Developmental commitment in Dictyostelium discoideum

Upon starvation, Dictyostelium discoideum cells halt cell proliferation, aggregate into multicellular organisms, form migrating slugs, and undergo morphogenesis into fruiting bodies while differentiating into dormant spores and dead stalk cells. At almost any developmental stage cells can be forced to dedifferentiate when they are dispersed and diluted into nutrient broth. However, migrating slugs can traverse lawns of bacteria for days without dedifferentiating, ignoring abundant nutrients and continuing development. We now show that developing Dictyostelium cells revert to the growth phase only when bacteria are supplied during the first 4 to 6 h of development but that after this time, cells continue to develop regardless of the presence of food. We postulate that the cells' inability to revert to the growth phase after 6 h represents a commitment to development. We show that the onset of commitment correlates with the cells' loss of phagocytic function. By examining mutant strains, we also show that commitment requires extracellular cyclic AMP (cAMP) signaling. Moreover, cAMP pulses are sufficient to induce both commitment and the loss of phagocytosis in starving cells, whereas starvation alone is insufficient. Finally, we show that the inhibition of development by food prior to commitment is independent of contact between the cells and the bacteria and that small soluble molecules, probably amino acids, inhibit development during the first few hours and subsequently the cells become unable to react to the molecules and commit to development. We propose that commitment serves as a checkpoint that ensures the completion of cooperative aggregation of developing Dictyostelium cells once it has begun, dampening the response to nutritional cues that might inappropriately block development.     

Habitat occupancy of sloth bear Melursus ursinus in Chitwan National Park,Nepal

Mammals have experienced a massive decline in their populations and geographic ranges worldwide. The sloth bear, Melursus ursinus (Shaw, 1791), is one of many species facing conservation threats. Despite being endangered in Nepal, decades of inattention to the situation have hindered their conservation and management. We assessed the distribution and patterns of habitat use by sloth bears in Chitwan National Park (CNP), Nepal. We conducted sign surveys from March to June, 2020, in 4 × 4 km grids (n = 45). We collected detection/non‐detection data along a 4‐km trail that was divided into 20 continuous segments of 200 m each. We obtained environmental, ecological, and anthropogenic covariates to understand determinants of sloth bear habitat occupancy. The data were analyzed using the single‐species single‐season occupancy method, with a spatially correlated detection. Using repeated observations, these models accounted for the imperfect detectability of the species to provide robust estimates of habitat occupancy. The model‐averaged occupancy estimate for the sloth bear was 69% and the detection probability was 0.25. The probability of habitat occupancy by sloth bears increased with the presence of termites and fruits and in rugged, dry, open, undisturbed habitats. Our results indicate that the sloth bear is elusive, functionally unique, and widespread in CNP. Future conservation interventions and action plans aimed at sloth bear management must adequately consider their habitat requirements.     

Akt and Ca2+ signaling in endothelial cells

The protein kinase Akt participates in such important functions of endothelial cells as nitric oxide production and angiogenesis, activities that involve changes in cytosolic Ca2+ concentration. However, it is not known if activation of Akt is itself involved in the regulation of Ca2+ signals produced in these cells. The objective of this study was to examine if Akt is involved in the regulation of Ca2+ signaling in endothelial cells. Agonist-stimulated Ca2+ signals, assessed using fura-2, were compared in porcine aortic endothelial cells under control conditions or conditions in which Akt was blocked either by different inhibitors of phosphatidylinositol 3-kinase (PI3 kinase)/Akt or by transient expression of a dominant-negative form of Akt (dnAkt). We found that the release of intracellular Ca2+ stores stimulated by bradykinin or thapsigargin is not affected by the PI3 kinase inhibitors LY294002 and wortmannin, or by expression of dnAkt. LY294002 dose-dependently inhibits store-operated Ca2+ entry, an effect not seen with wortmannin. Expression of dnAkt has no effect on store-operated Ca2+ entry. We conclude that Akt is not involved in the regulation of agonist-stimulated Ca2+ signals in endothelial cells. The compound LY294002 inhibits store-operated Ca2+ entry in these cells by a mechanism independent of PI3 kinase/Akt inhibition.     

