Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart

Changes in cytosolic free calcium concentration during myocardial ischemia were measured by 19F NMR in 5FBAPTA-loaded perfused rat hearts. The hearts were perfused with Krebs-Henseleit buffer containing 5 microM of the acetoxymethyl ester of 5FBAPTA, which was hydrolyzed by cytosolic esterases to achieve cytosolic concentrations of 5FBAPTA of 0.12 to 0.65 mM. Cytosolic free calcium concentrations were calculated as the product of the ratio of peak areas for bound and free 5FBAPTA in the NMR spectra and the dissociation constant (708 nM). The basal cytosolic calcium concentration, measured in potassium or magnesium arrested hearts, was 252 nM, and the time-average calcium concentration in beating hearts was 630 nM. Following the onset of total ischemia, there was no immediate substantial change in cytosolic calcium despite a rapid decline in creatine phosphate and ATP and a marked increase in inorganic phosphate as monitored by 31P NMR, but by 10 minutes, there was a substantial increase in free calcium concentration. The ratio of peak areas of bound and free 5FBAPTA returned to the preischemic value during reperfusion, and there was no detectable loss of 5FBAPTA from the heart. Creatine phosphate was also restored to its preischemic level during reperfusion. These results indicate that cytosolic free calcium increases during ischemia and is not immediately associated with lethal injury. This increase in cytosolic calcium may activate degradative enzymes that eventually could compromise myocyte viability.     

The effects of recombinant-DNA-derived interferons on the growth of myeloid progenitor cells

Interferons (IFNs) have been shown to have significant effects on hematopoietic cell growth. Previous studies defining these effects have utilized mouse and human alpha-, beta-, and gamma-IFN isolated from supernatants of stimulated cells. Despite purification, the possible presence of other lymphokines and soluble factors remains a concern. In this study, the effects of gene-cloned alpha- and gamma-IFN on colony- forming units of granulocyte/macrophage (CFU-GM) progenitors cultured from the peripheral blood of normal volunteers were examined. In addition, blast cell colonies from one patient with acute myelogenous leukemia (AML) were studied. The growth of normal CFU-GM and AML blast cell colonies was inhibited in a dose-dependent manner by gamma- and alpha-IFN. gamma-IFN was ten to 100 times more potent than alpha-IFN in that this species of IFN reduced colony formation by greater than 50% at concentrations of less than 15 antiviral U/mL. The effects of gamma- IFN were neutralized by a monoclonal antibody specific for gamma-IFN. These in vitro studies indicate that human gamma-IFN may be an important modulator of myelopoiesis. Although these data indicate a possible efficacy of gamma-IFN in the treatment of AML, the in vitro results should be considered for their in vivo significance.     

Evaluation of the echocardiogram as an epidemiologic tool in an asymptomatic population.

An asymptomatic adult population of 196 men and women was studied with the echocardiogram to derive age- and sex-specific "normal" values for a number of clinically used echocardiograhic variables. The results are in general agreement with previously published normal values. Body position during the examination, age and sex influence the echocardiographic results; body surface area correction normalized most of these effects. The prevalence of occult abnormalities determined by the echocardiogram is 7%; the most common finding was mitral valve prolapse. Inter- and intraobserver variability was assessed. The interobserver differences found on analysis are statistically, but not clinically , significant. The echocardiogram appears to be a suitable tool to use in epidemiologic studies to detect selected cardiac abnormalities, but is limited for this purpose because some subjects in such a population cannot be adequately examined.     

Cytokine production by primary bone marrow megakaryocytes

Primary human bone marrow megakaryocytes were studied for their ability to express and release cytokines potentially relevant to their proliferation and/or differentiation. The purity of the bone marrow megakaryocytes was assessed by morphologic and immunocytochemical criteria. Unstimulated marrow megakaryocytes constitutively expressed genes for interleukin-1 beta (IL-1 beta), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha), by the polymerase chain reaction (PCR) and Northern blot analysis. At the protein level, megakaryocytes secreted significant amounts of IL-1 beta (53.6 +/- 3.6 pg/mL), IL-6 (57.6 +/- 15.6 pg/mL), and GM-CSF (24 +/- 4 pg/mL) but not TNF-alpha. Exposure of human marrow megakaryocytes to IL-1 beta increased the levels of IL-6 (87.3 +/- 2.3 pg/mL) detected in the culture supernatants. Transforming growth factor- beta was also able to stimulate IL-6, IL-1 beta, and GM-CSF secretion, but was less potent than stimulation with phorbol-12-myristate-13- acetate (PMA). The secreted cytokines acted additively to maintain and increase the number of colony-forming unit-megakaryocytes colonies (approximately 35%). These studies demonstrate the production of multiple cytokines by isolated human bone marrow megakaryocytes constitutively or stimulated in vitro. The capacity of human megakaryocytes to synthesize several cytokines known to modulate hematopoietic cells supports the concept that there may be an autocrine mechanism operative in the regulation of megakaryocytopoiesis.     

