Calcium depletion dissociates and activates heterodimeric notch receptors.

Notch receptors participate in a highly conserved signaling pathway that regulates morphogenesis in multicellular animals. Maturation of Notch receptors requires the proteolytic cleavage of a single precursor polypeptide to produce a heterodimer composed of a ligand-binding extracellular domain (N(EC)) and a single-pass transmembrane signaling domain (N(TM)). Notch signaling has been correlated with additional ligand-induced proteolytic cleavages, as well as with nuclear translocation of the intracellular portion of N(TM) (N(ICD)). In the current work, we show that the N(EC) and N(TM) subunits of Drosophila Notch and human Notch1 (hN1) interact noncovalently. N(EC)-N(TM) interaction was disrupted by 0.1% sodium dodecyl sulfate or divalent cation chelators such as EDTA, and stabilized by millimolar Ca(2+). Deletion of the Ca(2+)-binding Lin12-Notch (LN) repeats from the N(EC) subunit resulted in spontaneous shedding of N(EC) into conditioned medium, implying that the LN repeats are important in maintaining the interaction of N(EC) and N(TM). The functional consequences of EDTA-induced N(EC) dissociation were studied by using hN1-expressing NIH 3T3 cells. Treatment of these cells for 10 to 15 min with 0.5 to 10 mM EDTA resulted in the rapid shedding of N(EC), the transient appearance of a polypeptide of the expected size of N(ICD), increased intranuclear anti-Notch1 staining, and the transient activation of an Notch-sensitive reporter gene. EDTA treatment of HeLa cells expressing endogenous Notch1 also stimulated reporter gene activity to a degree equivalent to that resulting from exposure of the cells to the ligand Delta1. These findings indicate that receptor activation can occur as a consequence of N(EC) dissociation, which relieves inhibition of the intrinsically active N(TM) subunit.

Protein mixed-disulfides in cardiac cells. S-thiolation of soluble proteins in response to diamide.

Protein mixed-disulfides in cultured rat heart cells were analyzed by gel electrophoresis under conditions that eliminated artifactual formation of these protein derivatives. Protein S-thiolation (protein mixed-disulfide formation) was detectable under normal culture conditions. Diamide oxidized intracellular glutathione in these cells and produced extensive protein S-thiolation. The specificity of this protein modification indicates a role in the regulation of cardiac metabolism.

A comparison of protein S-thiolation (protein mixed-disulfide formation) in heart cells treated with t-butyl hydroperoxide or diamide.

Beating neonatal heart cell cultures were treated with diamide or t-butyl hydroperoxide, and changes in glutathione oxidation, cell beating, and protein S-thiolation (protein mixed-disulfide formation) were examined. Both compounds caused extensive oxidation of glutathione. Cells treated with diamide stopped beating within 2 min, and beating returned to normal after 30-45 min. Cells stopped beating 25 min after the addition of t-butyl hydroperoxide, and beating did not resume. t-Butyl hydroperoxide caused S-thiolation of a variety of proteins, but only one protein, of molecular mass 23 kDa, was extensively modified. Diamide caused extensive modification of proteins with molecular masses of 97, 42 and 23 kDa as well as three proteins of about 35 kDa. Though the GSSG content of cell cultures returned to normal by 15 min after diamide treatment. S-thiolation of several proteins persisted. These studies show that S-thiolation of proteins is an important metabolic response in cells exposed to an oxidative challenge by t-butyl hydroperoxide or diamide, and that the specificity of the response depends on the agent used.

Apoptosis and the proteasome.

The ubiquitin-proteasome pathway is responsible for the regular turnover of a wide variety of proteins and is a critical regulator of many cellular processes. Although this pathway is abundant and ubiquitous, it is also discriminating. This specificity is achieved because there are multiple levels of regulation at work in the pathway. X-ray crystallographic data on the eukaryotic 20S proteasome suggest that substantial rearrangement of the alpha rings, probably mediated by the association of additional regulatory complexes, is required to allow access of substrates into the inner core of the complex. The associated complexes also confer a ubiquitin-dependence on the proteasome, requiring that potential substrates be tagged with chains of ubiquitin proteins. The presence of multiple ubiquitinating enzymes that favor distinct substrates provides a way for a cell to regulate what proteins are to be ubiquitinated. In some cases ubiquitination is not required, but we now know that other modifications, such as phosphorylation and protein-protein interactions, are also important for targeting proteins for degradation. Even with the existence of so many regulatory controls, it is difficult to imagine how one complex can perform so many tasks. As more information is gathered about the proteasome, we begin to understand that all proteasomes are not exactly the same. For example, there is strong evidence that proteasomes involved in antigen presentation differ in both composition and function from proteasomes involved in other processes. The past image of the proteasome as a static structure is being shed, and a new image is emerging that portrays the complex as dynamic and flexible, able to tailor its composition and function to meet a particular need. With this new image of the proteasome in mind, investigators are looking at the potential involvement of the proteasome in cell death. Inhibitor studies have demonstrated a requirement for proteasomes during apoptosis in noncycling and differentiated cells. Similar studies in cycling cells suggest that the proteasome may regulate a cell's decision to proliferate, differentiate, or die. It will be necessary in the future to supplement the peptide and lactacystin studies with work that is not inhibitor-driven since the specificity of an inhibitor for a particular protease is always in question. In addition, a real understanding of how proteasomes may regulate this process awaits the identification of its substrates. With cell death investigators showing increased interest in proteasomes, it may be possible in the next few years to determine the precise role of the proteasome in the pathways that lead to the death of a cell.

