Potential limitations in the use of KillerRed for fluorescence microscopy.

KillerRed, a bright red fluorescent protein, is a genetically encoded photosensitizer, which generates radicals and hydrogen peroxide upon green light illumination. The protein is a potentially powerful tool for selective light-induced protein inactivation and cell killing, and can also be used to study downstream effects of locally increased levels of reactive oxygen species. The initial aim of this study was to investigate whether or not KillerRed-mediated reactive oxygen species production inside peroxisomes could trigger the sequestration of these organelles into autophagosomes. Green fluorescent protein-tagged microtubule-associated protein 1 light chain 3 was used as autophagosome marker. We observed that KillerRed also emits weak green fluorescence upon excitation at 480 nm, and this may lead to erroneous data interpretation in conditions where green fluorophores are used. We discuss this potential pitfall of KillerRed for biological imaging and formulate recommendations to avoid misinterpretation of the data.

Farnesylation of Pex19p is not essential for peroxisome biogenesis in yeast and mammalian cells.

Pex19p exhibits a broad binding specificity for peroxisomal membrane proteins (PMPs), and is essential for the formation of functional peroxisomal membranes. Pex19p orthologues contain a C-terminal CAAX motif common to prenylated proteins. In addition, Saccharomyces cerevisiae and Chinese hamster Pex19p are at least partially farnesylated in vivo. Whether farnesylation of Pex19p plays an essential or merely ancillary role in peroxisome biogenesis is currently not clear. Here, we show that (i) nonfarnesylated and farnesylated human Pex19p display a similar affinity towards a select set of PMPs, (ii) a variant of Pex19p lacking a functional farnesylation motif is able to restore peroxisome biogenesis in Pex19p-deficient cells, and (iii) peroxisome protein import is not affected in yeast and mammalian cells defective in one of the enzymes involved in the farnesylation pathway. Summarized, these observations indicate that the CAAX box-mediated processing steps of Pex19p are dispensable for peroxisome biogenesis in yeast and mammalian cells.

A non-inflatable anti-shock garment for obstetric hemorrhage.

OBJECTIVES: Maternal death from hemorrhage in low resource settings is frequently due to long delays in transportation to referral centers and/or in obtaining blood and surgical interventions. This case series was designed to demonstrate the feasibility, efficacy and safety of the non-inflatable anti-shock garment (NI-ASG) for resuscitation and hemostasis in the initial management of obstetric hemorrhage and shock. METHODS: Fourteen cases of obstetric hemorrhage and hypovolemic shock at Memorial Christian Hospital, Sialkot, Pakistan were managed with a specific clinical protocol based on using NI-ASG as the primary intervention. RESULTS: The NI-ASG was used to resuscitate and stabilize women with hypovolemic shock from 18 to 57 h. Thirteen patients survived without evidence of morbidity, but one had prolonged shock followed by multiple organ failure and death. CONCLUSIONS: This study confirmed that the NI-ASG quickly restored the vital signs of most women in severe hemorrhagic shock and stabilized them while awaiting blood transfusion.

Human pex19p binds peroxisomal integral membrane proteins at regions distinct from their sorting sequences.

The molecular machinery underlying peroxisomal membrane biogenesis is not well understood. The observation that cells deficient in the peroxins Pex3p, Pex16p, and Pex19p lack peroxisomal membrane structures suggests that these molecules are involved in the initial stages of peroxisomal membrane formation. Pex19p, a predominantly cytosolic protein that can be farnesylated, binds multiple peroxisomal integral membrane proteins, and it has been suggested that it functions as a soluble receptor for the targeting of peroxisomal membrane proteins (PMPs) to the peroxisome. An alternative view proposes that Pex19p functions as a chaperone at the peroxisomal membrane. Here, we show that the peroxisomal sorting determinants and the Pex19p-binding domains of a number of PMPs are distinct entities. In addition, we extend the list of peroxins with which human Pex19p interacts to include the PMP Pex16p and show that Pex19p's CaaX prenylation motif is an important determinant in the affinity of Pex19p for Pex10p, Pex11pbeta, Pex12p, and Pex13p.

C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal.

The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human d-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released d-aspartate, as a peroxisome-targeting signal (PTS1) was investigated in vivo and in vitro. Bacterially expressed human d-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP-PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP-PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTS1 consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.

Identification and characterization of human PMP34, a protein closely related to the peroxisomal integral membrane protein PMP47 of Candida boidinii.

