Isolation of Temperature-Sensitive Membrane Mutants of Escherichia coli K-12

Mutants with impaired biosynthesis of unsaturated fatty acids or altered metabolism of the phospholipids were isolated at a rather high frequency from a set of temperature-sensitive lysis mutants. It is suggested that preselection for the lysis phenotype makes it possible to isolate several kinds of mutants affected in the integrity of the cytoplasmic membrane.     

Platelet-neutrophil interactions. (12S)-hydroxyeicosatetraen-1,20-dioic acid: a new eicosanoid synthesized by unstimulated neutrophils from (12S)-20-dihydroxyeicosatetraenoic acid

In the course of a cell-cell interaction, 12-HETE (12-hydroxy-5,8,10,14-eicosatetraenoic acid), the arachidonic acid lipoxygenase product released from stimulated platelets, is metabolized by a cytochrome P-450 enzyme system in unstimulated neutrophils to 12,20-DiHETE (12,20-dihydroxy-5,8,10,14-eicosatetraenoic acid). This report describes time-dependent formation of a new eicosanoid by unstimulated neutrophils exposed to 12-HETE, which is more polar than 12,20-DiHETE (reversed-phase high performance liquid chromatography). Time course studies indicated that the precursor compound of this new eicosanoid was 12,20-DiHETE. This was determined by incubation of purified 12,20-DiHETE with neutrophils, which resulted in a progressive decrease in 12,20-DiHETE as formation of the polar metabolite increased. In the absence of neutrophils, 12,20-DiHETE was quantitatively unchanged. The new metabolite of 12,20-DiHETE was identified as 12-hydroxyeicosatetraen-1,20-dioic acid, based upon its UV spectrum, co-chromatography with a chemically synthesized standard in both high performance liquid chromatography and thin layer chromatography systems, and gas chromatography-mass spectrometry. Formation of 12-HETE-1,20-dioic acid was partially inhibited by 20-hydroxy-LTB4. This indicated that the neutrophil dehydrogenase responsible for further metabolism of 12,20-DiHETE may also be involved in conversion of 20-hydroxy-LTB4 to 20-carboxy-LTB4. The 12,20-DiHETE dehydrogenase enzyme system specifically requires NAD as cofactor and has subcellular components in both cytosolic and microsomal fractions which are synergistic in their activity. These results provide additional evidence for the occurrence of multicellular metabolic events during hemostasis, thrombosis, and the inflammatory response.     

Phospholipid metabolism in human neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine. Degranulation is not required for release of arachidonic acid: studies with neutrophils and neutrophil-derived cytoplasts.

Neutrophils respond to chemoattractants by aggregating, degranulating, remodelling of phospholipids and releasing arachidonic acid. To determine whether ligand-induced remodelling of phospholipids depends on redistribution of intracellular organelles (degranulation), we compared phospholipid remodelling of human neutrophils with that of neutrophil-derived cytoplasts. Cytoplasts, organelle-depleted vesicles of cytosol surrounded by plasmalemma, cannot degranulate. Without a stimulus, [3H]arachidonate was incorporated preferentially into phosphatidylinositol (PI) and phosphatidylcholine (PC). Exposure of cytoplasts and neutrophils prelabelled with [3H]arachidonate or [14C]glycerol to fMet-Leu-Phe (10(-7) M) induced rapid changes in distribution of label and mass of individual phospholipids: [3H]arachidonate in phosphatidic acid (PA) increased 500% (120 s), [14C]glycerol incorporation and mass of PA approached 200% of unstimulated values, and [3H]arachidonate in PI decreased continuously; these data are compatible with activity of a PI/PA cycle. However, the mass of PI in both preparations and [14C]glycerol label in intact neutrophils increased initially (5 s), suggesting net synthesis and mobilization of more than one pool of PI. Heterogeneity of PC pools was also observed: [3H]arachidonate was lost from PC immediately upon addition of stimulus, whereas mass and [14C]glycerol values increased. Thus, net phospholipid synthesis, redistribution of arachidonate and activation of the PI/PA cycle are immediate responses of the neutrophil to receptor occupancy by chemoattractants. Furthermore, the similarity in response to fMet-Leu-Phe of neutrophils and granule-free cytoplasts indicates that these processes are independent of degranulation.     

