Expert opinion on the use of transvaginal sonography for presurgical staging and classification of endometriosis.

Gynecological ultrasonography plays a central role in the management of endometriosis. The rapid technical development as well as the currently increasing evidence for non-invasive diagnostic methods require an updated compilation of recommendations for the use of ultrasound in the management of endometriosis. The present work aims to highlight the accuracy of sonography for diagnosing and classifying endometriosis and will formulate the present list of key messages and recommendations. This paper aims to demonstrate the accuracy of TVS in the diagnosis and classification of endometriosis and to discuss the clinical applications and consequences of TVS findings for indication, surgical planning and assessment of associated risk factors. (1) Sophisticated ultrasound is the primary imaging modality recommended for suspected endometriosis. The examination procedure should be performed according to the IDEA Consensus. (2) Surgical intervention to confirm the diagnosis alone is not recommended. A preoperative imaging procedure with TVS and/or MRI is strongly recommended. (3) Ultrasound examination does not allow the definitive exclusion of endometriosis. (4) The examination is primarily transvaginal and should always be combined with a speculum and a bimanual examination. (5) Additional transabdominal ultrasonography may enhance the accuracy of the examination in case of extra pelvic disease, extensive findings or limited transvaginal access. (6) Sonographic assessment of both kidneys is mandatory when deep endometriosis (DE) and endometrioma are suspected. (7) Endometriomas are well defined by sonographic criteria. When evaluating the ovaries, the use of IOTA criteria is recommended. (8) The description of sonographic findings of deep endometriosis should be systematically recorded and performed using IDEA terminology. (9) Adenomyosis uteri has sonographically well-defined criteria (MUSA) that allow for detection with high sensitivity and specificity. MRI is not superior to differentiated skilled ultrasonography. (10) Classification of the extent of findings should be done according to the #Enzian classification. The current data situation proves the best possible prediction of the intraoperative situs of endometriosis (exclusive peritoneum) for the non-invasive application of the #Enzian classification. (11) Transvaginal sonographic examination by an experienced examiner is not inferior to MRI diagnostics regarding sensitivity and specificity in the prediction of the extent of deep endometriosis. (12) The major advantage of non-invasive imaging and classification of endometriosis is the differentiated planning or possible avoidance of surgical interventions. The recommendations represent the opinion of experts in the field of non-invasive and invasive diagnostics as well as therapy of endometriosis. They were developed with the participation of the following national and international societies: DEGUM, ÖGUM, SGUM, SEF, AGEM/DGGG, and EEL.

Lymph node dissection in endometrial cancer and clinical outcome: A population-based study in 5546 patients.

BACKGROUND: According to current treatment guidelines, comprehensive surgical staging procedures in endometrial cancer confined to the uterus depend on uterine risk factors: a systematic lymph node dissection (LND) is recommended in high risk patients and should be omitted in low risk patients. Its role in intermediate and high intermediate risk patients is inconclusive. The aim of this analysis was to review the implementation of this risk-adopted strategy. MATERIALS AND METHODS: Data were provided by the population-based Munich Cancer Registry. Patients with endometrial cancer diagnosed between 1998 and 2016 were included. RESULTS: Of 5446 eligible patients, 58.5%, 30.1% and 11.4% belonged to the low risk, intermediate/high-intermediate and high risk group, respectively. Lymph node dissection was performed in 20.2%, 53.0% and 63.7% within these groups. Lymph node involvement was diagnosed in 1.7%, 9.6% and 19.3%, respectively. Within these risk groups, there was no significant difference in the time to local recurrence, lymph node recurrence or distant metastases between patients with and without LND. After adjusting for age and comorbidity-status, no significant difference in overall survival was found. CONCLUSIONS: The application of a risk-adopted management of LND in early endometrial cancer in real-life is associated with a high rate of surgical under- and overtreatment. Corresponding survival data do not show a significant benefit of a systematic lymph node dissection. In order to improve the management and outcome of early endometrial cancer in the future, prospective trials, new surgical concepts and prognostic markers will be primary and necessary.

Effect of Histophilus somni on Heart and Brain Microvascular Endothelial Cells.