Molecular epidemiology of Escherichia coli O157:H7 strains by bacteriophage lambda restriction fragment length polymorphism analysis: application to a multistate foodborne outbreak and a day-care center cluster.

Genomic DNAs prepared from 168 isolates of Escherichia coli O157:H7 were analyzed for restriction fragment length polymorphisms on Southern blots probed with bacteriophage lambda DNA. The isolates analyzed included strains from a recent large multistate outbreak of E. coli O157:H7 infection associated with consumption of poorly cooked beef in restaurants, a day-care center cluster, and temporally and geographically unrelated isolates. E. coli O157:H7 isolates recovered from the incriminated meat and from 61 (96.8%) of 63 patients from Washington and Nevada possessed identical lambda restriction fragment length patterns. The lambda restriction fragment length polymorphisms observed in 11 (91.7%) of 12 day-care center patients were identical, but they differed from that of the strain associated with the multistate outbreak. E. coli O157:H7 from 42 patients temporally or geographically unrelated to either cluster of infection possessed unique and different lambda restriction fragment length patterns, except for paired isolates from three separate clusters of infection. These data demonstrate that the hybridization of DNA digests of E. coli O157:H7 with radiolabelled bacteriophage lambda DNA can be a useful, stable, and discriminatory epidemiologic tool for analyzing the linkage between strains of E. coli O157:H7.

Proteasomes play an essential role in thymocyte apoptosis.

Cell death in many different organisms requires the activation of proteolytic cascades involving cytosolic proteases. Here we describe a novel requirement in thymocyte cell death for the 20S proteasome, a highly conserved multicatalytic protease found in all eukaryotes. Specific inhibitors of proteasome function blocked cell death induced by ionizing radiation, glucocorticoids or phorbol ester. In addition to inhibiting apoptosis, these signals prevented the cleavage of poly(ADP-ribose) polymerase that accompanies many cell deaths. Since overall rates of protein degradation were not altered significantly during cell death in thymocytes, these results suggest that the proteasome may either degrade regulatory protein(s) that normally inhibit the apoptotic pathway or may proteolytically activate protein(s) than promote cell death.

Mecillinam alone and in combination with ampicillin or moxalactam in experimental Escherichia coli meningitis.

The activity of mecillinam, ampicillin and moxalactam alone and in combination was determined in a lapin meningitis model and a mouse meningitis model against two Escherichia coli strains isolated from infants with meningitis. Both strains were highly susceptible in vitro to the antibiotics, and responded well in systemic mouse protection tests (PD50 less than 4 mg/kg). Continuous infusion of mecillinam in the lapin model over nine hours was effective in sterilizing the CSF of three of four animals infected with one strain. This prompt bacteriologic response to mecillinam alone precluded the possibility of constant infusion administration for synergy studies. Therefore, single dose administration was used to demonstrate the synergistic potential of mecillinam with ampicillin in the lapin meningitis model against the E. coli Kl # 2 strain. The combination of moxalactam and mecillinam was synergistic against the E. coli Kl # 2 strain in the mouse meningitis model. The synergistic potential of these combinations could not be reliably predicted by in vitro tests, time kill curves or systemic mouse protection tests.

Molecular epidemiology of a fast-food restaurant-associated outbreak of Escherichia coli O157:H7 in Washington State.

We studied the molecular epidemiology of the recent fast-food restaurant chain-associated Escherichia coli O157:H7 outbreak in Washington State. Genomic DNAs prepared from strains isolated from 433 patients were probed with radiolabelled Shiga-like toxin (SLT) I and SLT II genes and bacteriophage lambda DNA and were subsequently analyzed for their restriction fragment length polymorphism (RFLP) patterns. The SLT RFLP and lambda RFLP profiles of an E. coli O157:H7 strain isolated from the incriminated beef and prototype patient were compared with those of the patient isolates for determination of the concordance between patterns. Of the 377 patients with primary and secondary cases of infection epidemiologically linked to the outbreak, isolates from 367 (97.3%) of the patients displayed SLT RFLP and lambda RFLP profiles identical to those of the outbreak strains. Isolates from 10 of the 377 (2.6%) patients possessed SLT RFLP and lambda RFLP profiles different from those of the outbreak strains, and the patients from whom those isolates were obtained were subsequently characterized as having non-outbreak-related infections. The E. coli O157:H7 strains isolated from 31 of 44 (70.4%) patients who were epidemiologically excluded from the outbreak were linked to the outbreak by RFLP typing. Our results indicate that SLT RFLP and lambda RFLP analyses are stable and sensitive methods, and when they are used in conjunction with an epidemiological investigation they could result in an earlier recognition of outbreaks and their sources, hence prompting measures to prevent the continued transmission of E. coli O157:H7.