The Effects of Hemin and Double-Stranded RNA on α and β Globin Synthesis in Reticulocyte and Krebs II Ascites Cell-Free Systems and the Relationship of These Effects to an Initiation Factor Preparation

Protein synthesis in reticulocyte lysates ceases abruptly in the absence of added hemin or in the presence of double-stranded RNA. A similar effect of double-stranded RNA is observed in Krebs II ascites cell-free systems translating exogenous globin mRNA. The shut-off of protein synthesis is due to inhibition of initiation and can be prevented or reversed by addition of the initiation factor preparation M(3). Preparations of M(1), M(2), and dissociation factor are ineffective under these conditions. The effects of added hemin, M(3), and globin mRNA on the synthesis of alpha and beta globin chains have been studied in the reticulocyte and ascites cell extracts. When the concentration of M(3) is rate limiting, the synthesis of beta chains exceeds that of alpha chains. When the concentration of mRNA is rate limiting, synthesis of alpha and beta chains is more nearly equal.     

Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase: Inhibition of interaction of Met-tRNAfMet binding factor with another initiation factor in formation of Met-tRNAfMet·40S ribosomal subunit complexes

Protein synthesis in reticulocytes and their lysates is regulated by heme. In heme deficiency a heme-regulated translational inhibitor (HRI) that blocks initiation of polypeptide chains is activated. HRI is a protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNA(f) (Met) binding factor (IF), which forms a ternary complex with Met-tRNA(f) (Met) and GTP, a finding that suggests that the inhibition by HRI involves the phosphorylation of IF.We have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNA(f) (Met) to 40S ribosomal subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNA(f) (Met) to 40S subunits. The inhibitory effect of HRI requires ATP. Under these conditions HRI phosphorylates only the 38,000-dalton subunit of IF.The TDF preparations not only promote the binding of the ternary complex to 40S subunits but also promote the dissociation of the ternary complex in the presence of 5 mM Mg(2+) at 0 degrees . The preincubation of purified IF alone with low concentrations of HRI and ATP does not significantly affect its capacity to form the ternary complex; however, the TDF-promoted dissociation of the ternary complex is inhibited. The nonhydrolyzable analog adenosine 5'-[beta,gamma-imido]triphosphate does not substitute for ATP. These findings suggest that phosphorylation causes a conformational modification in IF, which results in inhibition of the interaction between the ternary complex and TDF that is required for the binding of the ternary complex to 40S subunits.     

Regulation of protein synthesis: Activation by double-stranded RNA of a protein kinase that phosphorylates eukaryotic initiation factor 2

Incubation of reticulocyte lysates or isolated crude ribosomes with low levels of double-stranded RNA (0.1-10 ng/ml) induces the formation of an inhibitor of protein synthesis initiation similar to that observed in heme deficiency. The inhibitor is associated with a cyclic AMP-independent protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) that phosphorylates the small polypeptide (38,000 daltons) of the eukaryotic initiation factor eIF-2. Activation of the inhibitor requires ATP in addition to double-stranded RNA and is accompanied by the phosphorylation of a 67,000-dalton polypeptide of unknown function. The inhibitor remains associated with the ribosomes during high-speed sedimentation. Once formed, the ribosome-associated inhibitor phosphorylates eIF-2 and inhibits protein synthesis in the absence of double-stranded RNA. Inhibition is prevented by exogenous eIF-2. The bound inhibitor can be solubilized by extraction with 0.5 M KCl. The soluble inhibitor preparation retains the ability to phosphorylate the small polypeptide of eIF-2 and to inhibit protein synthesis. Untreated crude ribosomes also contain cyclic AMP-independent protein kinase activities that phosphorylate the middle polypeptide (49,000 daltons) of eIF-2 and several polypeptide subunits of eIF-3 (160,000, 125,000, and 65,000 daltons); these kinase activities are not affected by double-stranded RNA and do not inhibit protein synthesis.     

Placebo use in vaccine trials: Recommendations of a WHO expert panel

Vaccines are among the most cost-effective interventions against infectious diseases. Many candidate vaccines targeting neglected diseases in low- and middle-income countries are now progressing to large-scale clinical testing. However, controversy surrounds the appropriate design of vaccine trials and, in particular, the use of unvaccinated controls (with or without placebo) when an efficacious vaccine already exists. This paper specifies four situations in which placebo use may be acceptable, provided that the study question cannot be answered in an active-controlled trial design; the risks of delaying or foregoing an efficacious vaccine are mitigated; the risks of using a placebo control are justified by the social and public health value of the research; and the research is responsive to local health needs. The four situations are: (1) developing a locally affordable vaccine, (2) evaluating the local safety and efficacy of an existing vaccine, (3) testing a new vaccine when an existing vaccine is considered inappropriate for local use (e.g. based on epidemiologic or demographic factors), and (4) determining the local burden of disease.     

Artificial Intelligence and Black‐Box Medical Decisions: Accuracy versus Explainability

Although decision‐making algorithms are not new to medicine, the availability of vast stores of medical data, gains in computing power, and breakthroughs in machine learning are accelerating the pace of their development, expanding the range of questions they can address, and increasing their predictive power. In many cases, however, the most powerful machine learning techniques purchase diagnostic or predictive accuracy at the expense of our ability to access “the knowledge within the machine.” Without an explanation in terms of reasons or a rationale for particular decisions in individual cases, some commentators regard ceding medical decision‐making to black box systems as contravening the profound moral responsibilities of clinicians. I argue, however, that opaque decisions are more common in medicine than critics realize. Moreover, as Aristotle noted over two millennia ago, when our knowledge of causal systems is incomplete and precarious—as it often is in medicine—the ability to explain how results are produced can be less important than the ability to produce such results and empirically verify their accuracy.