Type beta transforming growth factor is a potent inhibitor of murine megakaryocytopoiesis in vitro

To investigate the potential role of platelets in the inhibition of megakaryocytopoiesis, freeze-thawed extracts of human platelets were added to serumless liquid cultures of murine marrow. When acetylcholinesterase (AchE), a marker of megakaryocytic differentiation in mice, was assayed, a significant inhibition of enzymatic activity was noted in cultures containing the equivalent of greater than 5 X 10(6) solubilized platelets per milliliter. Freeze-thawed extracts of granulocytes had significantly less inhibitory effect than did platelets. Transforming growth factor beta (TGF-beta), a growth factor known to be inhibitory to some cell lineages and to be found at relatively high concentrations in platelets, was then added to liquid marrow cultures. A similar inhibition of AchE activity was detected when cultures were stimulated with mitogen-stimulated conditioned medium. The effect was potent with 50% inhibition of AchE activity observed at 4 pmol TGF-beta/L. To determine if TGF-beta inhibited specifically one aspect of megakaryocytic differentiation, the factor was added to isolated single megakaryocytes in serumless culture induced by interleukin 3 (IL3) to increase in size. The number of megakaryocytes increasing in size in response to IL 3 exposure was reduced from 68% to 20% when both factors were simultaneously added to cultures. Colony assays showed that megakaryocytic and granulocyte- macrophage colony detection was inhibited at picomolar concentrations of the factor. These data suggest that TGF-beta is a potent in vitro inhibitor of the murine megakaryocytic lineage, although its effects are not limited to this lineage.     

Drug-induced endocytosis of neonatal erythrocytes

The erythrocytes of the newborn infant have many properties that distinguish them from those of adults, and their membranes are also different from those of adult erythrocytes. We compared the ability of adult and neonatal RBCs to undergo endocytosis on exposure to drugs. Using a quantitative method, we showed that neonatal erythrocytes undergo a greater degree of endocytosis than do adult RBCs in response to primaquine, vinblastine, and chlorpromazine, and are sensitive to lower concentrations of the drugs. Some forms of drug-induced endocytosis are red cell age-dependent; when RBCs were separated by density gradient centrifugation, the membranes of the younger, less dense populations of both the neonatal and adult RBCs were capable of more extensive internalization than those of the denser, older RBCs. Neonatal RBCs of a given density undergo more endocytosis than do adult RBCs of the same density, suggesting that the membrane of the neonatal RBC is less stable and capable of more of the reorganization reflected in endocytosis than is the adult RBC membrane.     

Hereditary antithrombin deficiency: heterogeneity of the molecular basis and mortality in Dutch families

We studied the molecular basis and genetic heterogeneity of hereditary antithrombin (III) deficiency in nine Dutch families. Polymerase chain reaction (PCR) amplification and direct sequencing of all antithrombin gene exons and flanking intronic regions identified mutations in eight families. Given the opportunity to correlate the molecular basis with survival, we addressed the relevance of molecular defects to mortality in inherited antithrombin deficiency. The defects included single nucleotide deletions (7671 del G, 7768-69 del G) and insertions (5501 ins A, 2463 G-->TC) that lead to frameshifts, a single base substitution [5381 C-->T (129Arg-->stop)] leading to a premature termination codon, and single base substitutions resulting in amino acid substitutions [2652 A-->C (63Tyr-->Ser), 13380 T-->C (421Ile-- >Thr), and 13407 G-->T (430Cys-->Phe)]. All affected individuals were heterozygous for the defects. Previously we found in Dutch families that antithrombin deficiency did not lead to higher mortality compared with the general population. In accordance with these findings, we observed no excess mortality in the nine families [Observed:Expected, 52:52.6; standardised mortality ratio (SMR) 1.0, 95% confidence interval (CI), 0.7-1.3]. Our findings confirmed a considerable genetic heterogeneity underlying antithrombin deficiency. We therefore concluded that the lack of excess mortality in these families is not caused by a Dutch mild defect. We suggest that the longevity is not affected by molecular defects in the antithrombin gene and hypothesize that differences in mortality or natural history between families most likely result from other (genetic) risk factors.     

A frameshift mutation leading to type 1 antithrombin deficiency and thrombosis

Type 1 antithrombin III (ATIII) deficiency, which is the commonest form of inherited ATIII defect, is characterized by a quantitative reduction in both immunologically and functionally detectable protein. This condition is associated with a high incidence of thromboembolic disorder. Previous investigations have shown that the ATIII genes in the majority of cases are grossly intact, but the precise underlying molecular defects remain unknown. We have investigated the molecular basis of a type 1 ATIII deficiency in an Italian kindred by enzymatic amplification of the ATIII gene sequences in affected family members and direct sequencing of the amplified genomic DNA. A novel mutation, the deletion of a single T in the second position of codon 119, was identified in each of the affected individuals. The resulting frameshift leads to a premature termination in codon 126, effectively resulting in a null allele.     

