance response to attack by an avirulent pathogen.

Neurotoxicity of advanced glycation endproducts during focal stroke and neuroprotective effects of aminoguanidine.

Cerebral infarction (stroke) is a potentially disastrous complication of diabetes mellitus, principally because the extent of cortical loss is greater in diabetic patients than in nondiabetic patients. The etiology of this enhanced neurotoxicity is poorly understood. We hypothesized that advanced glycation endproducts (AGEs), which have previously been implicated in the development of other diabetic complications, might contribute to neurotoxicity and brain damage during ischemic stroke. Using a rat model of focal cerebral ischemia, we show that systemically administered AGE-modified bovine serum albumin (AGE-BSA) significantly increased cerebral infarct size. The neurotoxic effects of AGE-BSA administration were dose- and time-related and associated with a paradoxical increase in cerebral blood flow. Aminoguanidine, an inhibitor of AGE cross-linking, attenuated infarct volume in AGE-treated animals. We conclude that AGEs may contribute to the increased severity of stroke associated with diabetes and other conditions characterized by AGE accumulation.

Degradation of the cleaved leader peptide of thiolase by a peroxisomal proteinase.

A peroxisomal location for insulin-degrading enzyme (IDE) has been defined by confocal immunofluorescence microscopy of stably transfected CHO cells overexpressing IDE and digitonin-permeabilization studies in normal nontransfected fibroblasts. The functional significance of IDE in degrading cleaved leader peptides of peroxisomal proteins targeted by the type II motif was evaluated with a synthetic peptide corresponding to the type II leader peptide of prethiolase. The peptide effectively competed for degradation and cross-linking of the high-affinity substrate 125I-labeled insulin to IDE. Direct proteolysis of the leader peptide of prethiolase was confirmed by HPLC; degradation was inhibited by immunodepletion with an antibody to IDE. Phylogenetic analysis of proteinases related to IDE revealed sequence similarity to mitochondrial processing peptidases.

The murine stk gene product, a transmembrane protein tyrosine kinase, is a receptor for macrophage-stimulating protein.

Macrophage-stimulating protein (MSP) was originally identified as an inducer of murine resident peritoneal macrophage responsiveness to chemoattractants. We recently showed that the product of RON, a protein tyrosine kinase cloned from a human keratinocyte library, is the receptor for MSP. Similarity of murine stk to RON led us to determine if the stk gene product is the murine receptor for MSP. Radiolabeled MSP could bind to NIH 3T3 cells transfected with murine stk cDNA (3T3/stk). Binding was saturable and was inhibited by unlabeled MSP but not by structurally related proteins, including hepatocyte growth factor and plasminogen. Specific binding to STK was demonstrated by cross-linking of 125I-labeled MSP to membrane proteins of 3T3/stk cells, which resulted in a protein complex with a molecular mass of 220 kDa. This radiolabeled complex comprised 125I-MSP and STK, since it could be immunoprecipitated by antibodies to the STK beta chain. Binding of MSP to stk cDNA-transfected cells induced tyrosine phosphorylation of the 150-kDa STK beta chain within 1 min and caused increased motile activity. These results establish the murine stk gene product as a specific transmembrane protein tyrosine kinase receptor for MSP. Inasmuch as the stk cDNA was cloned from a hematopoietic stem cell, our data suggest that in addition to macrophages and keratinocytes, a cell in the hematopoietic lineage may also be a target for MSP.