Protein synthesis and storage in human platelets: a defective storage of fibrinogen in platelets in Glanzmann's thrombasthenia

In vivo metabolic labelling experiments were performed to investigate the ability of human platelets to synthesize and store fibrinogen and thrombospondin. Newly synthesized proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Results were compared with those obtained for the platelets of a patient with Glanzmann's thrombasthenia where endogenous fibrinogen levels were severely reduced. Normal human platelets were able to synthesize the different subunits of fibrinogen and thrombospondin and to assemble them into native fibrinogen and thrombospondin molecules. This synthesis was inhibited by cycloheximide. Synthesis of both fibrinogen and thrombospondin was observed in the platelets of the Glanzmann's thrombasthenia patient. However, radiolabelled fibrinogen was no longer detected after an 18-h non-radioactive chase, although it was retained in the control platelets. Neosynthesized thrombospondin of the patient was normally preserved during the same chase period. When the fate of the radioactive fibrinogen was studied, it was found to be degraded in Glanzmann's thrombasthenia platelets to the same extent as neosynthesized cytoplasmic proteins, whereas in control platelets less degradation had occurred. We conclude that human platelets maintain a residual capacity to synthesize fibrinogen and that its deficiency in Glanzmann's thrombasthenia results from a storage abnormality and not from a synthesis defect.     

Critical ontologies: rethinking relations to other-than-humans from the Bolivian Andes

Drawing from fieldwork and archival research carried out in Bolivia between 2010 and 2017, this article undertakes a rethinking of Indigenous ontologies in light of Bolivian interlocutors’ efforts to navigate deeply precarious ties to named places and saints. Attention to such instabilities challenges romantic accounts of ontology that presume a stable domain of materiality or religiosity outside of practice. During fieldwork in central Bolivia, I learned about the ways that Quechua farmers negotiated the relational and ecological effects of a divisive history of indentured labour and sexual violence through acts of devotion including paraman purina (‘walking for rain’), feasting, flute-play, dance, and chapel prayer each February for the Patron Saint La Virgen de la Candelaria, named places, and the Pachamama. These practices sought to rebuild ties to named places that were interrupted by the forbidding of offerings by the prior hacienda master and reshaped by state projects of Indigenous revivalism. These devotional practices, and participants’ narrations of them, offer insight into the political workings of Indigenous ontologies in twenty-first-century Bolivia. I propose critical ontologies as a scholarly lens that insists upon placing relations with other-than-humans within broader fields of legal and political contestation over rights, nature, and Indigeneity.     

Design and preclinical testing of an anti-CD41 CAR T cell for the treatment of acute megakaryoblastic leukaemia

Acute megakaryoblastic leukaemia (AMkL) is a rare subtype of acute myeloid leukaemia (AML) representing 5% of all reported cases, and frequently diagnosed in children with Down syndrome. Patients diagnosed with AMkL have low overall survival and have poor outcome to treatment, thus novel therapies such as CAR T cell therapy could represent an alternative in treating AMkL. We investigated the effect of a new CAR T cell which targets CD41, a specific surface antigen for M7-AMkL, against an in vitro model for AMkL, DAMI Luc2 cell line. The performed flow cytometry evaluation highlighted a percentage of 93.8% CAR T cells eGFP-positive and a limited acute effect on lowering the target cell population. However, the interaction between effector and target (E:T) cells, at a low ratio, lowered the cell membrane integrity, and reduced the M7-AMkL cell population after 24 h of co-culture, while the cytotoxic effect was not significant in groups with higher E:T ratio. Our findings suggest that the anti-CD41 CAR T cells are efficient for a limited time spawn and the cytotoxic effect is visible in all experimental groups with low E:T ratio.     

