Simultaneous production of IFN-gamma, IFN-alpha/beta and nitric oxide in peritoneal macrophages from TDM-treated mice

Peritoneal macrophages (PM) were isolated from mice treated with Dimycolate of Trehalose (TDM), a glycolipid extracted from the cell wall of Mycobacterium tuberculosis. PM from TDM-treated mice (TDM-PM) were shown to secrete consistent amount of IFN-gamma, which was not detectable in control Resident-PM (Res-PM), as revealed by ELISA. In addition, biologically active IFN was detected in the supernatants of TDM-PM, whereas no IFN production was found in those of control Res-PM. The addition of specific antisera to PM cultures revealed the simultaneous production of both type I and II IFNs in TDM-PM cultures. No reciprocal regulation in the production of IFN-gamma and IFN-alpha/beta was found in these cultures. In parallel, nitric oxide (NO) production was measured in TDM-PM cultures by detecting nitrites (NO2-). TDM-PM cultures accumulated high amounts of NO2- which decreased to the level of Res-PM in the presence of NMMA, an inhibitor of NO-synthases. In vitro, neither type I nor type II IFNs were involved in the stimulation of NO production. The capacity of macrophages to simultaneously secrete IFN-gamma, IFN-alpha/beta and NO upon in vivo TDM-treatment could be of particular relevance for the defense process of innate immunity in which macrophages play a crucial role.     

Molecular systematics and paleobiogeography of the South American sigmodontine rodents [published erratum appears in Mol Biol Evol 1998 Feb;15(2):224]

The murid rodent subfamily Sigmodontinae contains 79 genera which are distributed throughout the New World. The time of arrival of the first sigmodontines in South America and the estimated divergence time(s) of the different lineages of South American sigmodontines have been controversial due to the lack of a good fossil record and the immense number of extant species. The "early-arrival hypothesis" states that the sigmodontines must have arrived in South America no later than the early Miocene, at least 20 MYA, in order to account for their vast present-day diversity, whereas the "late-arrival hypothesis" includes the sigmodontines as part of the Plio-Pleistocene Great American Interchange, which occurred approximately 3.5 MYA. The phylogenetic relationships among 33 of these genera were reconstructed using mitochondrial DNA (mtDNA) sequence data from the ND3, ND4L, arginine tRNA, and ND4 genes, which we show to be evolving at the same rate. A molecular clock was calibrated for these genes using published fossil dates, and the genetic distances were estimated from the DNA sequences in this study. The molecular clock was used to estimate the dates of the South American sigmodontine origin and the main sigmodontine radiation in order to evaluate the "early-" and "late-arrival" scenarios. We estimate the time of the sigmodontine invasion of South America as between approximately 5 and 9 MYA, supporting neither of the scenarios but suggesting two possible models in which the invading lineage was either (1) ancestral to the oryzomyines, akodonts, and phyllotines or (2) ancestral to the akodonts and phyllotines and accompanied by the oryzomyines. The sigmodontine invasion of South America provides an example of the advantage afforded to a lineage by the fortuitous invasion of a previously unexploited habitat, in this case an entire continent.      

The abundance of additional N-acetyllactosamine units in N-linked tetraantennary oligosaccharides of human Tamm-Horsfall glycoprotein is a donor-specific feature

Previously, treatment of Tamm-Horsfall glycoprotein (THp) from different donors with endo-beta-galactosidase has been shown to liberate a tetra- and a Sd(a)-active pentasaccharide, concluding the presence of N-linked carbohydrate chains containing additional N - acetyllactosamine units. These type of oligosaccharides were not found in a detailed structure elucidation of the carbohydrate moiety of THp of one male donor, suggesting a donor-specific feature for these type of structures. Therefore, THp was isolated from four healthy male donors and each subjected to endo-beta-galactosidase treatment in order to release these tetra- and Sd(a)-active pentasaccharide. Differences were observed in the total amount of released tetra- and Sda-active pentasaccharide of the used donors (42, 470, 478, 718 microg/100 mg THp), indicating that the presence of repeating N-acetyllactosamine units incorporated into the N-glycan moiety of THp is donor specific. Furthermore, a higher expression of the Sd(a) determinant on antennae which display N-acetyllactosamine elongation was observed, suggesting a better accessibility for the beta-N-acetylgalactosaminyltransferase. In order to characterize the N-glycans containing repeating N- acetyllactosamine units, carbohydrate chains were enzymatically released from THp and isolated. The tetraantennary fraction, which accounts for more than 33% of the total carbohydrate moiety of THp, was used to isolate oligosaccharides containing additional N - acetyllactosamine units. Five N-linked tetraantennary oligosaccharides containing a repeating N-acetyllactosamine unit were identified, varying from structures bearing four Sd(a) determinants to structures containing no Sd(a) determinant (see below). One compound was used in order to specify the branch location of the additional N- acetyllactosamine unit, and it appeared that only the Gal-6' and Gal-8' residues were occupied by a repeating N -acetyllactosamine unit.      

Embryonic induction: Is the Nieuwkoop centre a useful concept?

