Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.     

The importance of mosaic genes to trypanosome survival

African trypanosomes possess several elegant ways to evade the immune defenses o f mammalian hosts. They have an extensive repertoire o f genes for a variant surface antigen and recent data show that this finite repertoire can be further amplified by mosaic gene formation and point mutation, producing an almost limitless capacity to vary. The significance of these mechanisms of antigenic diversity to trypanosome biology and the parasite-host relationship in general are discussed here by Anthony Barbet and Sondra Kamper.     

Assay of hybrid ribonuclease using a membrane filter-immobilized synthetic hybrid: application to the human leukemic cell

A method for assaying hybrid ribonuclease has been devised which utilizes as substrate the synthetic hybrid [3H]polyriboadenylic acid [poly(rA)]:polydeoxythymidylic acid [poly(dT)] immobilized on the solid matrix of nitrocellulose filters. The hybridization on filter of [3H]poly(rA) to poly(dT) has been explored in terms of efficacy of the process and the response of the product to RNase H. A pulse of uv irradiation of poly(dT) while in dry state on the filter increased its firm binding to the filter in a concentration-dependent manner, resulting in a concomitant increase of the yield of hybrid formation. The filter-immobilized hybrid was 95% resistant to RNase A but sensitive to RNase H. When stored in toluene in the cold the hybrid maintained its stability for over 6 months, as judged by its resistance to RNase A. The method offers a number of advantages over assays that use solution hybrids as substrates and was readily applicable in the screening of leukemic patients, in the leukocytes of which it has demonstrated increased RNase H levels.     

Factors that drive the increasing use of FFPE tissue in basic and translational cancer research

The decision to use 10% neutral buffered formalin fixed, paraffin embedded (FFPE) archival pathology material may be dictated by the cancer research question or analytical technique, or may be governed by national ethical, legal and social implications (ELSI), biobank, and sample availability and access policy. Biobanked samples of common tumors are likely to be available, but not all samples will be annotated with treatment and outcomes data and this may limit their application. Tumors that are rare or very small exist mostly in FFPE pathology archives. Pathology departments worldwide contain millions of FFPE archival samples, but there are challenges to availability. Pathology departments lack resources for retrieving materials for research or for having pathologists select precise areas in paraffin blocks, a critical quality control step. When samples must be sourced from several pathology departments, different fixation and tissue processing approaches create variability in quality. Researchers must decide what sample quality and quality tolerance fit their specific purpose and whether sample enrichment is required. Recent publications report variable success with techniques modified to examine all common species of molecular targets in FFPE samples. Rigorous quality management may be particularly important in sample preparation for next generation sequencing and for optimizing the quality of extracted proteins for proteomics studies. Unpredictable failures, including unpublished ones, likely are related to pre-analytical factors, unstable molecular targets, biological and clinical sampling factors associated with specific tissue types or suboptimal quality management of pathology archives. Reproducible results depend on adherence to pre-analytical phase standards for molecular in vitro diagnostic analyses for DNA, RNA and in particular, extracted proteins. With continuing adaptations of techniques for application to FFPE, the potential to acquire much larger numbers of FFPE samples and the greater convenience of using FFPE in assays for precision medicine, the choice of material in the future will become increasingly biased toward FFPE samples from pathology archives. Recognition that FFPE samples may harbor greater variation in quality than frozen samples for several reasons, including variations in fixation and tissue processing, requires that FFPE results be validated provided a cohort of frozen tissue samples is available.     

AGE-DEPENDENT TOXIC PROPERTIES OF ACTINOMYCIN D AND X-RAYS IN CULTURED CHINESE HAMSTER CELLS

A comparative study was made of the toxic properties of actinomycin D and X-rays using synchronized populations of Chinese hamster cells cultured in vitro. X-irradiated cells are most resistant in the latter half of the DNA synthetic period (late S). While cells treated with actinomycin D appear to go through a survival maximum at the same age, they are most resistant after the completion of DNA synthesis; i.e. in G₂ (or G₂-mitosis). In spite of these differences, we found that actinomycin D damage in late S cells interacts with X-ray damage. Thus, a common locus for the site of actions of both agents is suggested which may be in or around the genome of a cell in view of the well-known DNA binding properties of actinomycin D.     

