The possible biological and reproductive functions of ubiquitin.

The protein ubiquitin (Ub) appears to be present in all eukaryotic cells. Its widespread presence and extremely conserved structure indicate that it may play a vital role in cell metabolism. The roles of Ub are mediated by its covalent attachment to target proteins, a process known as ubiquitylation, a form of protein modification which may lead to degradation of the modified protein. A number of proteins with similar structure to Ub but varying in function have been isolated. Recently, there has been much interest in the role of Ub and its related proteins in reproductive processes. Ub and Ub-related proteins may be involved in gametogenesis, modulation of steroid receptor concentrations, placental development and endometrial modification at the beginning of pregnancy. These wide-ranging effects have led to extensive research which will be reviewed in this article.

Endoscopic ultrasound of the upper gastrointestinal tract.

Endoscopic ultrasound (EUS) is an evolving technique used by gastroenterologists to examine lesions that are located either within or adjacent to the walls of the upper gastrointestinal (GI) tract; this topic is relatively unknown to most radiologists. Proper use of this modality is benefited by a cooperative effort between gastroenterologists and radiologists specializing in ultrasound and cross-sectional imaging. This article informs radiologists of the applications of this procedure. Most patients are examined with EUS after a biopsy of a mucosal tumor has been performed. A smaller number are performed to evaluate submucosal masses or when pancreatic disease is suspected but not diagnosed. The examinations can be performed either with dedicated flexible echoendoscopes or with catheter-based probes passed through a conventional endoscope. The exact location of abnormalities associated with the upper GI tract can be observed. Known anatomic landmarks are sought. Abnormalities of structures outside the upper GI tract will occasionally be found during these examinations. The specific layers of the walls of the gut are examined, and the T and N-classification of upper GI tumors can be determined accurately. The performance of an EUS examination requires advanced skills, and in many medical centers, it is the imaging modality of choice to stage cancers, to evaluate submucosal masses, and to investigate both malignant and benign pancreaticobiliary disease. Endoscopic ultrasound is sensitive but not specific, and biopsy is necessary to establish a diagnosis. Therapeutic applications of EUS are evolving. Specialized applications with catheter-based probes are also being developed.

The progesterone receptor and ubiquitin are differentially regulated within the endometrial glands of the natural and stimulated cycle.

The initiation of human pregnancy requires precisely timed development of the endometrium to receive the implanting blastocyst. The ovarian steroid hormones are essential for development and maintenance of a hospitable uterine environment. The hormonal regimes employed in assisted reproduction procedures are known to alter the abundance of specific endometrial receptors for these steroids. Since, in the presence of ligand, the progesterone receptor (PR) is known to be modified by the small intracellular protein ubiquitin, we have investigated the localization of ubiquitin and PR within the endometrial glands of 28 fertile women during a monitored menstrual cycle and also during a stimulated cycle prior to oocyte donation. We have also observed the number of gland cells undergoing cell division as demonstrated by the presence of Ki67 immunostaining. We demonstrate that the percentage of ubiquitin-positive nuclei increases from day four post-ovulation to day 10 post-ovulation in the natural cycle, but that this increase is not seen during a stimulated cycle. The presence of PR within glandular epithelium and the proliferation of gland cells were only observed during the early secretory phase and did not appear to vary significantly between the two cycles. We conclude that ubiquitin may play an important role in endometrial development and that perturbation of ubiquitin may be related to the lower implantation rate seen in the stimulated cycle.

Ubiquitin and ubiquitin-protein conjugates are present in human cytotrophoblast throughout gestation.

Ubiquitin is a small protein involved in many intracellular processes. We have previously shown that levels of ubiquitin change during the process of decidualisation in the human uterus at the beginning of pregnancy. Other workers have shown that the ubiquitin system may be essential for normal murine placental development. In this investigation we employed immunohistochemistry and immunoblotting techniques to study the distribution and abundance of ubiquitin and ubiquitin-protein conjugates within human placental specimens from throughout gestation. Trophoblast from two pathological conditions, ectopic pregnancy and pregnancy-induced hypertension (PIH), was also investigated. Ubiquitin was detected within both the cytoplasm and nucleus of the cytotrophoblast layer only. Both monomeric and conjugated forms of ubiquitin were detected. The relative abundance of ubiquitin did not change through gestation or in the two disorders of pregnancy studied. Ubiquitin cross-reactive protein was not detected in the tissues of interest. This is the first report to demonstrate the cell-specific localisation of ubiquitin and ubiquitin-protein conjugates in the human cytotrophoblast and provides supportive evidence that ubiquitin may be important during placental development.

Ubiquitin cross-reactive protein gene expression is increased in decidualized endometrial stromal cells at the initiation of pregnancy.

