Characterization of antigen receptor response elements within the interleukin-2 enhancer.

T-cell activation and induction of interleukin-2 (IL-2) expression in human T lymphocytes require both interaction of foreign antigen with the T-cell antigen receptor and protein kinase C (PKC) stimulation. Agents such as phorbol 12-myristate 13-acetate (PMA) that stimulate PKC augment the effects of antigen but are not sufficient for IL-2 activation. By analysis of deletion mutants, we identified three DNA sequences extending from -73 to -89, -217 to -255, and -263 to -279, designated IL-2 sites A, D, and E, respectively, that are required for maximal induction of IL-2 expression. One of these regions, site E, interacted with a protein (NF-IL-2E) present only in the nuclei of cells which have been stimulated. The other two sequences interacted with a protein (NF-IL-2A) that is constitutively expressed in T cells. When multiple tandem copies of either the E site or the A site were placed upstream of the gamma-fibrinogen promoter, they activated expression via this promoter in response to signals initiated at the antigen receptor but not following PMA stimulation. For this reason, we denoted them antigen receptor response elements. The uncoupling of antigen receptor and PKC requirements in these studies indicates that these signal pathways are, at least in part, distinct and integrated at the level of the gene.     

Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis.

Rotaviruses are icosahedral viruses with a segmented, double-stranded RNA genome. They are the major cause of severe infantile infectious diarrhea. Rotavirus growth in tissue culture is markedly enhanced by pretreatment of virus with trypsin. Trypsin activation is associated with cleavage of the viral hemagglutinin (viral protein 3 [VP3]; 88 kilodaltons) into two fragments (60 and 28 kilodaltons). The mechanism by which proteolytic cleavage leads to enhanced growth is unknown. Cleavage of VP3 does not alter viral binding to cell monolayers. In previous electron microscopic studies of infected cell cultures, it has been demonstrated that rotavirus particles enter cells by both endocytosis and direct cell membrane penetration. To determine whether trypsin treatment affected rotavirus internalization, we studied the kinetics of entry of infectious rhesus rotavirus (RRV) into MA104 cells. Trypsin-activated RRV was internalized with a half-time of 3 to 5 min, while nonactivated virus disappeared from the cell surface with a half-time of 30 to 50 min. In contrast to trypsin-activated RRV, loss of nonactivated RRV from the cell surface did not result in the appearance of infection, as measured by plaque formation. Endocytosis inhibitors (sodium azide, dinitrophenol) and lysosomotropic agents (ammonium chloride, chloroquine) had a limited effect on the entry of infectious virus into cells. Purified trypsin-activated RRV added to cell monolayers at pH 7.4 medicated 51Cr, [14C]choline, and [3H]inositol released from prelabeled MA104 cells. This release could be specifically blocked by neutralizing antibodies to VP3. These results suggest that MA104 cell infection follows the rapid entry of trypsin-activated RRV by direct cell membrane penetration. Cell membrane penetration of infectious RRV is initiated by trypsin cleavage of VP3. Neutralizing antibodies can inhibit this direct membrane penetration.     

The role of blood platelets in nucleoside metabolism: assay, cellular location and significance of thymidine phosphorylase in human blood

The enzyme thymidine phosphorylase (thymidine: orthophosphate deoxyribosyltransferase, EC 2.4.2.4.), which plays a crucial role in nucleic acid metabolism in both prokaryotic and eukaryotic cells by regulating the availability of thymidine, is present in mammalian blood. Here we describe a simple, rapid HPLC-based micromethod for the assay of blood thymidine phosphorylase. We have arbitarily defined 1 unit of blood thymidine phosphorylase activity as the activity required to produce a 1-nM increment in the plasma concentration of thymine after incubation for 1 h at 37°C with a saturating concentration of exogenous thymidine.

In normal adults, whole (peripheral venous) blood thymidine phosphorylase activity with blood cells intact was 64 ± 11 units (mean ± S.D., n =20, range 45–89). The apparent Michaelis constant for thymidine was of the order to 10⁻⁴ M but varied nearly 5-fold between different individuals. Activity increased when blood cells were permeabilised or lysed with non-ionic detergents, implying that thymidine phosphorylase is an intracellular enzyme which may be influenced by exogenous as well as intracellular factors. When blood from normal donors was fractionated, thymidine phosphorylase activity consistently co-isolated with platelets. Whole-blood thymidine phosphorylase activity correlated well with platelet parameters. Although thymidine phosphorylase activity was also detected in plasma and serum, the small size and notorious fragility of platelets suggest its platelet origin.

Blood from leukaemic donors showed significantly increased thymidine phosphorylase activity compared to normal controls (mean activity ± S.D. was 96 ± 27 units; range 58–140, n = 8).

Thymine formation from thymidine was temperature- and pH-depdendent in whole blood. 2′-Deoxyuridine and 3 of its 5-halogenated analogues (but not 3′-azido-3′-deoxythymidine (AZT), were catabolised by blood thymidine phosphorylase, even during blood clotting at room temperature. Assumptions about in vivo concentrations of these compounds should therefore be interpreted cautiously.

In the presence of high concentrations of thymine and suitable deoxyribose donors, small amounts of thymidine were formed in some blood samples, so it is conceivable that thymidine catabolism may be reversible in vivo under some circumstances.     

Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.     

"Rates" of birth defects

The proportion of children born with a particular defect is not a "birth defect rate" but, rather, a prevalence proportion. The implications of confusing a rate and a proportion are discussed in terms of the interpretation of birth defect data. It is recommended that "prevalence proportion" or "prevalence" be used to report the frequency of various defects rather than the often-used "prevalence rate."     

A distributed knowledge-based approach to flexible automation: The contract net framework

This article applied distributed artificial intelligence to the real-time planning and control of flexible manufacturing systems (FMS) consisting of asynchronous manufacturing cells. A knowledge-based approach is used to determine the course of action, resource sharing, and processor assignments. Within each cell there is an embedded automatic planning system that executes dynamic scheduling and supervises manufacturing operations. Because of the decentralized control, real-time task assignments are carried out by a negotiation process among cell hosts. The negotiation process is modeled by augmented Petri nets —the combination of production rules and Petri nets—and is excuted by a distributed, rule-based algorithm.