The cDNA of a human protein displaying 33% identity to the peroxisomal integral membrane protein PMP47 of Candida boidinii (CbPMP47) was cloned. It encodes a protein of 307 amino acids, with a calculated molecular mass of 34,566 Da. The protein, which we name HsPMP34, contains six membrane spans and belongs, like PMP47, to the family of mitochondrial solute carriers. Based on overlapping available expressed sequence tags (ESTs) combined with the sequencing of one EST, the amino acid sequence of the mouse counterpart (molecular mass of 34,422 Da) could also be deduced. By means of reverse-transcription PCR, the presence of PMP34 mRNA was revealed in liver of man and in liver and other tissues of mouse and rat. The human gene is localized to chromosome 22q13. Given the similarity of HsPMP34 not only to CbPMP47 but also to known or putative mitochondrial transporters, its subcellular localization was investigated. Fluorescence microscopy of HepG2 cells or mouse fibroblasts transfected with a plasmid encoding a HsPMP34/green fluorescent fusion protein revealed a punctate pattern, reminiscent of a peroxisomal distribution. By indirect immunofluorescence, the HsPMP34/green fluorescent fusion protein clearly colocalized with the dimeric peroxisomal thiolase. Upon transfection of mouse fibroblasts lacking functional peroxisomes due the knock-out of PEX5 [Baes, M., Gressens, P., Baumgart, E., Carmeliet, P., Casteels, M., Fransen, M., Evrard, P., Fahimi, D., Declercq, P. E., Collen, D., Van Veldhoven, P. P. & Mannaerts, G. P. (1997) Nat. Genet. 17, 49-53], fluorescence was associated with larger and less-abundant structures. Taken together, the data indicate that HsPMP34 is a peroxisomal membrane protein and is either the human counterpart of CbPMP47 or closely related to it. According to its structure, the protein is most probably involved in transport.

The visualization of peroxisomal proteins containing a C-terminal targeting sequence on western blot by using the biotinylated PTS1-receptor.

A procedure to visualize proteins containing a C-terminal peroxisomal targeting signal (PTS1) in complex protein mixtures was developed using a bacterially expressed, biotinylated form of the human PTS1-receptor. The binding of this fusion product to purified PTS1-containing proteins that were separated by SDS-PAGE and blotted onto nitrocellulose was detected by means of streptavidin-alkaline phosphatase and shown to be both saturable and specific. When applied to total tissue extracts, in addition to PTS1-containing proteins various endogenous biotinylated proteins were visualized. Therefore, a two-step staining procedure was optimized whereby the endogenous biotinylated proteins were shielded with a blue precipitate, followed by incubation with the biotinylated receptor and detection of the resulting PTS1-receptor/PTS1-protein complexes with a phosphatase reaction coupled to the formation of a red-colored precipitate. This relatively inexpensive, simple, and fast technique enabled us to visualize a variety of PTS1-containing proteins. In addition, the information presented in this study can be used to facilitate the identification and characterization of receptor-ligand interaction in general and to eliminate interference by endogenous biotinylated proteins intrinsic to the streptavidin-biotin detection system.

Rupture of the external iliac artery during pregnancy: a case of type IV Ehlers-Danlos syndrome.

Ehlers-Danlos Syndrome is a group of connective tissue disorders involving defects in collagen synthesis. Ten different types have been described. The most lethal of these is type IV, with up to 25% maternal mortality described. The defect in type III collagen can lead to rupture of arteries, bowel, or uterus. We present a maternal mortality that occurred in a patient not previously known to have Ehlers-Danlos syndrome. She presented at 29 weeks gestational age with a ruptured external iliac artery. Further complications, including jejunal rupture and aortic disruption ultimately led to her demise. Postmortem examination ultimately confirmed the diagnosis of type IV Ehlers-Danlos syndrome. Although type IV Ehlers-Danlos syndrome is rare, it carries a high risk for maternal morbidity and mortality. Therefore, it is important to be aware of the diagnosis prior to pregnancy and to have a high index of suspicion for arterial or viscus perforation in the face of acute abdominal findings.

Identification and characterization of the putative human peroxisomal C-terminal targeting signal import receptor.

To identify proteins interacting with the C-terminal peroxisomal targeting signal (PTS1), we screened a human liver cDNA library by means of a Saccharomyces cerevisiae genetic system, known as the two-hybrid system. We isolated a cDNA encoding a protein that specifically bound the PTS1 topogenic signal in the intact yeast cell but also in vitro after bacterial expression and purification. Sequence analysis of the full-length cDNA revealed the presence of an open reading frame encoding a 70-kDa polypeptide that belongs to the tetratricopeptide repeat family and that is homologous to the PAS8 and PAS10 gene products, which are required for the formation of normal peroxisomes in yeast. Subcellular fractionation of human liver and immunofluorescence studies on HepG2 cells demonstrated that this PTS1-binding protein is present exclusively in peroxisomes and that the PTS1-binding domain is located to the cytosolic side of the peroxisomal membrane. All available evidence indicates that the PTS1-binding protein is part of the peroxisomal protein import machinery and most probably is the long sought after human PTS1 import receptor.

