IL-4 inhibits the costimulatory activity of IL-2 or picolinic acid but not of lipopolysaccharide on IFN-gamma-treated macrophages

We reported previously that IL-2 induces tumoricidal activity in IFN-gamma-treated murine macrophages. The present study was performed to investigate the regulation of IL-2-dependent tumoricidal activity in murine macrophage cell lines. The v-raf/v-myc-immortalized murine macrophage cell lines ANA-1, GG2EE, and HEN-CV did not express constitutive levels of cytotoxic activity against P815 mastocytoma cells. Moreover, these macrophage cell lines did not become tumoricidal after exposure to IL-4, IFN-gamma, IL-2 or LPS. However, these macrophages developed cytotoxic capabilities after incubation with either IFN-gamma plus IL-2 or IFN-gamma plus LPS. IL-4 inhibited IFN-gamma plus IL-2- but not IFN-gamma plus LPS-induced tumoricidal activity. This effect of IL-4 was not restricted to v-raf/v-myc-immortalized macrophage cell lines because similar results were obtained by using a macrophage cell line that was established from a spontaneous histiocytic sarcoma. The suppressive activity of IL-4 on the ANA-1 macrophage cell line was dose-dependent (approximately 12-200 U/ml) and was neutralized by the addition of anti-IL-4 mAb. IL-4 decreased the IFN-gamma-induced expression of mRNA for the p55 (alpha) subunit of the IL-2R in ANA-1 macrophages. Therefore, at least one mechanism by which IL-4 may have inhibited IFN-gamma plus IL-2-induced tumoricidal activity was by reducing macrophage IL-2R alpha mRNA expression. We have previously reported that picolinic acid, a tryptophan metabolite, is a costimulator of macrophage tumoricidal activity. We now report that IL-4 also inhibited IFN-gamma plus picolinic acid-induced cytotoxicity in ANA-1 macrophages. We propose that IL-2 and picolinic acid may have a common mechanism of action that is susceptible to IL-4 suppression.     

Enterocytozoon salmonis n. sp.: an intranuclear microsporidium from salmonid fish

The developmental stages of a recently described microsporidian from the nucleus of hematopoietic cells of salmonid fish were found to be unique among the Microsporida. All observed stages, including meronts, sporonts, and spores were in direct contact with the host cell nucleus (principally hematopoietic cells) of chinook salmon (Oncorhynchus tshawytscha). There is no parasitophorous vacuole and sporogony does not involve formation of a pansporoblastic membrane as with other members of the suborder Apansporoblastina. The extrusion apparatus differentiates prior to division of sporogonial plasmodia. The spores are ovoid (1 x 2 microns) and uninucleate, and possess a coiled polar tube with 8-12 turns. Developmental stages of the salmonid microsporidian are similar to those described for Enterocytozoon bieneusi as found in the intestinal mucosa of human AIDS patients. However, the intranuclear development, different cell types, and host infected clearly separate the salmonid and human parasites. Accordingly, the intranuclear parasite of salmonids is given the name Enterocytozoon salmonis n. sp. within the suborder Apansporoblastina.     

Spatial and seasonal changes in the species composition of armored dinoflagellates in the Southwestern Atlantic Ocean

Summary To compare the spatial and temporal (seasonal) distribution of dinoflagellates, vertical net hauls were taken along similar cruise tracks in the Scotia Sea, Weddell Sea and across the Polar Front Zone in the austral spring and the austral fall. Sixty-three species of armored dinoflagellates were identified and enumerated. Chisquare and hierarchical cluster analyses were performed to define spatial and seasonal patterns in genera and species assemblages. The dominant genera were Protoperidinium, Dinophysis and Ceratium. The Polar Front Zone was an important biogeographical barrier with Blepharocysta, Gonyaulax, Heteroschisma, Oxytoxum and Podolampas occurring mainly north of the Front. Species found primarily in the austral spring were Ceratium fusus, Ceratium lineatum, Dinophysis antarctica, Dinophysis simplex, Gonyaulax digitale, Protoperidinium pyriforme and Protoperidinium variegatum. Austral fall species included Dinophysis tuberculata and Protoperidinium elegantissum. Distribution of armored dinoflagellates in the Southwestern Atlantic Ocean is influenced at the generic level by spatial considerations, particular with relation to the Polar Front Zone, whereas species composition can be effected by both region and season.     

Interspersed repetitive element polymerase chain reaction product mapping using a mouse interspecific backcross

We have developed a rapid method of generating and simultaneously mapping interrepeat polymerase chain reaction products using DNA from interspecific backcross animals derived from mating C57BL/6J and Mus spretus mice. This method is based on the high degree of B1, B2, and L1 dispersed repeat position polymorphism found between these two species of mouse. We have mapped 13 new loci to 9 different chromosomes and have found no evidence of clustering among these loci. The advantages of this approach are that no prior knowledge of sequence is required, a single PCR reaction generates many markers which can be mapped simultaneously, and only 50 ng of each backcross DNA (a finite resource) is required. We anticipate that many more markers remain to be characterized in this valuable new source of polymorphism.     

