Differences among immune complexes: association of C1q in SLE immune complexes with renal disease

Studies that made use of multiple assay systems demonstrated increased levels of immune complexes (IC) in patients with systemic lupus erythematosus (SLE), but no consistent correlations of IC concentration to patterns or activity of disease have been observed. Furthermore, consistent associations between qualitative differences in IC and disease manifestations have been elusive. IC interaction with erythrocytes and mononuclear phagocytic cells is another variable in SLE that may also mediate some of the biological effects of IC. The present report concerns studies of the composition of purified IC obtained from individuals with SLE and other rheumatic diseases; a 64,000 dalton component identified as the A-B subunit of C1q was detected in purified IC from 27 of 51 SLE patients (53%). The presence of this 64,000 dalton component was not related to either IC concentration or to the serum C1q level. However, the presence of the C1q component in isolated SLE IC did correlate with the presence of renal disease (p less than 0.02). These observations are interpreted relative to a recently described kinetic model of IC clearance.     

Detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts,ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20–24 dyn/cm² were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells’ ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor-derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell’s ability to metastasize.     

Inhibition of the mitochondrial Ca2+ uniporter by pure and impure ruthenium red

Commercial ruthenium red is often purified by a single recrystallization as described by Luft, J.H. (1971) Anat Rec 171, 347–368, which yields small amounts of material having an apparent molar extinction coefficient of 67,400 at 533 nm. A simple modification to the procedure dramatically improves the yield, allowing crystallization to be repeated. Three times recrystallized ruthenium red has an apparent extinction coefficient of 85,900, the highest value reported to date. Both crude and highly purified ruthenium red can be shown to inhibit reverse activity of the mitochondrial Ca²⁺ uniporter (uncoupled mitochondria), provided that care is taken to minimize and account for Ca²⁺ release through the permeability transition pore. Crude ruthenium red is 7–10 fold more potent than the highly purified material in this regard, on an actual ruthenium red concentration basis. The same relative potency is seen against forward uniport (coupled mitochondria), however, the I₅₀ values are 10 fold lower for both the crude and purified preparations. These data demonstrate unambiguously that the energy state of mitochondria affects the sensitivity of the Ca²⁺ uniporter to ruthenium red preparations, and that both the forward and reverse reactions are subject to complete inhibition. The data suggest, however, that the active inhibitor may not be ruthenium redper se, but one or more of the other ruthenium complexes which are present in ruthenium red preparations.Abbreviations CCP carbonyl cyanide p-chlorophenylhydrazone - CSA cyclosporin A - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid     

Reduction in animal abundance and oxygen availability during and after the end-Triassic mass extinction

The end-Triassic biodiversity crisis was one of the most severe mass extinctions in the history of animal life. However, the extent to which the loss of taxonomic diversity was coupled with a reduction in organismal abundance remains to be quantified. Further, the temporal relationship between organismal abundance and local marine redox conditions is lacking in carbonate sections. To address these questions, we measured skeletal grain abundance in shallow-marine limestones by point counting 293 thin sections from four stratigraphic sections across the Triassic/Jurassic boundary in the Lombardy Basin and Apennine Platform of western Tethys. Skeletal abundance decreased abruptly across the Triassic/Jurassic boundary in all stratigraphic sections. The abundance of skeletal organisms remained low throughout the lower-middle Hettangian strata and began to rebound during the late Hettangian and early Sinemurian. A two-way ANOVA indicates that sample age (p < .01, η² = 0.30) explains more of the variation in skeletal abundance than the depositional environment or paleobathymetry (p < .01, η² = 0.15). Measured I/Ca ratios, a proxy for local shallow-marine redox conditions, show this same pattern with the lowest I/Ca ratios occurring in the early Hettangian. The close correspondence between oceanic water column oxygen levels and skeletal abundance indicates a connection between redox conditions and benthic organismal abundance across the Triassic/Jurassic boundary. These findings indicate that the end-Triassic mass extinction reduced not only the biodiversity but also the carrying capacity for skeletal organisms in early Hettangian ecosystems, adding to evidence that mass extinction of species generally leads to mass rarity among survivors.     