Nonrandom location of IS1 elements in the genomes of natural isolates of Escherichia coli

We have studied the spatial distribution of IS1 elements in the genomes of natural isolates comprising the ECOR reference collection of Escherichia coli. We find evidence for nonrandomness at three levels. Many pairs of IS1 elements are in much closer proximity (< 10 kb) than can be accounted for by chance. IS1 elements in close proximity were identified by long-range PCR amplification of the genomic sequence between them. Each amplified region was sequenced and its map location determined by database screening of DNA hybridization. Among the ECOR strains with at least two IS1 elements, 54% had one or more pairs of elements separated by < 10 kb. We propose that this type of clustering is a result of "local hopping," in which we assume that a significant proportion of tranposition events leads to the insertion of a daughter IS element in the vicinity of the parental element. A second level of nonrandomness is found in strains with a modest number of IS1 elements that are mapped through the use of inverse PCR to amplify flanking genomic sequences: in these strains, the insertion sites tend to be clustered over a smaller region of chromosome than would be expected by chance. A third level of nonrandomness is observed in the composite distribution of IS elements across strains: among 20 mapped IS1 elements, none were found in the region of 48-77 minutes, a significant gap. One region of the E. coli chromosome, at 98 min, had a cluster of IS1 elements in seven ECOR strains of diverse phylogenetic origin. We deduce from sequence analysis that this pattern of distribution is a result of initial insertion in the most recent common ancestor of these strains and therefore not a hot spot of insertion. Analysis using long- range PCR with primers for IS2 and IS3 also yielded pairs of elements in close proximity, suggesting that these elements may also occasionally transpose by local hopping.      

Effects of unilateral lesions of retrotrapezoid nucleus on breathing in awake rats

Akilesh, Manjapra R., Matthew Kamper, Aihua Li, and EugeneE. Nattie. Effects of unilateral lesions of retrotrapezoid nucleuson breathing in awake rats. J. Appl.Physiol. 82(2): 469-479, 1997.In anesthetizedrats, unilateral retrotrapezoid nucleus (RTN) lesions markedlydecreased baseline phrenic activity and the response toCO₂ (E. E. Nattie and A. Li.Respir. Physiol. 97: 63-77,1994). Here we evaluate the effects of such lesions on restingbreathing and on the response to hypercapnia and hypoxia inunanesthetized awake rats. We made unilateral injections [24 ± 7 (SE) nl] of ibotenic acid (IA; 50 mM), an excitatoryamino acid neurotoxin, in the RTN region(n = 7) located by stereotaxic coordinates and by field potentials induced by facial nervestimulation. Controls (n = 6) receivedRTN injections (80 ± 30 nl) of mock cerebrospinal fluid. A secondcontrol consisted of four animals with IA injections (24 ± 12 nl)outside the RTN region. Injected fluorescent beads allowed anatomicidentification of lesion location. Using whole body plethysmography, wemeasured ventilation in the awake state during room air, 7%CO₂ in air, and 10%O₂ breathing before and for 3 wkafter the RTN injections. There was no statistically significant effectof the IA injections on resting room air breathing in the lesion groupcompared with the control groups. We observed no apnea. The response to7% CO₂ in the lesion groupcompared with the control groups was significantly decreased, by 39%on average, for the final portion of the 3-wk study period. There wasno lesion effect on the ventilatory response to 10%O₂. In this unanesthetized model,other areas suppressed by anesthesia, e.g., the reticular activatingsystem, hypothalamus, and perhaps the contralateral RTN, may providetonic input to the respiratory centers that counters the loss of RTNactivity.

     

Dialysis-Requiring Acute Kidney Injury in Denmark 2000-2012: Time Trends of Incidence and Prevalence of Risk Factors—A Nationwide Study

Introduction

Dialysis-requiring acute kidney injury is a severe illness associated with poor prognosis. However, information pertaining to incidence rates and prevalence of risk factors remains limited in spite of increasing focus. We evaluate time trends of incidence rates and changing patterns in prevalence of comorbidities, concurrent medication, and other risk factors in nationwide retrospective cohort study.

Materials and Methods

All patients with dialysis-requiring acute kidney injury were identified between January 1^st 2000 and December 31^st 2012. By cross-referencing data from national administrative registries, the association of changing patterns in dialysis treatment, comorbidity, concurrent medication and demographics with incidence of dialysis-requiring acute kidney injury was evaluated.

Results

A total of 18,561 adult patients with dialysis-requiring AKI were identified between 2000 and 2012. Crude incidence rate of dialysis-requiring AKI increased from 143 per million (95% confidence interval, 137–144) in 2000 to 366 per million (357–375) in 2006, and remained stable hereafter. Notably, incidence of continuous veno-venous hemodialysis (CRRT) and use of acute renal replacement therapy in elderly >75 years increased substantially from 23 per million (20–26) and 328 per million (300–355) in 2000, to 213 per million (206–220) and 1124 per million (1076–1172) in 2012, respectively. Simultaneously, patient characteristics and demographics shifted towards increased age and comorbidity.

Conclusions

Although growth in crude incidence rate of dialysis-requiring AKI stabilized in 2006, continuous growth in use of CRRT, and acute renal replacement therapy of elderly patients >75 years, was observed. Our results indicate an underlying shift in clinical paradigm, as opposed to unadulterated growth in incidence of dialysis-requiring AKI.