Histophilus somni is a pathogenic gram-negative bacterium responsible for pneumonia and septicemia in cattle. Sequelae include infectious thrombotic meningoencephalitis (ITME), myocarditis, arthritis, and abortion. These syndromes are associated with widespread vasculitis and thrombosis, implicating a role for endothelium in pathogenesis. Histopathologic and immunohistochemical investigation of 10 natural cases of bovine H. somni myocarditis and 1 case of ITME revealed intravascular H. somni in large biofilm-like aggregates adherent to the luminal surface of microvascular endothelium. Ultrastructurally, bacterial communities were extracellular and closely associated with degenerating or contracted endothelial cells. Histophilus somni was identified by bacterial culture and/or immunohistochemistry. Western blots of the bacterial isolates revealed that they expressed the immunodominant protective 40 kDa OMP and immunoglobulin-binding protein A (IbpA) antigens. The latter is a large surface antigen and shed fibrillar antigen with multiple domains. The cytotoxic DR2Fic domain of IbpA was conserved as demonstrated by polymerase chain reaction. Treatment of endothelial cells in vitro with IbpA in crude culture supernatants or purified recombinant GST-IbpA DR2Fic (rDR2) cytotoxin induced retraction of cultured bovine brain microvascular endothelial cells. By contrast, no retraction of bovine endothelium was induced by mutant rDR2H/A with an inactive Fic motif or by a GST control, indicating that the cytotoxic DR2Fic motif plays an important role in endothelial cell retraction in vasculitis. The formation of biofilm-like aggregates by H. somni on bovine microvascular endothelium may be fundamental to its pathogenesis in heart and brain.

Intracellular colocalization of variant surface glycoprotein and transferrin-gold in Trypanosoma brucei.

Endocytosis and intracellular transport has been studied in the bloodstream forms of Trypanosoma brucei by light and electron microscopy, using colloidal gold coupled to bovine transferrin (transferrin-gold). The endocytosed transferrin-gold, visualized by silver intensification for light microscopy, was present in vesicular structures between the cell nucleus and flagellar pocket of the organism. At the ultrastructural level, transferrin-gold was present after a 10-min incubation in the flagellar pocket, coated vesicles, cisternal networks, and lysosomelike structures. Endocytosis and intracellular processing of T. brucei variable surface glycoprotein (VSG) was studied using two preparations of affinity-purified rabbit IgG directed against different parts of the VSG. One preparation of IgG was directed against the cross-reacting determinant (CRD): a complex glycolipid side chain covalently linked to the COOH-terminus of the VSG molecule. The other was directed against determinants on the rest of the VSG molecule. When the two IgG preparations were used on thawed, thin cryosections of trypanosomes that had been incubated in transferrin-gold before fixation, the organelles involved with transferrin-gold endocytosis labeled with both antibodies, as well as many vesicular, tubular, and vacuolar structures that did not contain endocytosed transferrin-gold. Both antibodies also labeled the cell surface. In double-labeling experiments both antibodies were closely associated except that IgG directed against the VSG molecule labeled all the cisternae of the Golgi apparatus, whereas anti-CRD IgG was shown to label only half of the Golgi apparatus. Evidence for sorting of VSG molecules from endocytosed transferrin-gold was found. Double-labeling experiments also showed some tubular profiles which labeled on one side with anti-CRD IgG and on the other side with anti-VSG IgG, suggesting a possible segregation of parts of the VSG molecule.

Function of a calmodulin in postsynaptic densities. II. Presence of a calmodulin-activatable protein kinase activity.

Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of coomassie blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [gamma-(32)P]ATP and Mg(2+) alone. Addition of Ca(2+) alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). Similar results were also obtained in sonicated or freeze thawed native PSD preparations by addition of Ca(2+) alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg(2+)-dependent activity, by the Ca(2+) chelator, EGTA, which also removes the calmodulin from the structure (26), and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca(2+). In addition, when a calmodulin-deficient PSD preparation was prepared (26), sonicated, and incubated with [gamma-(32)P]ATP, Mg(2+) and Ca(2+), one could not induce a Ca(2+)-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg(2+)-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate protein I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter two proteins.

Function of calmodulin in postsynaptic densities. I. Presence of a calmodulin-activatable cyclic nucleotide phosphodiesterase activity.

The postsynaptic density (PSD) fraction from canine cerebra cortex was found to contain an endogenous cyclic nucleotide-phosphodiesterase activity that was independent on Mn2+ and/or Mg2+ but not on Ca2+. Maximal activity was obtained at 1 micrometer Mn2+. This cyclic nucleotide phosphodiesterase activity was not decreased upon removal of the calmodulin from the PSD fraction, nor was it increased by the addition of calmodulin to a postsynaptic density fraction deficient in calmodulin. The enzymatic activity could be extracted by sonication, with the soluble enzyme having properties similar to those found in the native structure. Two peaks of cyclic nucleotide phosphodiesterase activities could be obtained after S-300 Sephacryl column chromatography of this soluble fraction: fraction I (excluded peak) and fraction II (215,000 mol wt). The fraction I activity preferred cyclic AMP over cyclic GMP and was not activated by calmodulin. The fraction II activity has an approximately fourfold lower Km for cyclic GMP over cyclic AMP. This fraction II activity was activatable by calmodulin, which increased the Vmax and decreased the Km in the case of both cyclic nucleotides. We conclude that two activities are present in the PSD, one activatable, and one not activatable, by calmodulin.