Proteolytic processing of the vitellogenin precursor in the boll weevil,Anthonomus grandis

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M_rs of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M_r honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M_r boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10–15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis

The Staphylococcus epidermidis genes icaABC are involved in the synthesis of the polysaccharide intercellular adhesin (PIA), which is located mainly on the cell surface, as shown by immunofluorescence studies with PIA-specific antiserum. PIA was shown to be a linear β-1,6-linked glucosaminoglycan composed of at least 130 2-deoxy-2-amino-D-glucopyrano-syl residues of which 80–85% are N-acetylated, the rest being non-N-acetylated and positively charged. A transposon insertion in the icaABC gene cluster (ica, intercellular adhesion) led to the loss of several traits, such as the ability to form a biofilm on a polystyrene surface, cell aggregation, and PIA production. The mutant could be complemented by transformation with the IcaABC-carrying plasmid pCN27. Transfer of pCN27 into the heterologous host Staphylococcus carnosus led to the formation of large cell aggregates, the formation of a biofilm on a glass surface, and PIA expression. The nucleotide sequence of icaABC suggests that the three genes are organized in an operon and that they are co-transcribed from the mapped ica A promoter. Ica A contains four potential transmembrane helices, indicative of a membrane location. The deduced Ica A sequence shows similarity to those of polysaccharide-polymerizing enzymes, the most pronounced being with a Rhizobium meliloti N-acetylglucosaminyltransferase involved in lipo-chitin biosynthesis (22.5% overall identity and 37.4% overall similarity). This similarity suggests that Ica A has N-acetylglucosaminyltransferase activity in the formation     

Effect of canine surfactant protein (SP-A) on the respiratory burst of phagocytic cells

Cells obtained from bronchoalveolar lavage, or neutrophils of peripheral blood of dog, were incubated with the canine surfactant-associated protein A (SP-A). A significant decrease of the production of Superoxide anion was observed after subsequent stimulation with phorbol-12-myristate-13-acetate (PMA) as measured by the lucigenin-dependent chemiluminesence (CL). Several other proteins used for control experiments did not decrease lucigenin-dependent CL, indicating a specific effect of SP-A on phagocytes. Treatment of SP-A with collagenase prior to incubation with neutrophils destroyed the depleting effect on oxygen radical production of PMA-stimulated cells. We propose that SP-A acts as a regulatory factor of the respitatory burst of alveolar macrophages and neutrophils in the lungs. The inhibitory effect of SP-A is down-regulated by collagenase released from stimulated alveolar macrophages.     

The spatial organization of the active sites of the bifunctional oligomeric enzyme tryptophan synthase: Cross-linking by a novel method

To achieve specific cross-linking between the active sites of the non-identical subunits tryptophan synthase from E. coli was modified by a novel method. After reaction with bifunctional reagents of the isolated subunits at their active sites, the tetrameric complex was formed and the free ends of the reagent molecules reacted with each other forming a covalent bridge between the subunits. The distance between the amino acid side chains involved in the cross-linking should not exceed approx. 1.8 nm. A distance much shorter than that is unlikely since all attempts to cross-link the active sites with different shorter bifunctional reagents failed. The implications of these results in the mechanism of action of the enzyme are discussed.     

Dependency on serine concentration of the activity of tryptophan synthase. Cooperative properties

The activity of the enzyme tryptophan synthase from Escherichia coli was tested as a function of the concentration of L-serine which serves as a substrate in the indole to tryptophan reaction as well as for the L-serine deaminase activity. L-Serine binding was found to follow the pattern of negative cooperativity both by kinetic and by equilibrium methods. The enzyme kinetic data support the view that a rapid equilibration model for the enzyme . substrates complex formation is not strictly obeyed.     

The effect of extracorporeal blood radiotherapy on the peripheral blood T and B-lymphocytes in chronic lymphadenitis]

B-lymphocytes and T-lymphocytes were determined before and after therapy in 12 patients with chronic lymphatic leukemia (CLL) treated with extracorporal blood radiation (ECIB). There was a significant decrease of B-lymphocytes (p less than 0.001) from 85.3 +/- 6.55% to 71.55 +/- 10.60% by ECIB, whereas no significant changes could be found in T-lymphocytes. 12 untreated CLL patients, whose B-lymphocytes amounted 83.25 +/- 6.79% with 7.5 +/- 3.42% of T-lymphocytes, were examined as group of comparison. From these findings ECIB is concluded to cause a decrease of accumulated B-cells.