Regionalisation of the amphibian embryo is classically thought to involve induction by the Spemann organiser, itself induced by the Nieuwkoop centre. This model has now been extended to teleosts, with the identification of a gene that appears to define the zebrafish equivalent of the Nieuwkoop centre.     

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

Coexpression of mutant and wild type protein kinase in lymphoma cells resistant to dibutyryl cyclic AMP.

A mutant clone resistant to dibutyryl cyclic AMP was isolated from S49 mouse lymphoma cells. The mutant expressed a form of cyclic AMP-dependent protein kinase distinguishable from wild type kinase by its decreased sensitivity to activation by cyclic AMP and its increased thermal lability. Hybrids formed between mutant and wild type cells were resistant to dibutyryl cyclic AMP and expressed both mutant and wild type activities in about equal amount. The parent mutant cells also appeared to express wild type kinase activity, but at a lower level. We conclude that wild type S49 cells have and express two identical alleles for the regulatory subunit of protein kinase, one of which has undergone mutation in the mutant cells.     

Membrane compartmentalized ATP and its preferential use by the Na, K-ATPase of human red cell ghosts

This paper describes work which begins to define the molecular organization in the region of the membrane that comprises the functional domain of the Na:K pump. The membrane-bound phosphoglycerate kinase (PGK) and Na, K-ATPase appear to be directly linked via a compartmentalized form of ATP. Evidence for the membrane pool of ATP is based on the labeling characteristics of the phosphoproteins by [γ-(32)P]ATP of ghosts incubated under various conditions. Preincubation of ghosts in the presence of ATP at 37 degrees C, but not at 0 degrees C, completely obscures the formation of the Na-phosphoprotein in ghosts washed and subsequently incubated in the presence of [gamma-(32)P]ATP. In contrast to the Na component, the Mg component of phosphorylation is only slightly altered by preincubation with ATP. ATPase activity measured as (32)P(i) liberated during the subsequent incubation at 0 degrees C, reflects completely the differential effects of preincubation with ATP on (32)P incorporation into phosphoprotein. ATP placed within the pool by preincubation can be removed by operating the Na, K-ATPase or the PGK reaction in the reverse direction by use of exogenous substrates. Alternatively, the membrane pool of ATP can be formed also from exogenous substrates by running the PGK reaction in the forward direction. These results, while providing direct support for a membrane compartment of ATP, also indicate the location of this compartment in relation to the PGK and the Na, K-ATPase. In addition, these results also imply that the Mg and Na components are different enzymatic entities since substrate ATP can be derived from separate sources.     

Cyclic AMP-induced cytolysis in S49 cells: selection of an unresponsive "deathless" mutant.

Wild-type S49 lymphoma cells respond to cyclic adenosine 3', 5'-monophosphate (cAMP) by inducing cAMP phosphodiesterase, halting growth in the G1 phase of the cell cycle and subsequently dying. By using a counter selection procedure, we have isolated a new class of mutants of S49 cells termed "deathless" that are resistant to cytolysis, but otherwise respond like the wild-type cells to cAMP. Upon removal of the cyclic nucleotide, D-cells resume their normal growth. Unlike all other cAMP-resistant mutants of S49 cells isolated until now, the D- mutant has a functionally normal cAMP-dependent protein kinase and retains normal ability to induce phosphodiesterase and arrest cell growth in G1. It is probable that the altered gene product of the D- mutant is distal to protein kinase and in a biochemical pathway separate from that of cAMP induction of phosphodiesterase or growth arrest. The D- mutant may facilitate studies of the mechanism of cAMP-induced cytolysis and growth regulation in S49 cells.     

Differential cytostatic effects of NO donors and NO producing cells.

A 3-h exposure to NO donors (spermine-NO, DETA-NO, or SNAP), or to NOS II-expressing cells (activated macrophages or EMT6 cells) reversibly inhibited DNA synthesis in K562 tumor cells. In GSH-depleted K562 cells, cytostasis remained reversible when induced by DETA-NO or NOS II activity, but became irreversible after exposure to spermine-NO or SNAP. Only SNAP and spermine-NO efficiently inhibited GAPDH, an enzyme with a critical thiol, in GSH-depleted cells. Thus, the irreversible cytostasis induced in GSH-depleted cells by spermine-NO or SNAP can be tentatively attributed to S-nitrosating or oxidizing species derived from NO. However, these species did not contribute significantly to the early antiproliferative effects of macrophages. Ribonucleotide reductase, a key enzyme in DNA synthesis. has been shown to be inhibited by NO. Supplementation of the medium with deoxyribonucleosides to bypass RNR inhibition restored DNA synthesis in target cells exposed to DETA-NO and NO-producing cells, but was inefficient for GSH-depleted cells previously submitted to spermine-NO or SNAP. These cells also exhibited a persistent depletion of the dATP pool. In conclusion, GSH depletion reveals striking qualitative differences in the nature of the toxic effectors released by various NO sources, questioning the significance of S-nitrosating or oxidizing nitrogen oxides in NOS II-dependent cytostasis.