Effect of the Osmotic Pressure of the Growth Medium on fabB Mutants of Escherichia coli

Temperature-sensitive, unsaturated fatty acid (fabB) auxotrophs of Escherichia coli can grow at the restrictive temperature in the absence of unsaturated fatty acid in a medium with a high osmotic pressure. If a mutant culture was starved for unsaturated fatty acids and harvested just before the lysis started, the fatty acid composition of the cells was the same as that of cells grown until late log phase in a high-osmotic medium. Evidence is presented that the in vivo unsaturated fatty acid biosynthesis is significantly increased in a high osmotic medium. The increase is probably due to a partial activation of the temperature-sensitive fabB product. Besides the stimulation of the temperature-sensitive fabB product, a minimal osmotic pressure of the medium appeared to be necessary to allow growth of cells containing lipids with a changed fatty acid composition. fabA mutants are unable to grow in a high-osmotic medium in the absence of unsaturated fatty acids. No increase in the in vivo unsaturated fatty acid biosynthesis could be detected in the temperature-sensitive fabA mutants.     

HomeRange: A global database of mammalian home ranges

Motivation

Home range is a common measure of use of space by animals because it provides ecological information that is useful for conservation applications. In macroecological studies, values are typically aggregated to species means to examine general patterns of use of space by animals. However, this ignores the environmental context in which the home range was estimated and does not account for intraspecific variation in home range size. In addition, the focus of macroecological studies on home ranges has historically been biased towards terrestrial mammals. The use of aggregated numbers and the terrestrial focus limit our ability to examine home-range patterns across different environments, their variation in time and variation between different levels of organization. Here, we introduce HomeRange, a global database with 75,611 home-range values across 960 different species of mammals, including terrestrial, aquatic and aerial species.

Main types of variables contained

The dataset contains estimates of home ranges of mammals, species names, methodological information on data collection, method of home-range estimation, period of data collection, study coordinates and name of location, in addition to species traits derived from the studies, such as body mass, life stage, reproductive status and locomotor habit.

Spatial location and grain

The collected data are distributed globally. Across studies, the spatial accuracy varies, with the coarsest resolution being 1°.

Time period and grain

The data represent information published between 1939 and 2022. Across studies, the temporal accuracy varies; some studies report start and end dates specific to the day, whereas for other studies only the month or year is reported.

Major taxa and level of measurement

Mammalian species from 24 of the 27 different taxonomic orders. Home-range estimates range from individual-level values to population-level averages.

Software format

Data are supplied as a comma-delimited text file (.csv) and can be loaded directly into R using the “HomeRange” R package ( https://github.com/SHoeks/HomeRange ).     

Intratumor heterogeneity of biomarker expression in breast carcinomas

Small biopsy samples are used increasingly to assess the biomarker expression for prognostic information and for monitoring therapeutic responses prior to and during neoadjuvant therapy. The issue of intratumor heterogeneity of expression of biomarkers, however, has raised questions about the validity of the assessment of biomarker expression based on limited tissue samples. We examined immunohistochemically the expression of HER-2neu (p185^erbB-2), epidermal growth factor receptor (EGFR), Bcl-2, p53, and proliferating cell nuclear antigen (PCNA) in 30 breast carcinomas using archived, paraffin embedded tissue and determined the extent of intratumor heterogeneity. Each section was divided into four randomly oriented discrete regions, each containing a portion of the infiltrating carcinoma. For each tumor, the entire lesion and four regions were analyzed for the expression of these markers. Scores of both membrane and cytoplasmic staining of HER-2neu and EGFR, scores of cytoplasmic staining of Bcl-2, and scores of nuclear staining of both p53 and PCNA were recorded. The intensity of staining and the proportion of immunostained cells were determined. A semiquantitative immunoscore was calculated by determining the sum of the products of the intensity and corresponding proportion of stained tumor cells. We analyzed both invasive (IDC) and in situ (DCIS) carcinomas. The Wilcoxon signed-rank test was used for paired comparisons between overall and regional immunoscores and between overall and regional percentages of stained cells. Spearman's correlation coefficients were used to assess the level of agreement of overall biomarker expression with each of the regions. Generalized linear models were used to assess overall and pair-wise differences in the absolute values of percent changes between overall and regional expression of biomarkers. For IDCs, there were no statistically significant differences in the expression of the biomarkers in terms of either the percentage of cells staining or the immunoscores when comparing the entire tumor with each region except for the lower EGFR expression of arbitrarily selected region 1 and lower p53 expression of region 1 compared to that of the entire tumor section. For DCIS, there were no statistically significant differences in the expression of the biomarkers between the entire tumor and each region except in PCNA of region 2 compared to that of entire tumor section. Positive correlation of immunoscores was observed between the entire tumor and each region as well as across all four regions for IDC. Similar observations were noted with DCIS except for HER-2neu and PCNA. No statistically significant differences were observed in the absolute values of percent changes of biomarker expression between overall and the four regions for both DCIS and IDC. Therefore, no significant intratumor heterogeneity in the expression of HER-2neu, Bcl-2, and PCNA was observed in IDC. Minor regional variations were observed for EGFR and p53 in IDC. Similarly, no significant regional variation in the expression of markers was observed in DCIS except for PCNA.     