Purification and characterization of a co(II)-sensitive alpha-mannosidase from Ginkgo biloba seeds

An alpha-mannosidase was purified from developing Ginkgo biloba seeds to apparently homogeneity. The molecular weight of the purified alpha-mannosidase was estimated to be 120 kDa by SDS-PAGE in the presence of 2-mercaptoethanol, and 340 kDa by gel filtration, indicating that Ginkgo alpha-mannosidase may function in oligomeric structures in the plant cell. The N-terminal amino acid sequence of the purified enzyme was Ala-Phe-Met-Lys-Tyr-X-Thr-Thr-Gly-Gly-Pro-Val-Ala-Gly-Lys-Ile-Asn-Val-His-Leu-. The alpha-mannosidase activity for Man(5)GlcNAc(1) was enhanced by the addition of Co(2+), but the addition of Zn(2+), Ca(2+), or EDTA did not show any significant effect. In the presence of cobalt ions, the hydrolysis rate for pyridylaminated Man(6)GlcNAc(1) was significantly faster than that for pyridylaminated Man(6)GlcNAc(2), suggesting the possibility that this enzyme is involved in the degradation of free N-glycans occurring in developing plant cells (Kimura, Y., and Matsuo, S., J. Biochem., 127, 1013-1019 (2000)). To our knowledge, this is the first report showing that plant cells contain an alpha-mannosidase, which is activated by Co(2+) and prefers the oligomannose type free N-glycans bearing only one GlcNAc residue as substrate.     

Regeneration and maturation of daughter cell walls in the autospore-forming green alga<Emphasis Type="Italic"> Chlorella vulgaris</Emphasis> (Chlorophyta,Trebouxiophyceae)

Cell-wall synthesis in Chlorella vulgaris, an autospore-forming alga, was observed using the cell wall-specific fluorescent dye Fluostain I. The observation suggested two clearly distinguishable stages in cell-wall synthesis: moderate synthesis during the cell-growth process and rapid synthesis at the cell-division stage. We used electron microscopy to examine the structural changes that occurred with growth in the premature daughter cell wall during the cell-growth and cell-division phases. The cell began to synthesize a new daughter cell wall shortly after its release from the autosporangium. A very thin daughter cell wall, with a thickness of about 2 nm, was formed inside the mother cell wall and completely enveloped the outer surface of the plasma membrane of the cell. The daughter cell wall gradually increased in thickness from 2 to 3.8 nm. During the protoplast-division phase in the cell-division stage, the daughter cell wall expanded on the surface of the invaginating plasma membrane of the cleavage furrow, accompanied by active synthesis of the cell wall, which increased in thickness from 3.8 to 6.1 nm. The daughter cell matured into an autospore while completely enclosed by its own thickening (from 6.1 to 17 nm) wall. Finally, the released daughter cell was enclosed by its own cell wall after the mother cell wall burst. The daughter cell with mature wall thickness (17–21 nm) emerged as a small, but complete, autospore.     

Decreased mRNA expression of the PTH/PTHrP receptor and type II sodium-dependent phosphate transporter in the kidney of rats fed a high phosphorus diet accompanied with a decrease in serum calcium concentration

This study investigates the phosphorus (P) homeostasis in the process of an altered parathyroid hormone (PTH) action in the kidney of rats fed a high P diet. Four-week-old male Wistar strain rats were fed diets containing five different P levels (0.3, 0.6, 0.9, 1.2 and 1.5%) for 21 days. The serum PTH concentration and urinary excretion of P were elevated with increasing dietary P level. Compared to rats fed the 0.3% P diet, the serum calcium (Ca) concentration remained unchanged, while the serum 1,25(OH)(2)D(3) concentration and urinary excretion of cAMP were elevated with increasing dietary P level in rats fed the high P diets containing 0.6-0.9% P. On the other hand, a lower serum Ca concentration was observed in rats fed the high P diets containing 1.2% or greater P. The serum 1,25(OH)(2)D(3) concentration remained unchanged in rats fed the high P diets containing 1.2% or greater P, comparison with rats fed the 0.3% P diet. The urinary excretion of cAMP and PTH/PTH-related peptide (PTHrP) receptor and type II sodium-dependent phosphate transporter (NaPi-2) mRNA in the kidney were both decreased in rats fed the high P diets containing 1.2% or greater P. In conclusion, a high P diet with subsequent decrease in serum Ca concentration suppressed the PTH action in the kidney due to PTH/PTHrP receptor mRNA down-regulation. Furthermore, an increase in the urinary excretion of P might have been caused by decreased NaPi-2 mRNA expression without the effects of PTH and 1,25(OH)(2)D(3).