Function of a calmodulin in postsynaptic densities. III. Calmodulin-binding proteins of the postsynaptic density.

A method has been developed for binding calmodulin, radioiodinated by the lactoperoxidase method, to denaturing gels and has been used to attempt to identify the calmodulin-binding proteins of cerebral cortex postsynaptic densities (PSDs). Calmodulin primarily bound to the major 51,000 Mr protein in a saturatable manner; secondarily bound to the 60,000 Mr region, 140,000 Mr region, and 230,000 Mr protein; and bound in lesser amounts to a number of other proteins. The major 51,000 Mr calmodulin-binding protein is one of unknown identity. Binding of iodinated calmodulin to these proteins was blocked by EDTA, EGTA, chlorpromazine, and preincubation with unlabeled calmodulin. Calmodulin iodinated by the chloramine-T method, which inactivates calmodulin did not bind to the PSD but bound nonspecifically to histone. Calmodulin did not bind to proteins from a variety of sources for which calmodulin interactions have not been found. Except for three proteins, all of the proteins of synaptic membranes that bind calmodulin could be accounted for by proteins of the PSD which are a part of the synaptic membrane fraction. The major 51,000 M, protein and the corresponding iodinated calmodulin binding were greatly reduced in cerebellar PSDs and this difference between cerebral cortex and cerebellar PSDs is discussed in light of the possible function of calmodulin in synaptic excitatory responses.

Glycosyltransferase activities in Golgi complex and endoplasmic reticulum fractions isolated from African trypanosomes.

Highly enriched Golgi complex and endoplasmic reticulum fractions were isolated from total microsomes obtained from Trypanosoma brucei, Trypanosoma congolense, and Trypanosoma vivax, and tested for glycosyltransferase activity. Purity of the fractions was assessed by electron microscopy as well as by biochemical analysis. The relative distribution of all the glycosyltransferases was remarkably similar for the three species of African trypanosomes studied. The Golgi complex fraction contained most of the galactosyltransferase activity followed by the smooth and rough endoplasmic reticulum fractions. The dolichol-dependent mannosyltransferase activities were highest for the rough endoplasmic reticulum, lower for the smooth endoplasmic reticulum, and lowest for the Golgi complex. Although the dolichol-independent form of N-acetylglucosaminyltransferase was essentially similar in all the fractions, the dolichol-dependent form of this enzyme was much higher in the endoplasmic reticulum fractions than in the Golgi complex fraction. Inhibition of this latter activity in the smooth endoplasmic reticulum fraction by tunicamycin A1 suggests that core glycosylation of the variable surface glycoprotein may occur in this organelle and not in the rough endoplasmic reticulum as previously assumed.

Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities.

Postsynaptic densities (PSDs) have been isolated from cerebral cortex, midbrain, cerebellum, and brain stem by the Triton X-100 method previously used in the isolation of cerebral PSDs (Cohen et al., 1977, J. Cell Biol. 74:181). These PSDs have been compared in protein composition, protein phosphorylation, and morphology. Thin-section electron microscopy revealed that cerebral cortex and midbrain PSDs were identical, being approximately 57 nm thick and composed of apparent aggregates 20-30 nm in diameter. Isolated cerebellar PSDs appeared thinner (33 nm) than cerebral cortex PSDs and lacked the apparent 20- to 30-nm aggregates, but had a latticelike structure. In unidirectional and rotary-shadowed replicas, the cerebrum and midbrain PSDs were circular in shape with a large central perforation or hole in the center of them. Cerebellum PSDs did not have a large perforation, but did have numerous smaller perforations in a lattice like structure. Filaments (6-9 nm) were observed connecting possible 20- to 30-nm aggregates in cerebrum PSDs and were also observed radiating from one side of the PSD. Both cerebral cortex and midbrain PSDs exhibited identical protein patterns on SDS gel electrophoresis. In comparison, cerebellar PSDs (a) lacked the major 51,000 Mr protein, (b) contained two times less calmodulin, and (c) contained a unique protein at 73,000 Mr. Calcium plus calmodulin stimulated the phosphorylation of the 51,000 and 62,000 Mr bands in both cerebral cortex and midbrain PSDs. In cerebellar PSDs, only the 58,000 and 62,000 Mr bands were phosphorylated. In the PSDs from all brain regions, cAMP stimulated the phosphorylation of Protein Ia (73,000 Mr), Protein Ib (68.000 Mr), and a 60,000 Mr protein, although cerebrum and midbrain PSDs contained very much higher levels of phosphorylated protein than did the cerebellum. On the basis of the morphological criteria, it is possible that PSDs isolated from cerebrum and midbrain were derived from the Gray type I, or asymmetric, synapses, whereas cerebellum PSDs were derived from the Gray type II, or symmetric, synapses. Since there is some evidence that the type I synapses are involved in excitatory mechanisms while the type II are involved in inhibitory mechanisms, the role of the PSD and of some of its proteins in these synaptic responses is discussed.