Computer-assisted assignment of 2D1H NMR spectra of proteins: Basic algorithms and application to phoratoxin B

Summary A suite of computer programs (CLAIRE) is described which can be of assistance in the process of assigning 2D¹H NMR spectra of proteins. The programs embody a software implementation of the sequential assignment approach first developed by Wüthrich and co-workers (K. Wüthrich. G. Wider, G. Wagner and W. Braun (1982)J. Mol. Biol. 155, 311). After data-abstraction (peakpicking), the software can be used to detect patterns (spin systems), to find cross peaks between patterns in 2D NOE data sets and to generate assignments that are consistent with all available data and which satisfy a number of constraints imposed by the user. An interactive graphics program calledCONPAT is used to control the entire assignment process as well as to provide the essential feedback from the experimental NMR spectra. The algorithms are described in detail and the approach is demonstrated on a set of spectra from the mistletoe protein phoratoxin B, a homolog of crambin. The results obtained compare well with those reported earlier based entirely on a manual assignment process.     

Differential expression of TGF beta 1, beta 2 and beta 3 genes during mouse embryogenesis

We have examined by Northern analysis and in situ hybridisation the expression of TGF beta 1, beta 2 and beta 3 during mouse embryogenesis. TGF beta 1 is expressed predominantly in the mesodermal components of the embryo e.g. the hematopoietic cells of both fetal liver and the hemopoietic islands of the yolk sac, the mesenchymal tissues of several internal organs and in ossifying bone tissues. The strongest TGF beta 2 signals were found in early facial mesenchyme and in some endodermal and ectodermal epithelial cell layers e.g., lung and cochlea epithelia. TGF beta 3 was strongest in prevertebral tissue, in some mesothelia and in lung epithelia. All three isoforms were expressed in bone tissues but showed distinct patterns of expression both spatially and temporally. In the root sheath of the whisker follicle, TGF beta 1, beta 2 and beta 3 were expressed simultaneously. We discuss the implication of these results in regard to known regulatory elements of the TGF beta genes and their receptors.     

Monomers,dimers, and tetramers of 4-n-propyl-5-ethyl farnesyl bacteriochlorophyll c in dichloromethane and carbon tetrachloride

Absorption (ABS) and circular dichroism (CD) spectra were recorded for 6 concentrations (2.0–290 M) of bacteriochlorophyll (BChl) c in each solvent. Monomer spectra were obtained by adding methanol (1:200) to each sample. The monomer showed an ABS peak and a CD trough at 664 nm in CH₂Cl₂ (ABS peak at 665 nm in CCl₄). Dimer-plus-monomer spectra were obtained by subtracting high concentration (e.g., 290 M) spectra appropriately scaled from lower concentration (e.g., 26 M) spectra. Pure dimer spectra were then obtained by subtracting monomer spectra appropriately scaled from dimer-plus-monomer spectra. The dimer showed an ABS peak at 679 nm in both CH₂Cl₂ and CCl₄ and a CD trough at ca. 670 nm in CH₂Cl₂. The optical properties of the dimer do not agree with the model for bacteriochlorophyllide d [Smith KM, Bobe FW, Goff DA and Abraham RJ (1986) J Am Chem Soc 108: 1111–1120]. Higher aggregate spectra were obtained by subtracting appropriately scaled monomer and dimer spectra from high concentration (e.g., 290 M) spectra. The aggregate showed ABS shoulders at ca. 636 and 678 nm with a peak at 702 nm in CH₂Cl₂ and at 708 nm in CCl₄; the CD spectrum in either solvent showed peaks at 638 and 679 nm with troughs at 658 and ca. 710 nm. These spectra are consistent with an excitonic interaction between 4 chromophores in the aggregate. Each of the 12 original ABS spectra was deconvoluted in terms of the appropriate monomer, dimer and aggregate spectra, and the concentrations of each component were determined. Plots of log aggregate concentration vs. log dimer concentration lay on or near a line of slope 1.9 for CH₂Cl₂ and on or near a line of slope 2.1 for CCl₄. The aggregate was thus shown to be a tetramer. The theoretical relationship between dimers and monomers (slope 2.0) was not observed in all cases.Abbreviations ABS absorbance - BChl bacteriochlorophyll - CD circular dichroism - Chl chlorophyll - DNS data not shown - PEF 4-n-propyl-5-ethyl farnesyl     

Precision of DNA flow cytometry in inter-institutional analyses

A Bladder Cancer Flow Cytometry Network study has been carried out to further identify and quantify sources of inter- and intra-laboratory variability. Replicate samples containing four mixtures of peripheral blood lymphocytes and aneuploid cell lines were distributed together with reference standards to six laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Two of each of the four sample types and a reference standard were analyzed by each laboratory on 3 separate days to obtain cellular DNA distributions. DNA index (DI) and hyperdiploid fraction (HDF) were calculated for each histogram using an automated technique. The results showed significant inter- and intra-laboratory differences. Results were evaluated by a two-way analysis of variance to estimate components of the overall variation attributable to individual sources. Error variation was found to be the major component of random variation. Specimen means were also compared for each laboratory. No significant differences were noted in mean DI for similar specimens; however, agreement in HDF between similar specimens was lacking in most laboratories. Prediction intervals were computed to estimate the range of values expected for a single specimen based on the analysis of the previous six. Prediction intervals for DI were quite good while those for HDF were troublesome due to wide variation. The results of these studies indicate that intra- and inter-laboratory variability are high enough that results for a single sample may not be sufficiently precise to allow comparison to results obtained in other laboratories.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of the dihydrolipoamide branched-chain transacylase gene (DBT) of the human branched-chain keto acid dehydrogenase complex to chromosome 1

The gene coding for the transacylase subunit (DBT) of the human branched-chain keto acid dehydrogenase complex was localized to chromosome 1 by probing panels of human x mouse chromosome hybrids with an E2 cDNA amplified by the polymerase chain reaction. Additional data with two hybrids containing chromosome 1 fragments suggest that the DBT gene is located on the short arm (1pter----p21) of the chromosome.