Mass spectrometric and mutational analyses reveal Lys-6-linked polyubiquitin chains catalyzed by BRCA1-BARD1 ubiquitin ligase

The breast and ovarian cancer suppressor BRCA1 acquires significant ubiquitin ligase activity when bound to BARD1 as a RING heterodimer. Although the activity may well be important for the role of BRCA1 as a tumor suppressor, the biochemical consequence of the activity is not yet known. Here we report that BRCA1-BARD1 catalyzes Lys-6-linked polyubiquitin chain formation. K6R mutation of ubiquitin dramatically reduces the polyubiquitin products mediated by BRCA1-BARD1 in vitro. BRCA1-BARD1 preferentially utilizes ubiquitin with a single Lys residue at Lys-6 or Lys-29 to mediate autoubiquitination of BRCA1 in vivo. Furthermore, mass spectrometry analysis identified the Lys-6-linked branched ubiquitin fragment from the polyubiquitin chain produced by BRCA1-BARD1 using wild type ubiquitin. The BRCA1-BARD1-mediated Lys-6-linked polyubiquitin chains are deubiquitinated by 26 S proteasome in vitro, whereas autoubiquitinated CUL1 through Lys-48-linked polyubiquitin chains is degraded. Proteasome inhibitors do not alter the steady state level of the autoubiquitinated BRCA1 in vivo. Hence, the results indicate that BRCA1-BARD1 mediates novel polyubiquitin chains that may be distinctly edited by 26 S proteasome from conventional Lys-48-linked polyubiquitin chains.     

Three molecular forms of a rat asialoglycoprotein receptor

It has been reported that a rat asialoglycoprotein receptor is composed of three polypeptide chains with molecular masses of 43, 54, and 64 kilodaltons (43, 54, and 64-Kd forms) and that the first has a different primary structure from the latter two forms. Incorporation of [3H]leucine into these forms showed that no precursor-product relationship is found between the 54-Kd and 64-Kd forms. The half-life of the 43-Kd form (25 h) was shorter than those of the 54-Kd and 64-Kd forms (66 and 70 h, respectively). Glycopeptides of the three forms were prepared from rat livers previously labeled in vivo with [3H]glucosamine. Gel filtration analysis of the glycopeptides before and after endo H treatment revealed that they were all resistant to endo H. Alkali treatment did not change the elution position appreciately. These results indicate that the three molecular forms contained only complex oligosaccharide chains. The receptor was prepared from rat livers previously treated with tunicamycin in vivo and subjected to SDS-PAGE. A distinct band with a molecular mass of 33 Kd was observed. The receptor was also immunoprecipitated from rat hepatocytes in primary culture previously labeled with [35S]methionine and analysed by SDS-PAGE and fluorography. In addition to the major 43-Kd form, a band with a molecular mass of 41 Kd was found and tunicamycin treatment gave rise to a 33-Kd component, which is in good agreement with the receptor purified from tunicamycin treated rats. It is suggested that the 43-Kd form is synthesized as a 33 Kd polypeptide, cotranslationally glycosylated to form the 41 Kd component and then processed to the final 43-Kd form. We also think that the 43-Kd form could bind to asialoorosomucoid-Sepharose 4B without its carbohydrate chains.     

Sequential morphologic changes in adult Echinococcus granulosus during complement-mediated lysis In vitro

Herd R. P., Ko L., Weisbrode S. E. and Heath D. D. 1984. Sequential morphologic changes in adult Echinococcus granulosus during complement-mediated lysis in vitro. International Journal for Parasltology14:141–149. Sequential changes (5,10, 20, 30,40, 50 min, 1, 2, 3, 4, 5, 6, 7, 8, h) were observed by light, scanning and transmission electron microscopy after 38-day-old adult Echinococcus granulosus were exposed to 50% guinea pig serum in vitro. Early changes within 3 h included contraction of worms, fusion of microtriehes, vacuolization and vesiculization of the distal cytoplasm, followed by rupture of vesicles leading to erosion and loss of the distal cytoplasm. This was most marked in the terminal proglottid but ultimately there was complete erosion of the distal cytoplasm of all proglottids and the scolex. After 3 h there was loss of definition of organelles, apparent edema of the perinuclear cytoplasm and, in some instances, rupture of the circular muscle layer with extrusion of parenchyma. Adult tape-worms exposed to heat-inactivated complement showed none of these changes. Lysis and death of the parasite was attributed to osmotic changes subsequent to the formation of trans-membrane channels induced by complement-mediated attack of the tegument after activation of the alternate pathway by factors present in the cestode tegument.     

Lectinomics I. Relevance of exogenous plant lectins in biomedical diagnostics

This review focuses on utilization of plant lectins as medical diagnostic reagents and tools. The lectin-related diagnostic is aimed at detection of several diseases connected to alteration of the glycosylation profiles of cells and at identification of microbial and viral agents in clinical microbiology. Certain lectins, proposed for or used as diagnostic tools could even recognize those cellular determinants, which are not detected by available antibodies. Broad information is presented on the lectinomics field, illustrating that lectin diagnostics might become practical alternative to antibody-based diagnostic products. In addition, the rising trend of lectin utilization in biomedical diagnostics might initiate a development of innovative methods based on better analytical technologies. Lectin microarray, a rapid and simple methodology, can be viewed as an example for such initiative. This technology could provide simple and efficient screening tools for analysis of glycosylation patterns in biological samples (cellular extracts, tissues and the whole cells), allowing thus personalized detection of changes associated with carbohydrate-related diseases.     

Scanning electron microscope study of the labyrinth of Macaca mulatta under hypokinetic conditions

Rhesus monkeys have been kept in horizontal position under klinostatic or antiorthostatic hypokinetic conditions for 7 and 19 days. Using scanning electron microscope, studies were made of the otolithic membrane of the utricle, the receptor surface of the utricle, crista ampullaris of the lateral semicircular canals, the organ of Corti, the stria vascularis and spiral ligament. No significant differences were found between control and experimental animals.     

An Automated System for Monitoring and Regulating the pH of Bicarbonate Buffers

The bicarbonate buffer is considered as the most biorelevant buffer system for the simulation of intestinal conditions. However, its use in dissolution testing of solid oral dosage forms is very limited. The reason for this is the thermodynamic instability of the solution containing hydrogen carbonate ions and carbonic acid. The spontaneous loss of carbon dioxide (CO₂) from the solution results in an uncontrolled increase of the pH. In order to maintain the pH on the desired level, either a CO₂ loss must be completely avoided or the escaped CO₂ has to be replaced by quantitative substitution, i.e. feeding the solution with the respective amount of gas, which re-acidifies the buffer after dissociation. The present work aimed at the development of a device enabling an automatic pH monitoring and regulation of hydrogen carbonate buffers during dissolution tests.