Subcellular localization of a variable surface glycoprotein phosphatidylinositol-specific phospholipase-C in African trypanosomes.

African trypanosomes contain a membrane-bound enzyme capable of removing dimyristylglycerol from the membrane-attached form of the variable surface glycoprotein (mfVSG; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968). Although mfVSG phospholipase-C has been implicated in the removal of the VSG from the trypanosome surface (Cardoso de Almeida, M. L., and M. J. Turner, 1983, Nature (Lond.)., 302:349-352; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968), its precise function and subcellular location have not been determined. We have developed a procedure for the separation of the cell fractions and organelles of Trypanosoma brucei brucei (and other trypanosome species) by differential sucrose and isopycnic PercollR centrifugation. These fractions were tested for mfVSG phospholipase activity using Trypanosoma brucei mfVSG labeled with 3H-myristic acid as substrate. The highest enzyme-specific activity was associated with the flagella and evidence is presented to suggest that it is localized in the flagellar pocket. Some activity was also associated with the Golgi complex. These results suggest that the mfVSG phospholipase is localized primarily in the membrane of the flagella pocket and possibly other membrane organelles derived from and associated with this structure, and may be part of the VSG-membrane recycling system in African trypanosomes. The activity of mfVSG phospholipase amongst various trypanosome species was determined. We show that, in contrast to the bloodstream forms of Trypanosoma brucei, cultured procyclic Trypanosoma brucei and bloodstream Trypanosoma vivax had little or no mfVSG phospholipase activity. The activity found in bloodstream forms of Trypanosoma congolense was intermediate between Trypanosoma vivax and Trypanosoma brucei.

Subcellular distribution in cerebral cortex of two proteins phosphorylated by a cAMP-dependent protein kinase.

The subcellular distribution of Proteins Ia and Ib, two proteins which serve as specific substrates for protein kinases present in mammalian brain, was studied in the dog cerebral cortex. Proteins Ia and Ib were found to be most highly enriched in synaptic vesicle fractions; they were also present in postsynaptic density and synaptic membrane fractions in significant amounts. Proteins Ia and Ib present in the synaptic vesicle fraction appear to be similar, if not identical, to those present in the postsynaptic density fraction as judged by several criteria: (a) the ability to serve as substrate for cAMP-dependent protein kinase, (b) electrophoretic mobility in the presence of sodium dodecyl sulfate, (c) extractability with NH4Cl or EGTA, and (d) fragmentation to electrophoretically similar peptides by a purified Staphylococcus aureus protease. In addition, the postsynaptic density fraction has been found to contain cAMP-dependent Protein Ia and Protein Ib kinase activity. The subcellular localization of Proteins Ia and Ib suggests a role for these proteins in the physiology of the synapse.

Cancer of the ovary, fallopian tube, and peritoneum: a population-based comparison of the prognostic factors and outcomes.

