In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum‐infected red blood cells

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle‐like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake byP. falciparum‐infected erythrocytes shows that at R and S stages, a time‐increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time‐increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.     

The classical nuclear localization signal receptor, importin-alpha, is required for efficient transition through the G1/S stage of the cell cycle in Saccharomyces cerevisiae

There is significant evidence linking nucleocytoplasmic transport to cell cycle control. The budding yeast, Saccharomyces cerevisiae, serves as an ideal model system for studying transport events critical to cell cycle progression because the nuclear envelope remains intact throughout the cell cycle. Previous studies linked the classical nuclear localization signal (cNLS) receptor, importin-alpha/Srp1, to the G(2)/M transition of the cell cycle. Here, we utilize two engineered mutants of importin-alpha/Srp1 with specific molecular defects to explore how protein import affects cell cycle progression. One mutant, Srp1-E402Q, is defective in binding to cNLS cargoes that contain two clusters of basic residues termed a bipartite cNLS. The other mutant, Srp1-55, has defects in release of cNLS cargoes into the nucleus. Consistent with distinct in vivo functional consequences for each of the Srp1 mutants analyzed, we find that overexpression of different nuclear transport factors can suppress the temperature-sensitive growth defects of each mutant. Studies aimed at understanding how each of these mutants affects cell cycle progression reveal a profound defect at the G(1) to S phase transition in both srp1-E402Q and srp1-55 mutants as well as a modest G(1)/S defect in the temperature-sensitive srp1-31 mutant, which was previously implicated in G(2)/M. We take advantage of the characterized defects in the srp1-E402Q and srp1-55 mutants to predict candidate cargo proteins likely to be affected in these mutants and provide evidence that three of these cargoes, Cdc45, Yox1, and Mcm10, are not efficiently localized to the nucleus in importin-alpha mutants. These results reveal that the classical nuclear protein import pathway makes important contributions to the G(1)/S cell cycle transition.     

N-phenylglycolhydroxamate production by the action of transketolase on nitrosobenzene.

The incubation of nitrosobenzene with yeast transketolase and D-xylulose 5-phosphate resulted in the production of N-phenylglycolhydroxamic acid. The addition of D-ribose 5-phosphate decreased the amount of hydroxamic acid that was produced. This conversion of nitrosobenzene into the glycollic acid-derived hydroxamic acid was shown to be an enzymic process, and a chemical mechanism for the conversion was proposed.     

Distinct roles for classical nuclear import receptors in the growth of multinucleated muscle cells

Proper muscle function is dependent on spatial and temporal control of gene expression in myofibers. Myofibers are multinucleated cells that are formed, repaired and maintained by the process of myogenesis in which progenitor myoblasts proliferate, differentiate and fuse. Gene expression is dependent upon proteins that require facilitated nuclear import, however little is known about the regulation of nucleocytoplasmic transport during the formation of myofibers. We analyzed the role of karyopherin alpha (KPNA), a key classical nuclear import receptor, during myogenesis. We established that five karyopherin alpha paralogs are expressed by primary mouse myoblasts in vitro and that their steady-state levels increase in multinucleated myotubes, suggesting a global increase in demand for classical nuclear import during myogenesis. We used siRNA-mediated knockdown to identify paralog-specific roles for KPNA1 and KPNA2 during myogenesis. KPNA1 knockdown increased myoblast proliferation, whereas KPNA2 knockdown decreased proliferation. In contrast, no proliferation defect was observed with KPNA4 knockdown. Only knockdown of KPNA2 decreased myotube growth. These results identify distinct pathways involved in myoblast proliferation and myotube growth that rely on specific nuclear import receptors suggesting that regulation of classical nuclear import pathways likely plays a critical role in controlling gene expression in skeletal muscle.     

Histological and immunohistochemical effects of Curcuma longa on activation of rat hepatic stellate cells after cadmium induced hepatotoxicity

The liver is a target for toxic chemicals such as cadmium (Cd). When the liver is damaged, hepatic stellate cells (HSC) are activated and transformed into myofibroblast-like cells, which are responsible for liver fibrosis. Curcuma longa has been reported to exert a hepato-protective effect under various pathological conditions. We investigated the effects of C. longa administration on HSC activation in response to Cd induced hepatotoxicity. Forty adult male albino rats were divided into: group 1 (control), group 2 (Cd treated), group 3 (C. longa treated) and group 4 (Cd and C. longa treated). After 6 weeks, liver specimens were prepared for light and electron microscopy examination of histological changes and immunohistochemical localization of alpha smooth muscle actin (αSMA) as a specific marker for activated HSC. Activated HSC with a positive αSMA immune reaction were not detected in groups 1 and 3. Large numbers of activated HSC with αSMA immune reactions were observed in group 2 in addition to Cd induced hepatotoxic changes including excess collagen deposition in thickened portal triads, interlobular septa with hepatic lobulation, inflammatory cell infiltration, a significant increase in Kupffer cells and degenerated hepatocytes. In group 4, we observed a significant decrease in HSC that expressed αSMA with amelioration of the hepatotoxic changes. C. longa administration decreased HSC activation and ameliorated hepatotoxic changes caused by Cd in adult rats.     

Millisecond measurement of transport during and after an electroporation pulse.

Electroporation involves the application of an electric field pulse that creates transient aqueous pathways in lipid bilayer membranes. Transport through these pathways can occur by different mechanisms during and after a pulse. To determine the time scale of transport and the mechanism(s) by which it occurs, efflux of a fluorescent molecule, calcein, across erythrocyte ghost membranes was measured with a fluorescence microscope photometer with millisecond time resolution during and after electroporation pulses several milliseconds in duration. One of four outcomes was typically observed. Ghosts were: (1) partially emptied of calcein, involving efflux primarily after the pulse; (2) completely emptied of calcein, involving efflux primarily after the pulse; (3) completely emptied of calcein, involving efflux both during and after the pulse; or (4) completely emptied of calcein, involving efflux primarily during the pulse. Partial emptying, involving significant efflux during the pulse, was generally not observed. We conclude that under some conditions transport caused by electroporation occurs predominantly by electrophoresis and/or electroosmosis during a pulse, although under other conditions transport occurs in part or almost completely by diffusion within milliseconds to seconds after a pulse.     

Abundance and distribution of sturgeon feeding pits in a Washington estuary

Sturgeon diet and feeding habitats are notoriously difficult to document. We mapped the locations of feeding pits in Willapa Bay, Washington, to characterize estuarine habitats used by sub-adult and adult sturgeon for infaunal feeding. Monthly summer surveys of intertidal plots revealed that feeding pit density was highest in July and August, when sturgeon occupy Willapa Bay. The ephemeral nature of feeding pits and high daily densities (> 1000 pits/ha) indicated intensive sturgeon feeding over unvegetated littoral mud flats during high tide. Feeding pit density was lowest in subtidal areas, over sand (grain sizes primarily >63 μ), and at sites with dense stands of non-indigenous seagrass, Zostera japonica. Sub-adult and adult sturgeon apparently used these habitats significantly less than would be predicted based on their availability. Feeding pit formation was negatively correlated with Z. japonica shoot dry weight and positively correlated with the abundance of thalassinid shrimp burrows. Experimental removal of Z. japonica resulted in increased sturgeon feeding, but experimental removal of burrowing shrimp did not significantly affect feeding pit formation. Aquaculture activities that harden substrate and proliferation of invasive seagrass both appear to produce estuarine substrates that are unsuitable for benthic feeding by sturgeon.