Evaluation of apoptosis markers in different cell lines infected with equine arteritis virus

Equine arteritis virus (EAV) induces apoptosis in infected cells. Cell death caused by EAV has been studied mainly using three cell lines, BHK-21, RK-13 and Vero cells. The mechanism of apoptosis varies among cell lines and results cannot be correlated owing to differences in EAV strains used. We evaluated different markers for apoptosis in BHK-21, RK-13 and Vero cell lines using the Bucyrus EAV reference strain. Acridine orange/ethidium bromide staining revealed morphological changes in infected cells, while flow cytometry indicated the extent of apoptosis. We also observed DNA fragmentation, but the DNA ladder was detected at different times post-infection depending on the cell line, i.e., 48, 72 and 96 h post-infection in RK-13, Vero and BHK-21 cells, respectively. Measurement of viral titers obtained with each cell line indicated that apoptosis causes interference with viral replication and therefore decreased viral titers. As an unequivocal marker of apoptosis, we measured the expression of caspase-3 and caspases-8 and -9 as extrinsic and intrinsic markers of apoptosis pathways, respectively. Caspase-8 in BHK-21 cells was the only protease that was not detected at any of the times assayed. We found that Bucyrus EAV strain exhibited a distinctive apoptosis pathway depending on the cell line.     

Pig Reticulocytes. III. Glucose permeability in naturally occurring reticulocytes and red cells from newborn piglets

The loss of facilitated glucose transport of red cells occurring in the newborn pig was monitored in 11 density-separated cells from birth to a 4 wk of age. At birth there was a threefold increase in glucose permeability from the lightest cells to the most dense, suggesting that cells having progressively less glucose permeability are released into the circulation as gestation proceeds. Because of extraordinary stimulation of erythropoietic activity, the uppermost top fraction constituting 2-3 percent of the total cells is composed purely of reticulocytes in the growing animal. The glucose permeability of these reticulocytes which at birth has a slow but significant rate of 3.7 μmol/ml cell x min at 25 degrees C is rapidly decreased within 3-4 days to the level of reticulocytes produced in the adult in response to phenylhydrazine assault. Moreover, reticulocytes themselves discard their membrane permeability to glucose in the course of maturation to red cells. Thus, even though reticulocytes at birth are permeable to glucose, they will become red cells practically impervious to glucose within a few days. These findings suggest that the transition from a glucose- permeable fetal state to a glucose-impermeable postnatal state is brought about by two mechanisms: (a) dilution of fetal cells by glucose-impervious cells produced coincidentally with or shortly after birth; and (b) elimination of fetal cells, which have a shorter half-life, from the circulation.     

Sequence variation in the guillemot (Alcidae: Cepphus) mitochondrial control region and its nuclear homolog

We describe sequence variation in the mitochondrial control region and its nuclear homolog in three species and seven subspecies of guillemots (Cepphus spp.). Nuclear homologs of the 5' end of the control region were found in all individuals. Nuclear sequences were approximately 50% divergent from their mitochondrial counterparts and formed a distinct phylogenetic clade; the mitochondrial-nuclear introgression event must have predated the radiation of Cepphus. As in other vertebrates, the guillemot control region has a relatively conserved central block flanked by hypervariable 5' and 3' ends. Mean pairwise interspecific divergence values among control regions were lower than those in other birds. All individuals were heteroplasmic for the number of simple tandem nucleotide repeats (A(n)C) at the 3' end of the control region. Phylogenetic analyses suggest that black guillemots are basal to pigeon and spectacled guillemots, but evolutionary relationships among subspecies remain unresolved, possibly due to incomplete lineage sorting. Describing molecular variation in nuclear homologs of mitochondrial genes is of general interest in phylogenetics because, if undetected, the homologs may confound interpretations of mitochondrial phylogenies.      

Detailed description of an implantable directional Doppler flowmeter.

A Doppler flowmeter and the necessary modifications for implantation are described in detail. Since only part of the electronics was implanted a phase-locked loop had to be introduced in order to keep the flow measurement directional. The proper working of the apparatus is demonstrated in vitro and in vivo. As an example the result of flow studies in the aorta and the pulmonary artery after homotransplantation of the lung in dogs are given.     

Binding studies of NADPH to NADP-specific L-glutamate dehydrogenase from Saccharomyces cerevisiae.

Optical characteristics of enzyme-reduced coenzyme complexes of yeast NADP-specific glutamate dehydrogenase have been investigated in the presence and absence of product (L-glutamate) and in the presence or absence of phosphate. The phosphate effect, pointed out in a previous work, is found again: inorganic phosphate (Pi) destabilizes the binary complex (E - NADPH), the dissociation constant of which is equal to 14 muM, a value much higher than that determined in Tris-HCl buffer: Kd = 0.9 muM. Concerning the role of phosphate some assumptions are drawn up with respect to a similar behaviour of Pi toward yeast glutamate dehydrogenase and ADP toward the beef liver enzyme. In the same way, L-glutamate induces a stabilization of the binary complex; this latter effect is unchanged in the presence of phosphate, yet it is less marked than in the case of beef liver glutamate dehydrogenase. Protein fluorescence, nucleotide fluorescence and circular dichroism measurements allowed the determination of three identical and independent NADPH binding sites per hexameric active unit. In analogy with beef liver enzyme, it seems that yeast glutamate dehydrogenase is a good model to study anticooperativity in ligand binding.     

Synthesis of protoporphyrin IX induced by 5-aminolevulinic acid in yeast cells in the presence of 2,2;-dipyridyl

5-Aminolevulinic acid (ALA), a precursor of porphyrin synthesis, increased the production of various porphyrin compounds in Candida guilliermondii cells. Metalloporphyrins and protoporphyrin IX (PPIX) were predominantly accumulated, respectively, at ALA concentrations of 0.2-0.4 mM and at those higher than 1.5 mM. 2,2;-Dipyridyl which complexed with bivalent metals significantly increased the content of endogenous PPIX even at ALA concentrations lower than 0.5 mM. Under these conditions, the yeast sensitivity to photodynamic effect of visible light (400-600 nm) dramatically increased due to photosensitization by endogenous PPIX.