Basal autophagy is involved in the degradation of the ERAD component EDEM1.

Little is known about the fate of machinery proteins of the protein quality control and endoplasmic reticulum(ER)-associated degradation (ERAD). We investigated the degradation of the ERAD component EDEM1, which directs overexpressed misfolded glycoproteins to degradation. Endogenous EDEM1 was studied since EDEM1 overexpression not only resulted in inappropriate occurrence throughout the ER but also caused cytotoxic effects. Proteasome inhibitors had no effect on the clearance of endogenous EDEM1 in non-starved cells. However, EDEM1 could be detected by immunocytochemistry in autophagosomes and biochemically in LC3 immuno-purified autophagosomes. Furthermore, influencing the lysosome-autophagy pathway by vinblastine or pepstatin A/E64d and inhibiting autophagosome formation by 3-methyladenine or ATGs short interfering RNA knockdown stabilized EDEM1. Autophagic degradation involved removal of cytosolic Triton X-100-insoluble deglycosylated EDEM1, but not of EDEM1-containing ER cisternae. Our studies demonstrate that endogenous EDEM1 in cells not stressed by the expression of a transgenic misfolded protein reaches the cytosol and is degraded by basal autophagy.

Golgi apparatus immunolocalization of endomannosidase suggests post-endoplasmic reticulum glucose trimming: implications for quality control.

Trimming of N-linked oligosaccharides by endoplasmic reticulum (ER) glucosidase II is implicated in quality control of protein folding. An alternate glucosidase II-independent deglucosylation pathway exists, in which endo-alpha-mannosidase cleaves internally the glucose-substituted mannose residue of oligosaccharides. By immunogold labeling, we detected most endomannosidase in cis/medial Golgi cisternae (83.8% of immunogold labeling) and less in the intermediate compartment (15.1%), but none in the trans-Golgi apparatus and ER, including its transitional elements. This dual localization became more pronounced under 15 degrees C conditions indicative of two endomannosidase locations. Under experimental conditions when the intermediate compartment marker p58 was retained in peripheral sites, endomannosidase was redistributed to the Golgi apparatus. Double immunogold labeling established a mutually exclusive distribution of endomannosidase and glucosidase II, whereas calreticulin was observed in endomannosidase-reactive sites (17.3% in intermediate compartment, 5.7% in Golgi apparatus) in addition to the ER (77%). Our results demonstrate that glucose trimming of N-linked oligosaccharides is not limited to the ER and that protein deglucosylation by endomannosidase in the Golgi apparatus and intermediate compartment additionally ensures that processing to mature oligosaccharides can continue. Thus, endomannosidase localization suggests that a quality control of N-glycosylation exists in the Golgi apparatus.

Effect on global and regional left ventricular functions by percutaneous transluminal coronary angioplasty in the chronic stage after myocardial infarction.

Data are reported on 145 consecutive patients with prior myocardial infarction who had successful percutaneous transluminal coronary angioplasty (PTCA) of the infarct-related artery (5 +/- 6 months after infarction), and left ventricular (LV) angiograms before PTCA and during follow-up (7 +/- 4 months). There was a significant long-term improvement in LV function, ejection fraction increased from 60 +/- 13% to 64 +/- 13% (p less than 0.001), and regional wall motion abnormalities decreased by 40%. Multivariate discriminant analysis identified reduced LV function and a high degree of stenosis before PTCA as predictors for improvement in LV function (ejection fraction less than 60%: ejection fraction from 48 +/- 9% to 57 +/- 14%, p less than 0.001; and stenosis greater than or equal to 90%: ejection fraction from 59 +/- 15% to 66 +/- 14%, p = 0.003). Restenosis greater than or equal to 90% in patients with initial stenosis less than 90% decreased ejection fraction from 59 +/- 16% to 51 +/- 14% (p less than 0.05). Other factors tested (treatment of infarction by thrombolysis, time between infarction and PTCA, and severity of angina pectoris) had no effect on long-term changes in LV function. It is concluded that successful elective PTCA of a high-grade stenosis in an infarct-related artery may improve LV ejection fraction and regional wall motion abnormalities, especially in patients with impaired LV function.

Cyclidium porcatum n. sp.: a Free-living anaerobic scuticociliate containing a stable complex of hydrogenosomes, eubacteria and archaeobacteria.

A new ciliate species (Cyclidium porcatum) is the first freshwater anaerobic scuticociliate to be cultured and described. It contains a unique tripartite structure consisting of hydrogenosomes (confirmed by cytochemical staining for hydrogenase), interspersed with methanogens (confirmed by auto fluorescence and in situ hybridisation with an archaeobacterial 16S rRNA-specific probe) and unidentified eubacteria (confirmed with a eubacterial 16S rRNA-specific probe). This complex structure is stable and persistent, indicating that it is an anaerobic symbiotic consortium incorporating three functional partners.