Fc gamma receptor III induces actin polymerization in human neutrophils and primes phagocytosis mediated by Fc gamma receptor II.

Human polymorphonuclear leukocytes (PMN) express two classes of Fc gamma R: Fc gamma RII the 42-kDa receptor with a traditional membrane spanning domain and cytoplasmic tail and Fc gamma RIIIPMN the 50- to 80-kDa receptor with a glycosyl-phatidylinositol membrane anchor expressed on PMN. To explore the capacity of Fc gamma RIIIPMN to generate intracellular signals, we have analyzed the ability of Fab and F(ab')2 anti-Fc gamma R mAb to induce actin filament assembly, a prerequisite for motile behaviors. Multivalent ligation of Fc gamma RIIIPMN, independent of Fc gamma RII, results in an increase in F-actin content that is [Ca2+]i dependent. Multivalent ligation of Fc gamma RII also initiates actin polymerization but uses a [Ca2+]i-independent initial pathway. In addition to providing a mechanism for Fc gamma RIIIPMN triggered effector functions, the increase in F-actin and [Ca2+]i generated by Fc gamma RIIIPMN ligation also serves as a "priming" signal to modify PMN responses to other stimuli. Experiments using erythrocytes specifically coated with anti-Fc gamma RII Fab demonstrate that cross-linking of Fc gamma RIIIPMN with anti-Fc gamma RIII F(ab')2 enhances phagocytosis mediated by Fc gamma RII. Thus, Fc gamma RIIIPMN, a glycosyl-phosphatidylinositol anchored protein, may contribute directly to an intracellular program of actin assembly that may trigger and prime neutrophil effector functions.     

Effects of excess selenomethionine on selenium status indicators in pregnant long-tailed macaques (Macaca fascicularis)

Forty pregnant long-tailed macaques were treated daily for 30 d with 0, 25, 150 or 300 μg selenium as L-selenomethionine/kg body weight. Erythrocyte and plasma selenium and glutathione peroxidase specific activities, hair and fecal selenium, and urinary selenium excretion were increased by and were linearly related to L-selenomethionine dose. Hair selenium was most sensitive to L-selenomethionine dose, with an 84-fold increase in the 300 μg selenium/(kg-d) group relative to controls (r=0.917). Daily urinary selenium excretion (80-fold,r=0.958), plasma selenium (22-fold,r=0.885), erythrocyte selenium (24-fold,r=0.920), and fecal selenium (18-fold,r=0.911) also responded strongly to L-selenomethionine. Erythrocyte and plasma glutathione peroxidase specific activities increased 154% and 69% over controls, respectively. Toxicity was associated with erythrocyte selenium >2.3 μg/mL, plasma selenium >2.8 μg/mL, and hair selenium >27 μg/g. Plasma, erythrocyte, and hair selenium concentrations may be useful for monitoring and preventing the toxicity of L-selenomethionine administered to humans in cancer chemoprevention trials.     

The accuracy of absolute differential abundance analysis from relative count data

Concerns have been raised about the use of relative abundance data derived from next generation sequencing as a proxy for absolute abundances. For example, in the differential abundance setting, compositional effects in relative abundance data may give rise to spurious differences (false positives) when considered from the absolute perspective. In practice however, relative abundances are often transformed by renormalization strategies intended to compensate for these effects and the scope of the practical problem remains unclear. We used simulated data to explore the consistency of differential abundance calling on renormalized relative abundances versus absolute abundances and find that, while overall consistency is high, with a median sensitivity (true positive rates) of 0.91 and specificity (1—false positive rates) of 0.89, consistency can be much lower where there is widespread change in the abundance of features across conditions. We confirm these findings on a large number of real data sets drawn from 16S metabarcoding, expression array, bulk RNA-seq, and single-cell RNA-seq experiments, where data sets with the greatest change between experimental conditions are also those with the highest false positive rates. Finally, we evaluate the predictive utility of summary features of relative abundance data themselves. Estimates of sparsity and the prevalence of feature-level change in relative abundance data give reasonable predictions of discrepancy in differential abundance calling in simulated data and can provide useful bounds for worst-case outcomes in real data.