Calcium distribution within human erythrocytes during endocytosis

In order to study 45Ca movements within erythrocytes, a method was devised that had minimal deleterious effect on the treated erythrocytes. Agents that induce endocytosis in intact erythrocytes (primaquine, vinblastine, and chlorpromazine) caused a prompt movement of 45Ca from cytosol to membrane-associated sites. This drug-induced movement of 45Ca to membrane sites was blocked by depleting erythrocytes of adenosine triphosphate (ATP) or by incubating them with known inhibitors of endocytosis, NaF of N-ethylmaleimide (NEM). It appears that endocytosis in intact human erythrocytes involves the movement and redistribution of 45Ca from cytosol to membrane-associated sites. Therefore, in the erythrocyte, as in perhaps other cells, movement of Ca from one site to another may modulate important cellular biologic functions.     

The polyadenylation site mutation in the alpha-globin gene cluster

In a previous study, we described a form of nondeletion alpha- thalassemia (alpha T Saudi alpha) found in subjects of Saudi Arabian origin. In the current study, using synthetic oligoprobe hybridization and restriction enzyme analysis, we have demonstrated that the molecular basis of alpha T Saudi alpha is due solely to a single base mutation (AATAAA----AATAAG) in the polyadenylation signal of the alpha 2 gene and that the frameshift mutation in codon 14 of the linked alpha 1 gene is the result of a cloning artefact. The alpha 2 polyadenylation signal mutation occurs in other Middle Eastern and the Mediterranean populations and is responsible for the clinical phenotype of Hb H disease in some Saudi Arabian individuals with five alpha genes (alpha T Saudi alpha/(alpha alpha alpha)T Saudi). Evidence suggests that the (alpha alpha alpha)T Saudi haplotype has arisen as a result of a recombination between two misaligned chromosomes bearing the alpha T Saudi alpha defect.     

Anti-My-28, an antigranulocyte mouse monoclonal antibody, binds to a sugar sequence in lacto-N-neotetraose

Anti-My-28 is an IgM kappa monoclonal antibody produced by a hybridoma prepared from spleen cells of a mouse immunized with normal human granulocytes. By immunofluorescence it binds to human granulocytes but not to monocytes and lymphocytes. However, after treating cells with neuraminidase, the antibody also binds to lymphocytes and monocytes and to many leukemic cell lines and patient leukemic blast cells. Anti-My- 28 binds to several neutral glycolipids and desialylated gangliosides of leukocytes and erythrocytes as detected by radioimmunoassay and immunostaining of thin-layer chromatograms. It recognizes a sugar sequence in lacto-N-neotetraose, Gal beta 1-4GlcNAc beta 1-3Gal beta 1- 4Glc. This tetrasaccharide occurs in the glycolipids paragloboside and sialosylparagloboside, and its distal trisaccharide sequence is found in higher glycolipids and in glycoproteins.     

Nurses' perspectives on whether medical aid in dying should be accessible to incompetent patients with dementia: findings from a survey conducted in Quebec,Canada

We conducted a survey in a random sample of 514 Quebec nurses caring for the elderly to assess their attitudes towards extending medical aid in dying to incompetent patients and to explore associated factors. Attitudes were measured using clinical vignettes featuring a hypothetical patient with Alzheimer disease. Vignettes varied according to the stage of the disease (advanced or terminal) and the presence or absence of a written request. Of the 291 respondents, 83.5% agreed with the current legislation that allows physicians to administer aid in dying to competent patients who are at the end of life and suffer unbearably. A similar proportion (83%, p?=?0.871) were in favor of extending medical aid in dying to incompetent patients who are at the terminal stage of Alzheimer disease, show signs of distress, and have made a written request before losing capacity.     

Resveratrol Protects Against Functional Impairment and Cardiac Structural Protein Degradation Induced by Secondhand Smoke Exposure

Background

Secondhand smoke (SHS) impairs cardiac function and resveratrol is cardioprotective, possibly via antioxidant and anti-inflammatory capabilities. Previously, it was shown that resveratrol protects against SHS-induced cardiac dysfunction, but the molecular mechanism is not clear.

Methods

We measured cardiac function in pigs exposed to SHS alone in a first experiment or with and without resveratrol (5 mg/kg/day) in a second experiment using echocardiography and compared this with proteomic changes.

Results

In the first experiment after 28 days, end-diastolic volume, end-systolic volume, and stroke volume were all impaired in SHS pigs compared with control pigs, with cardiac output significantly depressed as early as 14 days. Depressed function corresponded to increased inflammation, oxidative stress, and matrix metalloproteinase-2, but decreased intact myosin light chain 1 in SHS compared with control pigs at 28 days. In our second study after 14 days, two-dimensional electrophoresis detected 6 significantly increased protein spots in SHS compared with control pigs. Mass spectrometry identified 4 spots as fragments of sarcomeric protein (1 myosin light chain 1, 1 β-myosin heavy chain, and 2 myosin-7), and 2 spots as glucose metabolism enzymes (lactate and pyruvate dehydrogenases). Resveratrol normalized the fragmented protein levels, but not the metabolic enzymes. At 14 days, matrix metalloproteinase-2 activity almost doubled in cardiac tissue from SHS compared with control pigs, and resveratrol appeared to normalize it.

Conclusions

Thus, the ventricular differences in protein expression might explain the mechanism by which SHS reduces critical hemodynamic parameters through the degradation of sarcomeres, appearing to be prevented by resveratrol administration.