[Cicatricial bone (author's transl)]. / Der Narbenknochen

Critical comments were presented in regard to the etiology, frequency, symptomatology and radiological findings in cicatricial bone.

Microtubule centers and the interphase microtubule cytoskeleton in amoebae of the cellular slime molds (Mycetozoans) Acytostelium leptosomum and Protostelium mycophaga.

We investigated the microtubule (MT) cytoskeleton and microtubule centers (MTC) in undifferentiated amoebae by indirect immunofluorescence with six monoclonal antitubulin antibodies, and by transmission electron microscopy and immunogold ultracytochemistry. Interphase amoebae of both species contain a distinct cytoplasmic complex of MTs, which is more elaborate in Protostelium mycophaga. In Acytostelium leptosomum amoebae a single MTC is attached to each interphase nucleus at its pointed end, as in the other dictyostelid cellular slime molds Dictyostelium discoideum and Polysphondylium violaceum. Ultrastructurally, MTCs of A. leptosomum also resemble those of these two species: They consist of an electron-opaque core shaped like a stout rod, which is embedded, together with nodules, in a fuzzy matrix. The nodules are the points of origin of the MTs. In most amoebae of P. mycophaga there are two MTCs on opposite sides of and close to the nucleus, but many amoebae also contain a variable number of MTCs that are remote from the nucleus. Nucleus-associated and "remote" MTCs are structurally identical. They consist of a ring-shaped core with inner and outer diameters of ca. 130 nm and 340 nm. A plug sits in the ring, and satellites are connected to the core by fine fibrils. The satellites are the points of origin of MTs. New MTCs are apparently formed during mitosis, the parent MTC probably serving as a template for the genesis of a new ring. The results support the notion that phylogenetically related organisms have similarly constructed MTCs and that these are dissimilar in less closely related organisms.

Unconventional antigen retrieval for carbohydrate and protein antigens.

Aldehyde fixation of tissues often adversely affects the reactivity of cellular proteins with antibodies. A most commonly used retrieval technique in immunohistochemistry is high-temperature microwave heating of sections from formaldehyde-fixed and paraffin-embedded tissues. Here we report that pretreatment of paraffin and ultrathin cryosections with N-glycanase F to remove N-glycosidically linked oligosaccharides can result in a dramatic increase in specificity and intensity of immunogold labeling for sugar moieties present on O-glycosidically linked oligosaccharides. This is demonstrated in the immunolocalization of poly alpha2,8 KDN (KDN, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) of megalin in rat kidney. The mechanism of this retrieval procedure is most probably based on the elimination of sterical hindrance by large N-glycosidically linked oligosaccharides. Furthermore, we demonstrate that exposure of ultrathin cryosections to acidic conditions (pH 5.5) at ambient temperature prior to immunogold labeling can result in an increased labeling intensity. This effect was observed for megalin immunoreactive sites in proximal tubular epithelia of rat kidney. It is proposed that the mechanism of this retrieval procedure is based on the depolymerization of methylen and polymethylen bridges introduced by formaldehyde in the acidic milieu.

[Growing bone islands as differential diagnosis of osteoplastic metastases (author's transl)]. / Wachsende Kompaktainseln als Differentialdiagnose osteoplastischer Metastasen.

A case with two growing bone islands is reported. In one of the lesions a biopsy was performed, the other one showed a positive scintiscan. Metastases could be excluded. The most important differential diagnosis is osteoplastic metastases. The etiology of the growing bone islands is unknown.

Immunolocalization of UDP-glucose:glycoprotein glucosyltransferase indicates involvement of pre-Golgi intermediates in protein quality control.

The UDP-glucose:glycoprotein glucosyltransferase (GT) is a protein folding sensor and glycosyltransferase that constitutes an important component of the protein quality control machinery. With the use of quantitative immunogold electron microscopy, we established the subcellular distribution of GT in rat liver and pancreas and Drosophila melanogaster salivary gland as well as cell lines and correlated it with that of glucosidase II, calreticulin, and pre-Golgi intermediate markers. Labeling for GT, as well as for glucosidase II and calreticulin, was found in the endoplasmic reticulum (ER), including nuclear envelope and pre-Golgi intermediates located between ER and Golgi apparatus, and in the cell periphery. In the rough ER, labeling for GT was inhomogeneous, with variously sized labeled and unlabeled cisternal regions alternating, indicative of a meshwork of quality control checkpoints. Notably, labeling intensity for GT was highest in pre-Golgi intermediates, corresponding to twice that of rough ER, whereas the Golgi apparatus exhibited no specific labeling. These results suggest that protein quality control is not restricted to the ER and that the pre-Golgi intermediates, by virtue of the presence of GT, glucosidase II, and calreticulin, are involved in this fundamental cellular process.