PURPOSE: The objective was to compare the prognostic factors and outcomes among primary ovarian cancer (OC), fallopian tube cancer (FC), and peritoneal cancer (PC) patients in a population-based setting. METHODS: We analysed 5399 OC, 327 FC, and 416 PC patients diagnosed between 1998 and 2014 in the catchment area of the Munich Cancer Registry (meanwhile 4.8 million inhabitants). Tumour site differences were examined by comparing prognostic factors, treatments, the time to progression, and survival. The effect of the tumour site was additionally analysed by a Cox regression model. RESULTS: The median age at diagnosis, histology, and FIGO stage significantly differed among the tumour sites (p < 0.001); PC patients were older, more often diagnosed with a serous subtype, and in FIGO stage III or IV. The time to progression and survival significantly differed among the tumour sites. When stratified by FIGO stage, the differences in time to progression disappeared, and the differences in survival considerably weakened. The differences in the multivariate survival analysis showed an almost identical outcome in PC patients (HR 1.07 [0.91-1.25]) and an improved survival of FC patients (HR 0.63 [0.49-0.81]) compared to that of OC patients. CONCLUSION: The comparison of OC, FC, and PC patients in this large-scale population-based study showed differences in the prognostic factors. These differences primarily account for the inferior outcome of PC patients, and for the improved survival of FC compared to OC patients.

Anaplasma phagocytophilum-infected neutrophils enhance transmigration of Borrelia burgdorferi across the human blood brain barrier in vitro.

The manifestations of Lyme disease, caused by Ixodes spp. tick-transmitted Borrelia burgdorferi, range from skin infection to bloodstream invasion into the heart, joints and nervous system. The febrile infection human granulocytic anaplasmosis is caused by a neutrophilic rickettsia called Anaplasma phagocytophilum, also transmitted by Ixodes ticks. Previous studies suggest that co-infection with A. phagocytophilum contributes to increased spirochetal loads and severity of Lyme disease. However, a common link between these tick-transmitted pathogens is dissemination into blood or tissues through blood vessels. Preliminary studies show that B. burgdorferi binds and passes through endothelial barriers in part mediated by host matrix metalloproteases. Since neutrophils infected by A. phagocytophilum are activated to release bioactive metalloproteases and chemokines, we examined the enhanced B. burgdorferi transmigration through vascular barriers with co-infection in vitro. To test whether endothelial transmigration is enhanced with co-infection, B. burgdorferi and A. phagocytophilum-infected neutrophils were co-incubated with EA.hy926 cells (HUVEC-derived) and human brain microvascular endothelial cells in Transwell cultures. Transmigration of B. burgdorferi through endothelial cell barriers was determined and endothelial barrier integrity was measured by transendothelial electrical resistivity. More B. burgdorferi crossed both human BMEC and EA.hy926 cells in the presence of A. phagocytophilum-infected neutrophils than with uninfected neutrophils without affecting endothelial cell integrity. Such a mechanism may contribute to increased blood and tissue spirochete loads.

Differences in the acute response of the various segments ofrat intestine to treatment with the intestinal carcinogen, methylazoxymethanol acetate.

Previous work has shown that single injections of methylazoxymethanol acetate in rats induce tumors predominantly in the colon, occasionally in the duodenum, and rarely in the jejunum and ileum. These studies describe the acute pathological and biochemical, alterations induced by this carcinogen in the different segments of rat small intestine and colon. Karyorrhexis was found in crypts of duodenum, cecum, and all segments of colon at 6 hr after treatment. Much of the cellular debris was removed by 24 hr, although mitoses did not return to normal levels until the third day after treatment. No pathological alterations were found in jejunum or ileum, even as late as 24 hr after treatment. Studies of DNA synthesis at 24 hr after treatment indicated that jejunum and ileum were much less affected than were duodenum, cecum, or colon. In contrast, 5-fluorouracil and nitrogen mustard, agents that can inhibit proliferating cells but are not known to be intestinal carcinogens, affected all of the segments equally. The results indicate that a correlation exists between those segments of intestine acutely affected by methylazoxymethanol acetate and the sites of eventual tumor development. The level of deacetylase activity in the various intestinal segments did not correlate with sensitivity to methylazoxymethanol acetate-induced inhibition of DNA synthesis. We also found that methylazoxy-methanol acetate inhibited DNA synthesis in the duodenum and colon in rats with cannulated bile ducts. These data indicate that the carcinogen does not require biliary transport to the intestinal lumen to exert its biological effects. Mechanisms that might account for the observed selectivity in action of methylazoxymethanol acetate in the various rat intestinal segments are discussed.

Fibronectin-binding activity in Borrelia burgdorferi1.

