Programmed cell death in trypanosomatids and other unicellular organisms

In multicellular organisms, cellular growth and development can be controlled by programmed cell death (PCD), which is defined by a sequence of regulated events. However, PCD is thought to have evolved not only to regulate growth and development in multicellular organisms but also to have a functional role in the biology of unicellular organisms. In protozoan parasites and in other unicellular organisms, features of PCD similar to those in multicellular organisms have been reported, suggesting some commonality in the PCD pathway between unicellular and multicellular organisms. However, more extensive studies are needed to fully characterise the PCD pathway and to define the factors that control PCD in the unicellular organisms. The understanding of the PCD pathway in unicellular organisms could delineate the evolutionary origin of this pathway. Further characterisation of the PCD pathway in the unicellular parasites could provide information regarding their pathogenesis, which could be exploited to target new drugs to limit their growth and treat the disease they cause.     

Expression of calreticulin P-domain results in impairment of secretory pathway in Leishmania donovani and reduced parasite survival in macrophages

The secretory proteins of Leishmania are thought to be involved in the parasite survival inside the insect vector or mammalian host. It is clear from studies in higher eukaryotes that proper folding in the endoplasmic reticulum and targeting out of the endoplasmic reticulum is critical for the function of secretory proteins. The endoplasmic reticulum chaperones such as calreticulin play an important role in the quality control of secretory proteins. However, very little is known about the secretory pathway of trypanosomatid parasites such as Leishmania. In the present study, we show that overexpression of the P-domain of Leishmania donovani calreticulin in transfected L. donovani resulted in a significant reduction in the secretion of the parasite secretory acid phosphatases. This effect is associated with an intracellular accumulation of active enzyme in these transfected parasites. In addition, parasites expressing the P-domain calreticulin showed a significant decrease in survival inside human macrophages. This study suggests that altering the function of an endoplasmic reticulum chaperone such as calreticulin in Leishmania may affect the targeting of proteins that are associated with the virulence of the parasite during their trafficking through the parasite secretory pathway.     

Rubella virus cDNA. Sequence and expression of E1 envelope protein

A cDNA clone encoding the entire E1 envelope protein (410 amino acid residues) and a portion of the C-terminal end of the E2 envelope protein of the rubella virus has been isolated and characterized. DNA sequence analysis has revealed a region 20 nucleotides in length at the 3' end of the cloned cDNA which may be a replicase recognition site or a recognition site for encapsidation. The proteolytic cleavage site between the E1 and E2 proteins was localized based on the known amino-terminal sequence of the isolated E1 protein (Kalkkinen, N., Oker-Blom, C., and Pettersson, R. F. (1984) J. Gen. Virol. 65, 1549-1557) and the deduced amino acid sequence. The mature E1 protein is preceded by a set of 20 highly hydrophobic amino acid residues possessing characteristics of a signal peptide. This "signal peptide" is flanked on both sides by typical protease cleavage sites for trypsin-like enzyme and signal peptidase. The presence of a leader sequence in the E1 protein precursor may facilitate its translocation through the host cell membrane. The E1 protein of rubella virus shows no significant homology with alphavirus E1 envelope proteins. However, a stretch of 39 amino acids in the E1 protein of rubella virus (residues 262-300) was found to share a significant homology with the first 39 residues of bovine sperm histone. The position of 4 half-cystines and 8 arginines overlaps. The E1 protein of rubella virus has been successfully expressed in COS cells after transfecting them with rubella virus cDNA in simian virus 40-derived expression vector. This protein is antigenically similar to the one expressed by cells infected with rubella virus.     

Isolation, cDNA cloning, and characterization of an 18-kDa hemagglutinin and amebocyte aggregation factor from Limulus polyphemus.

An 18-kDa hemagglutinin which possesses the property of inducing both aggregation of amebocytes and agglutination of erythrocytes has been isolated from Limulus polyphemus amebocytes and purified by ion exchange chromatography. This nonglycosylated, single chain polypeptide with an M(r) of 18,506 and isoelectric point of 8.3 is stored exclusively in the large secretory granules of amebocytes. Based on the partial N-terminal amino acid sequence of 63 residues, DNA probes have been synthesized for screening a pBR322 cDNA library constructed from Limulus amebocytes. The cDNA coding for this protein reveals the presence of a 19-residue signal peptide preceding the 153-residue open reading frame. Northern blot analysis indicates the presence of a single mRNA species. The primary structure derived from the corresponding cDNA sequence reveals an internal homology consisting of two consensus sequences, Val-Asn-Asp/Ser-Trp-Asp and Glu-Asp-Arg-Arg-Trp. The formation of 5 disulfide bonds between 10 half-cysteines divides the molecule into three looped domains each containing the Glu-Asp-Arg-Arg-Trp repeating unit. One of the novel features of this protein is that it shares 37% identity with a 22-kDa mammalian extracellular matrix protein isolated from fetal bovine skin (Neame, P.J., Choi, H.U., and Rosenberg, L.C. (1989) J. Biol. Chem. 264, 5474-5479). The two proteins exhibit a similar pattern of looped domains, each domain containing a homologous consensus sequence (i.e. Glu-Asp-Arg-Arg-Trp). The overall structure of both proteins seems to be highly related, with the exception of an N-terminal tyrosine-rich region present only in the mammalian extracellular matrix protein. The functional properties of the two proteins are similar in that the Limulus 18-kDa protein agglutinates horse erythrocytes and aggregates Limulus amebocytes, and the mammalian 22-kDa protein is an effective adhesion promoter for dermal fibroblasts. On the basis of these unique properties, the newly characterized hemagglutinin has been termed Limulus 18K agglutination-aggregation factor (18K-LAF).     

