Activation of the stress-activated protein kinase (SAPK/JNK) by genotoxic agents is necessary for induction of apoptosis. We report here that ionizing radiation ionizing radiation exposure induces translocation of SAPK to mitochondria and association of SAPK with the anti-apoptotic Bcl-x L protein. SAPK phosphorylates Bcl-x L on threonine 47 (Thr-47) and threonine 115 (Thr-115) in vitro and in vivo. In contrast to wild-type Bcl-x L , a mutant Bcl-x L with the two threonines substituted by alanines (Ala-47, Ala-115) is a more potent inhibitor of ionizing radiation-induced apoptosis. These findings indicate that translocation of SAPK to mitochondria is functionally important for interactions with Bcl-x L in the apoptotic response to genotoxic stress.

Apoptosis is induced by the release of cytochrome c from mitochondria to the cytoplasm. The present studies demonstrate that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces translocation of protein kinase C (PKC) ␦ from the cytoplasm to mitochondria. The results also show that translocation of PKC␦ results in release of cytochrome c. The functional significance of this event is further supported by the demonstration that PKC␦ translocation is required for TPA-induced apoptosis. These findings demonstrate that translocation of PKC␦ to mitochondria is responsible, at least in part, for inducing cytochrome c release and apoptosis.

A new coronavirus has been implicated as the causative agent of severe acute respiratory syndrome (SARS). We have used convalescent sera from several SARS patients to detect proteins in the culture supernatants from cells exposed to lavage another SARS patient. The most prominent protein in the supernatant was identified by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a ϳ46-kDa species. This was found to be a novel nucleocapsid protein that matched almost exactly one predicted by an open reading frame in the recently published nucleotide sequence of the same virus isolate (>96% coverage). A second viral protein corresponding to the predicted ϳ139-kDa spike glycoprotein has also been examined by MALDI-TOF MS (42% coverage). After peptide N-glycosidase F digestion, 12 glycosylation sites in this protein were confirmed. The sugars attached to four of the sites were also identified. These results suggest that the nucleocapsid protein is a major immunogen that may be useful for early diagnostics, and that the spike glycoprotein may present a particularly attractive target for prophylactic intervention in combating SARS. Molecular & Cellular Proteomics 2: 346 -356, 2003.

ANG converting enzyme (ACE) 2 (ACE2) is a homologue of ACE, which is not blocked by conventional ACE inhibitors. ACE2 converts ANG 1-10 (ANG I) to ANG 1-9, which can be hydrolyzed by ACE to form the biologically active peptide ANG 1-7. ACE2 is expressed in the kidney, but its precise intrarenal localization is unclear, and the role of intrarenal ACE2 in the production of ANG 1-7 is unknown. The present studies determined the relative distribution of ACE2 in the rat kidney and defined its role in the generation of ANG 1-7 in proximal tubule. In microdissected rat nephron segments, semiquantitative RT-PCR revealed that ACE2 mRNA was widely expressed, with relatively high levels in proximal straight tubule (PST). Immunohistochemistry demonstrated ACE2 protein in tubular segments, glomeruli, and endothelial cells. Utilizing mass spectrometry, incubation of isolated PSTs with ANG I (10(-6) M) led to generation of ANG 1-7 (sensitivity of detection > 1 x 10(-9) M), accompanied by the formation of ANG 1-8 (ANG II) and ANG 1-9. The ACE2 inhibitor DX600 completely blocked ANG I-mediated generation of ANG 1-7. Incubation of PSTs with ANG 1-9 also led to generation of ANG 1-7, an effect blocked by the ACE inhibitor captopril or enalaprilat, but not by DX600. Incubation of PSTs with ANG II or luminal perfusion of ANG II did not result in detection of ANG 1-7. The results indicate that ACE2 is widely expressed in rat nephron segments and contributes to the production of ANG 1-7 from ANG I in PST. ANG II may not be a major substrate for ACE2 in isolated PST. The data suggest that ACE2-mediated production of ANG 1-7 represents an important component of the proximal tubular renin-ANG system.

Real‐time quaking‐induced conversion (RT‐QuIC) has been proposed as a sensitive diagnostic test for sporadic Creutzfeldt–Jakob disease; however, before this assay can be introduced into clinical practice, its reliability and reproducibility need to be demonstrated. Two international ring trials were undertaken in which a set of 25 cerebrospinal fluid samples were analyzed by a total of 11 different centers using a range of recombinant prion protein substrates and instrumentation. The results show almost complete concordance between the centers and demonstrate that RT‐QuIC is a suitably reliable and robust technique for clinical practice. Ann Neurol 2016;80:160–165