Ubiquitin cross-reactive protein (UCRP; also known as ISG15) is an interferon-upregulated protein implicated in the response to viral infection. An equivalent protein has been demonstrated within the bovine uterus in early pregnancy in response to conceptus-derived interferon-tau. We have previously shown the upregulation of UCRP within decidualized stromal cells of the human and non-human primate endometria. We now show that an increase in UCRP gene expression within the decidualized stromal cell accompanies these increased protein concentrations. Using Northern blotting techniques we demonstrate basal concentrations of URCP mRNA within the non-pregnant endometrium and an increase in signal within some decidual specimens of first trimester decidua. This signal may represent increased URCP transcription within the sub-population of stromal cells that undergo decidualization, since this cell type exhibits an increase in UCRP message as detected by in-situ hybridization.

Localization of ubiquitin and ubiquitin cross-reactive protein in human and baboon endometrium and decidua during the menstrual cycle and early pregnancy.

We have examined the distribution of ubiquitin and the related ubiquitin cross-reactive protein (UCRP) in paraffin-embedded sections of human and baboon endometrium and decidua by immunoperoxidase or immunofluorescence cytochemistry with antibodies raised against ubiquitin, UCRP, CD45, and insulin-like growth factor-binding protein-1. Anti-ubiquitin immunoreactivity was present in the nonpregnant endometrium, particularly in the glandular epithelial cells, and up-regulated in endometrial stromal cells as they decidualized at the beginning of pregnancy. Anti-UCRP immunoreactivity was absent from nonpregnant tissue but accumulated to high levels in decidual cells during pregnancy. Western blotting indicated that immunoreactivity was primarily due to the presence of ubiquitin and UCRP conjugated to other proteins, and that although levels of ubiquitin-protein conjugates do not change substantially during pregnancy, decidualization is accompanied by the appearance of conjugates of UCRP. Baboon uterine tissues demonstrated a similar distribution of the two proteins, which indicates that the baboon may be a useful model for study of the role of the ubiquitin system and UCRP in the establishment of pregnancy in humans.

Related organelles of the endosome-lysosome system contain a different repertoire of ubiquitinated proteins in Sf9 insect cells.

Two components of the endosomal/lysosomal compartment of Sf9 cells, multivesicular bodies (MVB) and light vacuoles with membrane complexes (LVMC) have been isolated and probed for ubiquitin protein conjugates with a specific antibody. Immunogold electron microscopy indicates that whereas ubiquitin-protein conjugates are localised to electron dense areas of MVB they are associated with the membranes of LVMC. Five ubiquitinated polypeptides are revealed in MVB by immunoblotting while numerous ubiquitinated species forming a smear following electrophoresis are present in LVMC. We suggest two possible routes for entry of ubiquitin-protein conjugates into these organelles, via the cell surface and via primary lysosomes.

Calcium promotes membrane association of reticulocyte 15-lipoxygenase.

The reticulocyte 15-lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12) is implicated in oxidative damage to reticulocyte mitochondria before their elimination by degradation during maturation to the erythrocyte. A proportion of the 15-lipoxygenase sediments with the mitochondrial-rich stromal fraction of density-gradient-fractionated rabbit reticulocytes suggesting a physical association with mitochondria before their elimination. Ca2+ promotes binding of reticulocyte 15-lipoxygenase to isolated rat liver and reticulocyte mitochondria and 15-lipoxygenase-mediated lipid peroxidation of mitochondrial lipids and free linoleic acid. Association of reticulocyte 15-lipoxygenase with isolated mitochondria is not simply a consequence of Ca(2+)-induced swelling, but implies that Ca2+ mediates translocation of soluble lipoxygenase to mitochondrial membranes. Therefore, Ca2+ may have an important physiological role in the regulation of 15-lipoxygenase-mediated targeting of reticulocyte mitochondria for degradation.

Immunogold localisation of ubiquitin-protein conjugates in Sf9 insect cells. Implications for the biogenesis of lysosome-related organelles.

Immunogold electron microscopy with antibodies which primarily detect ubiquitin-protein conjugates shows conjugate-specific gold particles enriched severalfold in acid phosphatase-positive lysosomes and multivesicular bodies in insect Sf9 cells. The observations demonstrate that ubiquitinated proteins are associated with small acid phosphatase-containing primary lysosomes (transport vesicles) and indicate a pathway in which primary lysosomes fuse with multivesicular bodies to generate mature lysosome-related structures.

Cystic testicular mass caused by dilated rete testis: sonographic findings in 31 cases.

We reviewed the scrotal sonograms of 31 patients who had a testicular mass consisting of multiple small spherical or tubular anechoic structures in the region of the mediastinum testis. The median age of the patients was 62 years (range, 31-76 years). The abnormality was unilateral in 22 patients and bilateral in nine. Thirty-four (85%) of the 40 involved testicles had coexisting epididymal abnormalities: 32 with epididymal cysts and two with epididymitis. Follow-up sonograms were available in five patients and showed no change up to 4.5 years after the initial diagnosis. Surgical and histologic findings were available in one other patient and showed dilatation of the rete testis. The sonographic appearance and location of the lesions, the frequent presence of an epididymal abnormality, and the surgical and histologic findings in one case suggest that the lesion is due to dilatation of the rete testis, probably associated with obstruction in the epididymis. Recognition of this entity on sonograms may prevent unnecessary orchiectomy.