Signs of stabilisation and stable coexistence

Many empirical studies motivated by an interest in stable coexistence have quantified negative density dependence, negative frequency dependence, or negative plant–soil feedback, but the links between these empirical results and ecological theory are not straightforward. Here, we relate these analyses to theoretical conditions for stabilisation and stable coexistence in classical competition models. By stabilisation, we mean an excess of intraspecific competition relative to interspecific competition that inherently slows or even prevents competitive exclusion. We show that most, though not all, tests demonstrating negative density dependence, negative frequency dependence, and negative plant–soil feedback constitute sufficient conditions for stabilisation of two‐species interactions if applied to data for per capita population growth rates of pairs of species, but none are necessary or sufficient conditions for stable coexistence of two species. Potential inferences are even more limited when communities involve more than two species, and when performance is measured at a single life stage or vital rate. We then discuss two approaches that enable stronger tests for stable coexistence‐invasibility experiments and model parameterisation. The model parameterisation approach can be applied to typical density‐dependence, frequency‐dependence, and plant–soil feedback data sets, and generally enables better links with mechanisms and greater insights, as demonstrated by recent studies.     

Site-directed mutagenesis of human endothelial cell ecto-ADPase/soluble CD39: requirement of glutamate 174 and serine 218 for enzyme activity and inhibition of platelet recruitment

Endothelial cell CD39/ecto-ADPase plays a major role in vascular homeostasis. It rapidly metabolizes ADP released from stimulated platelets, thereby preventing further platelet activation and recruitment. We recently developed a recombinant, soluble form of human CD39, solCD39, with enzymatic and biological properties identical to CD39. To identify amino acids essential for enzymatic/biological activity, we performed site-directed mutagenesis within the four highly conserved apyrase regions of solCD39. Mutation of glutamate 174 to alanine (E174A) and serine 218 to alanine (S218A) resulted in complete and approximately 90% loss of solCD39 enzymatic activity, respectively. Furthermore, compared to wild-type, S57A exhibited a 2-fold increase in ADPase activity without change in ATPase activity, while the tyrosine 127 to alanine (Y127A) mutant lost 50-60% of both ADPase and ATPase activity. The ADPase activity of wild-type solCD39 and each mutant, except for R135A, was greater with calcium as the required divalent cation than with magnesium, but for ATPase activity generally no such preference was observed. Y127A demonstrated the highest calcium/magnesium ADPase activity ratio, 2.8-fold higher than that of wild-type, even though its enzyme activity was greatly reduced. SolCD39 mutants were further characterized by correlating enzymatic with biological activity in an in vitro platelet aggregation system. Each solCD39 mutant was similar to wild-type in reversing platelet aggregation, except for E174A and S218A. E174A, completely devoid of enzymatic activity, failed to inhibit platelet responsiveness, as anticipated. S218A, with 91% loss of ADPase activity, could still reverse platelet aggregation, albeit much less effectively than wild-type solCD39. Thus, glutamate 174 and serine 218 are essential for both the enzymatic and biological activity of solCD39.     

Polymerase chain reaction detection of Leishmania DNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.