Recently, the term MSCRAMM (microbial surface components recognizing adhesive matrix molecules), has been introduced to describe microbial molecules that recognize extracellular matrix (ECM) [1]. Here we present evidence for the presence of fibronectin-binding molecules in Borrelia burgdorferi and several other Borrelia species. Immunofluorescence studies show that plasma fibronectin is bound uniformly over the cell surface of free swimming B. burgdorferi. In addition, the spirochetes are able to bind to plasma fibronectin-coated microwell plates, an interaction that is inhibited by anti-fibronectin antibody as well as exogenous plasma fibronectin. Taken together, the data suggest that fibronectin binds to the surface of the spirochete. On Western blot-like assays, B. burgdorferi and some B. afzelii strains express a major fibronectin-binding protein (Fn-BA) with an approximate molecular mass of 52 kDa. In addition, several other major Fn-BAs were found in B. hermsii (26, 31, 33, 39, 46, 54 and 58 kDa) and B. turicatae (39, 41, 45, 50, 56, 59 and 66 kDa). Preliminary evidence suggests that fibronectin (and Fn-BA) may play a role as a molecular bridge between the spirochete and other components of the extracellular matrix.

Mechanism of inhibition of hepatic protein synthesis in rats by the carcinogen, methylazoxymethanol acetate.

Treatment of rats with the carcinogen, methylazoxymethanol acetate, results in a rapid, marked inhibition of hepatic protein synthesis and disaggregation of polysomes. Studies were undertaken to learn the mechanism by which this carcinogen induces these effects in rat liver. The data show that the inhibition of endogenous protein synthesis is not due to an effect on the high speed supernatant 'factors' but rather at the level of the polysome, and that both free and membrane-bound polysomes are affected. Poly(U)-directed polyphenylalanine synthesis by native ribosomal subunits is greater in preparations isolated from rats treated with carcinogen than it is in controls. Moreover, the native ribosomal subunit fraction from treated livers in response to added rabbit globin mRNA is able to synthesize a protein similar in molecular weight to globin. These studies show that methylazoxymethanol acetate does not induce significant alterations of ribosomal subunits or of initiation factors and suggest that the inhibition of protein synthesis and disaggregation of polysomes may be the results of an alteration of cytoplasmic mRNA, or its association with ribosomes.

Endocytosed transferrin in African trypanosomes is delivered to lysosomes and may not be recycled.

It has been shown in mammalian systems that the passage of transferrin-colloidal gold (Tf-Au) through the endocytic system is influenced by the size of the gold colloid (Neutra, M. R. et al., J. Histochem. Cytochem. 33, 1134-1144 (1985); Woods, J. W. et al., Eur. J. Cell Biol. 50, 132-143 (1989)). However, in both Trypanosoma brucei brucei and Trypanosoma congolense, widely varying sizes of Tf-Au (Tf-Au5 and Tf-Au15) have been shown to proceed to lysosomes (Webster, P., Eur. J. Cell Biol. 49, 295-302 (1989); Webster, P., D. Grab, J. Cell Biol. 106, 279-288 (1988)). Using an affinity-purified anti-bovine transferrin IgG we have demonstrated that, in both T. brucei and T. congolense, native transferrin, like Tf-Au, is found in the flagellar pocket, coated vesicles, tubular structures, and lysosome-like organelles where it appears to be concentrated. The presence of Tf in the lysosomes was confirmed in colocalization experiments using T. congolense, where native bovine transferrin colocalized with a trypanosome lysosomal marker, a cysteine protease. The data suggest that, unlike the situation in mammalian cells where most transferrin is recycled to the cell surface, in African trypanosomes transferrin is routed into lysosomes and may not, therefore, be recycled.

Directional movement of variable surface glycoprotein-antibody complexes in Trypanosoma brucei.

Binding of antibody, antibody fragments (Fab and F(ab)2) and biotin molecules to variable surface glycoprotein (VSG) of Trypanosoma brucei was studied by both light microscopy and fluorescence activated cell sorter (FACS) analysis. Antibodies, antibody fragments and biotin molecules were distributed over the entire parasite surface after incubation at 0 degree C. Upon warming to 37 degrees C, surface bound Fab and F(ab)2 fragments showed different rates of clearance from the parasite surface. Clearance, which in both cases followed double exponential decay kinetics, resulted from a directional movement of VSG-bound antibody complexes from both the surface of the flagellum and the cell body towards the cellular site of active endocytosis, the flagellar pocket (FP), even in the absence of antibody-mediated crosslinking of VSG. Immunofluorescence on trypanosomes permeabilized after binding, clearance and internalization, indicated the location of small amounts of antibody intracellularly, between the nucleus and the flagellar pocket. However, if a cocktail of protease inhibitors was added to the medium, larger amounts of internalized antibody could be detected within vacuoles situated between the nucleus and the flagellar pocket. Movement of antibody-VSG complexes was reversibly inhibited at temperatures below 37 degrees C and by increasing the NaCl concentration in the medium to 200 mM.