Gallbladder visualization during post-therapy iodine-131 imaging of thyroid carcinoma.

A persistent, extra-thyroidal focus of 131I on whole-body imaging of thyroid carcinoma usually represents a functioning metastasis. We report a case of 131I localization within a septated gallbladder, initially mimicking an isolated hepatic metastasis. Adjunctive liver/spleen and hepatobiliary scintigraphy helped to elucidate the true nature of this 131I activity.

Immunogold localisation of ubiquitin-protein conjugates in primary (azurophilic) granules of polymorphonuclear neutrophils.

Ubiquitin-protein conjugates are found in the primary (azurophilic) lysosome-related granules but not in the secondary (specific) granules in mature polymorphonuclear neutrophils prepared from bone marrow. This is the first reported demonstration of ubiquitin-protein conjugates in lysosome-related membrane-bound vesicles in granulocytes and complements our previous findings of ubiquitinated proteins in lysosomes of fibroblasts. The significance of the selective presence of conjugates in only one of the two main types of neutrophil granules remains to be elucidated but may relate to the presence of the complement of acid hydrolases, including proteases, in the azurophilic granules compared to the specific granules. Ubiquitin-protein conjugates may enter the primary granules during neutrophil maturation by an autophagic process or by a heterophagic process during the fusion of phagosomes with primary granules. Alternatively protein ubiquitination may be involved in granule biogenesis.

The degradative fate of ubiquitin-protein conjugates in nucleated and enucleated cells.

Covalent ligation of multiple copies of ubiquitin to proteins is known to target intracellular proteins for degradation by large molecular weight cytosolic proteinase(s). Ubiquitin protein conjugates are found in cytosolic cell compartments suggesting that ubiquitination may have multiple roles. We have detected ubiquitinated proteins in the lysosomal apparatus of normal fibroblasts and fibroblasts treated with lysosomal proteinase inhibitors. In contrast rabbit reticulocytes lack lysosomes. We present here direct evidence for ubiquitination of mitochondrial proteins during rabbit reticulocyte maturation. In addition ubiquitination appears to be associated with the terminal differentiation of human keratinocytes. These results suggest that: 1. ubiquitin-protein conjugates may be degraded lysosomally 2. organellar proteins may be degraded by the ubiquitin system 3. ubiquitination is involved in the programmed elimination of proteins and organelles from several cell types during differentiation.

Ubiquitinated protein conjugates are specifically enriched in the lysosomal system of fibroblasts.

Ubiquitin-protein conjugates are found by immunogold electron microscopy to be enriched (12-fold) in the lysosomal compartment of 3T3-L1 fibroblasts. Treatment of fibroblasts with the cysteine protease inhibitor E-64 leads to an expansion of the lysosomal compartment and as a result an increase in the cellular content of ubiquitin-protein conjugates. There is no change in the specific enrichment of ubiquitin-protein conjugates in the lysosomal compartment following E-64 treatment. The results suggest that some ubiquitin-protein conjugates may normally be degraded lysosomally following sequestration by microautophagy and imply that protein ubiquitination may be one of the signals for protein uptake into lysosomes.

Insoluble disulfide cross-linked polypeptides accumulate in the functionally compromised lysosomes of fibroblasts treated with the cysteine protease inhibitor E-64.

Mouse fibroblasts (3T3-L1 cells) accumulate pulse-labeled long-lived polypeptides in detergent- and salt-insoluble aggregates when chased in the presence of inhibitors of lysosomal cysteine cathepsins, including E-64. Proteins found in the detergent- and salt-insoluble fraction include polypeptides which are disulfide cross-linked. E-64-induced polypeptide aggregates cofractionate with lysosomal enzyme markers on density gradients and are found in multivesicular dense bodies which by electron microscopy appear to be engaged in microautophagy. The results are discussed in relation to the possible role of polypeptide aggregation in the sequestration or trapping of cytoplasmic proteins by the lysosomal system.

Ubiquitin-protein conjugates accumulate in the lysosomal system of fibroblasts treated with cysteine proteinase inhibitors.

Mouse fibroblasts (3T3-L1 cells) accumulate detergent- and salt-insoluble aggregates of proteins conjugated to ubiquitin when incubated in the presence of inhibitors of lysosomal cysteine cathepsins, including E-64. These ubiquitin-protein conjugates co-fractionate with lysosomes on density gradients and are found in multivesicular dense bodies which by electron microscopy appear to be engaged in microautophagy. Both E-64 and ammonium chloride increase the intracellular concentration of free ubiquitin, but only E-64 leads to the formation of insoluble lysosomal ubiquitin-protein conjugates. The results are discussed in relation to the possible intracellular roles of ubiquitin conjugation.