A non-trophoblastic tumor co-existing with a triploid fetus.

Non-trophoblastic neoplasms are the most frequent, benign tumors of the placenta, occurring in approximately 1% of all placentas examined. A case is described of a 24-year-old woman who presented with severe, early-onset pre-eclampsia, high human chorionic gonadotropin (hCG) levels, and a triploid fetus and who was found to have a small choriohemangioma. The woman, gravida 2 para 1, was referred to our hospital for perinatal evaluation. The fetus, gestational age 18 weeks 3 days, had fetal growth restriction with multiple congenital anomalies. The fetal karyotype was 69,XXY. Compared with the normal range for this gestational age, the beta-hCG level was significantly elevated (1,054,000 mIU/ml) as was the maternal serum alpha-feto-protein measurement (539.1 ng/ml). Sonographically, the placenta appeared hydropic, irregularly shaped, and gelatinous. A suction dilatation and evacuation under sonographic guidance was performed. Histological examination of placental tissue revealed hydropic degeneration of the chorionic villi. The specific histological features of a partial molar pregnancy were not present. However, there were changes consistent with a choriohemangioma. Flow cytometric DNA analysis performed on formalin-fixed, paraffin-embedded tissue blocks of placenta showed triploidy. Immunohistochemical staining with human placental alkaline phosphatase was consistent with a hydropic degeneration pattern. We conclude, first, that triploidy does not always imply the presence of a partial mole. Second, the dictum, that pre-eclampsia, if it occurs under 20 weeks' gestation, must be associated with a molar pregnancy, may not hold when placental aneuploidy is present. Although the findings in this pregnancy could have been incidental, there may be an association between a choriohemangioma and polyploidy.

The 80 kDa cytosolic protein that binds the C-terminal part of rat acyl-CoA oxidase is not a peroxisomal import receptor but a prolyl-endopeptidase.

In an attempt to identify putative peroxisomal import receptors, we investigated the cross-linking of a radioiodinated peptide consisting of the 13 last amino acids of acyl-CoA oxidase and comprising the carboxy-terminal SKL-peroxisomal targeting motive, to proteins present in different subcellular fractions from rat liver. The radiolabeled peptide could be cross-linked to an 80 kDa protein present in the cytosol but not to proteins present in other subcellular fractions including highly purified peroxisomes. Binding was reversible, saturable and dependent on the presence of Mg2+ and ATP or GTP but hydrolysis of the nucleotides was not required. Binding was abolished by pretreatment of the cytosol--but not of the peptide--with N-ethylmaleimide. Binding was not specific for peptides containing the carboxy-terminal SKL-motive, since binding was competed for by the SKL-peptide from which the SKL-motive had been deleted, by the SKL-peptide with reversed sequence and by the SV40 T-antigen nuclear localisation signal peptide, but not by other peptides tested. The 80 kDa binding protein cross-reacted with a monoclonal antibody against hsp90. Purification and internal peptide sequencing of the binding protein revealed its identity as prolyl-endopeptidase. In retrospect, we realized that the SKL-peptide and all competing peptides contained a proline residue, which was not present in the non-competing peptides. In recent experiments in yeast McNew et al. (McNew, J.A., Sykes, K. and Goodman, J.M. (1993) Mol. Biol. Cell 4, 223-232) cross-linked a peroxisomal targeting peptide to a 20 kDa cytosolic protein that was identified as proline isomerase despite the fact that the peptide did not contain proline. The experiments by McNew et al. in yeast and our experiments in the rat suggest that the (peroxisomal) targeting sequence cross-linking approach may not be suited for the identification of (peroxisomal) import receptors.

D-aspartate oxidase, a peroxisomal enzyme in liver of rat and man.

By means of subcellular fractionation D-aspartate oxidase was shown to be localized in peroxisomes in rat and human liver. The oxidase from both sources was most active on D-aspartate and N-methyl-D-aspartate. In different rat tissues, the highest enzyme activity was found in kidney, followed by liver and brain. In these tissues, oxidase activities became detectable 1-4 days after birth, reaching adult values after 4 weeks. Analysis of liver samples from patients with Zellweger syndrome, a generalized peroxisomal dysfunction, demonstrated no significant deficiency of this particular oxidase.