Hemopoietic-specific Sf3b1-K700E knock-in mice display the splicing defect seen in human MDS but develop anemia without ring sideroblasts.

Heterozygous somatic mutations affecting the spliceosome gene SF3B1 drive age-related clonal hematopoiesis, myelodysplastic syndromes (MDS) and other neoplasms. To study their role in such disorders, we generated knock-in mice with hematopoietic-specific expression of Sf3b1-K700E, the commonest type of SF3B1 mutation in MDS. Sf3b1K700E/+ animals had impaired erythropoiesis and progressive anemia without ringed sideroblasts, as well as reduced hematopoietic stem cell numbers and host-repopulating fitness. To understand the molecular basis of these observations, we analyzed global RNA splicing in Sf3b1K700E/+ hematopoietic cells. Aberrant splicing was associated with the usage of cryptic 3' splice and branchpoint sites, as described for human SF3B1 mutants. However, we found a little overlap between aberrantly spliced mRNAs in mouse versus human, suggesting that anemia may be a consequence of globally disrupted splicing. Furthermore, the murine orthologues of genes associated with ring sideroblasts in human MDS, including Abcb7 and Tmem14c, were not aberrantly spliced in Sf3b1K700E/+ mice. Our findings demonstrate that, despite significant differences in affected transcripts, there is overlap in the phenotypes associated with SF3B1-K700E between human and mouse. Future studies should focus on understanding the basis of these similarities and differences as a means of deciphering the consequences of spliceosome gene mutations in MDS.

Detection of specific antibodies to Plasmodium falciparum in blood bank donors from malaria-endemic and non-endemic areas of Venezuela.

Malaria antibody detection is valuable in providing retrospective confirmation of an attack of malaria. Blood bank screening is another area were malaria serology is potentially useful. In the present study, we tested the presence of antibodies to Plasmodium falciparum in sera from blood bank donors of non-endemic and malaria-endemic areas of Venezuela. Sera from 1,000 blood donors were tested by an indirect immunofluorescent antibody (IFA) assay and an IgG-ELISA for the presence of malaria antibodies using a synchronized in vitro-cultured Venezuelan isolate of P. falciparum as the antigen source. A selected group of positive and negative sera (n = 100) was also tested by a dot-IgG-ELISA. Positive results (reciprocal titer > or = 40) were found in 0.8% and 3.8% of blood donors when tested by the IFA assay and in 0.8% and 2% (optical density > or = 0.2) when tested by the IgG-ELISA in Caracas (non-endemic area) and Bolivar City (endemic area), respectively. The presence of anti-malarial antibodies in some sera from non-endemic areas such as Caracas reflects the increased potential risk of post-transfusional malaria in those areas due to the mobility of the blood donors. The data obtained indicate the need to implement new blood donor policy in blood banks in developing areas. Our results also indicate that the IFA assay is the most reliable test to use in malaria serodiagnosis.

Changes in proteolytic activity during the growth of Trypanosoma cruzi epimastigotes.

The proteolytic pattern of Trypanosoma cruzi epimastigotes was studied using electrophoresis in gelatin containing SDS polyacrylamide gels. Under conditions that represent a nutritional stress such as the depletion of glucose in an axenic medium the results obtained showed a significant increase in the activity of two proteases of M(r) = 50,000 and M(r) = 60,000 as well as the appearance of higher molecular weight bands during the transition from log to stationary phase parasites. The use of different protease inhibitors strongly suggests that all these proteases are of the cysteine type, and their acid pH optimal would be in agreement with the possible localization in the parasite lysosomes. The reincubation of stationary phase parasites in fresh LIT medium or the addition of 0.4% glucose partially reverted the proteolytic pattern to that observed in log phase parasites. The regulation in the activity of these enzymes could be related to the well established use of amino acids for catabolic purpose after depletion of the medium glucose.

Mechanisms of protein degradation in Trypanosoma cruzi.

Proteolysis of endogenous proteins may play a key role in the adaptation of T. cruzi to the different host environments to which it is exposed during its complex life cycle. For this reason, we have attempted to study the intracellular pathways of protein degradation in the non infective epimastigotes form (EP strain) of T. cruzi. Following intracellular proteolysis by pulse chase experiments with 35 S methionine, we observed a significant inhibition (50%) of the degradation of endogenous proteins in log phase parasites in the presence of inhibitors of lysosomal functions, such as chloroquine and E 64. A significant increase in proteolysis was observed in stationary phase parasites which was reverted to log phase values by supplementing the chase medium with 0.5% glucose or 10% serum, or in the presence of chloroquine. Under this condition of nutritional stress, we could observe an increase in the activity of acid proteases. A significant increase in the degradation rates was observed when abnormal proteins were induced in the parasite by amino acid analogs and puromycin. This increase was not affected by E 64, suggesting the participation of non lysosomal mechanisms in the degradation of rapidly degradable abnormal proteins. Under these conditions, we could observe an increase in high molecular weight conjugates of ubiquitin with respect to endogenous proteins. These results suggest the importance of lysosomal mechanisms in the degradation of cellular proteins in nutritional optimal conditions and during nutritional deprivation, and the possible involvement of the ubiquitin system in the degradation of high turnover proteins.

A PC12 variant lacking regulated secretory organelles: aberrant protein targeting and evidence for a factor inhibiting neuroendocrine gene expression.

A variant of the PC12 pheochromocytoma cell line (termed A35C) has been isolated that lacks regulated secretory organelles and several constituent proteins. Northern and Southern blot analyses suggested a block at the transcriptional level. The proprotein-converting enzyme carboxypeptidase H was synthesised in the A35C cell line but was secreted by the constitutive pathway. Transient transfection of A35C cells with cDNAs encoding the regulated secretory proteins dopamine beta-hydroxylase and synaptotagmin I resulted in distinct patterns of mistargeting of these proteins. It is surprising that hybrid cells created by fusing normal PC12 cells with A35C cells exhibited the variant phenotype, suggesting that A35C cells express an inhibitory factor that represses neuroendocrine-specific gene expression.

Heat shock enhances transcriptional activation of the murine-inducible nitric oxide synthase gene.

There is considerable interest in determining the conditions leading to enhanced inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) biosynthesis. Using in vivo footprinting, we demonstrate that heat shock of murine macrophages concurrent with lipopolysaccharide (LPS) treatment stimulated changes in guanine methylation sensitivity at ?898/9, at a putative partial heat shock element (HSE) and at -893/4, a site bordering an E-box, within the iNOS gene enhancer, suggesting inducible occupation by transcription factors at these regions. LPS treatment accompanied by heat shock provoked increased iNOS gene transcription, increased levels of iNOS protein, and increased production of NO compared with LPS treatment alone. Electrophoretic mobility shift analysis revealed low constitutive levels of specific binding to an E-box and a partial HSE within the iNOS enhancer. Binding to the E-box was increased by LPS treatment or by heat shock, achieving a greater increase by a combination of both treatments. The proteins occupying this site were identified as belonging to the USF family of transcription factors. Heat shock or LPS increased binding to the HSE, and the factor responsible for this interaction was identified as heeat shock factor-1 (HSF-1). Mutations at the HSE revealed the importance of HSF-1 in the induction of iNOS by LPS. Thus, our data reveal two novel regulatory sites in the murine iNOS gene, one of which is implicated in enhancing iNOS expression via LPS stimulation, and provide the first evidence that heat shock enhances transcription of the iNOS gene. These results could have implications in the host response mechanism to fever-associated gram-negative infection.