A novel signal sequence negative multimeric glycosomal protein required for cell cycle progression of Leishmania donovani parasites

Expression of the intracellular form amastigote specific genes in the Leishmania donovani parasite plays a major role in parasite replication in the macrophage. In the current work, we have characterized a novel hypothetical gene, Ld30b that is specifically transcribed in the intracellular stage of the parasite. The recombinant Ld30b protein exists as a pentamer in solution as identified by native-PAGE and size exclusion gel chromatography. Structural analysis using circular dichroism and molecular modeling indicate that Ld30b belongs to family of cAMP-dependent protein kinase type I-alpha regulatory subunit. Co-localization immunofluorescence microscopy and western blot analyses (using anti-Ld30b antibody and anti-hypoxanthine-guanine phosphoribosyl transferase, a glycosome marker) on the isolated parasite glycosome organelle fractions show that Ld30b is localized in glycosome, though lacked a glycosome targeting PTS1/2 signal in the protein sequence. Episomal expression of Ld30b in the parasite caused the arrest of promastigotes and amastigotes growth in vitro. Cell cycle analysis using flow cytometry indicates that these parasites are arrested in ‘sub G0/G1’ phase of the cell cycle. Single allele knockout of Ld30b in the parasite similarly attenuated its growth by accumulation of cells in the S phase of cell cycle, thus confirming the probable importance of appropriate level of protein in the cells. Studying such intracellular stage expressing genes might unravel novel regulatory pathways for the development of drugs or vaccine candidates against leishmaniasis.     

Quantitation of milk proteins and their mRNAs in rat mammary gland at various stages of gestation and lactation.

The content of alpha-lactalbumin and three species of caseins, 42K, 29K, and 25K, have been measured along with the levels and activities of their mRNAs in the rat mammary gland. Changes in these values were followed during gestation and lactation. An increment of 3- to 4-fold over the virgin level was observed for both alpha-lactalbumin and 42K casein during the 1st day of gestation. From this point on, the level of 42K remained unchanged during the 1st week of gestation and increased thereafter. After the increment of the 1st day, the alpha-lactalbumin content decreased rapidly during the 2nd day of gestation, continued to decrease more slowly until the 12th day, and then started to increase thereafter. During the 2nd and 3rd week of gestation. the amounts of alpha-lactalbumin within the gland increased continuously but not uniformly and caseins accumulated rapidly with a tendency to plateau around the 13th to 16th day of gestation. The relative proportions remained, respectively, 42K greater than 29K greater than 25K greater than alpha-lactalbumin until parturition. At the onset of lactation, both alpha-lactalbumin and casein content increased sharply, the relative proportion for caseins changed to 42K greater than 25K greater than 29K greater than alpha-lactalbumin and remained so throughout the lactation period. alpha-Lactalbumin and casein mRNA activity, as judged by the wheat germ translational system, remained unchanged during the 1st week of gestation, then showed a steady but not uniform increase from the 7th day of gestation until parturition. These activities increased sequentially during lactation, alpha-lactalbumin reaching a plateau by the 1st week, caseins between the 1st and 2nd week, and other mRNAs by the end of the 2nd week of lactation. By the 21st day of lactation, the activity of all mRNA had declined. The levels of alpha-lactalbumin mRNA and 16 S doublet casein mRNA sequences measured with the cDNA probes increased by about 8-fold for alpha-lactalbumin mRNA and 6-fold for casein mRNA during the 1st week of gestation. These levels declined slightly early in the 2nd week and then continued to increase until parturition with a shoulder in the levels around the 13th to 16th day. During lactation, these levels increased until the 8th to 12th day and from then on declined. The content of alpha-lactalbumin and caseins, as well as the measurement of sequences and activities of their mRNAs, showed that in the rat mammary gland these differentiated functions are already expressed at the onset of gestation. Both concentration and activity of mRNA are out of phase with protein levels during the 1st week of gestation but they remain in phase thereafter.     

Structural analysis of human neutrophil migration: Centriole, microtubule, and microfilament orientation and function during chemotaxis

Orientation of nucleus, centriole, microtubules, and microfilaments within human neutrophils in a gradient of chemoattractant (5 percent Escherichia coli endotoxin-activated serum) was evaluated by electron microscopy. Purified neutropils (hypaque-Ficoll) were placed in the upper compartment of chemotactic chambers. Use of small pore (0.45 μm) micropore filters permitted pseudopod penetration, but impeded migration. Under conditions of chemotaxis with activated serum beneath the filter, the neutrophil population oriented at the filter surface with nuclei located away from the stimulus, centrioles and associated radial array of microtubules beneath the nuclei, and microfilament-rich pseudopods penetrating the filter pores. Reversal of the direction of the gradient of the stimulus (activated serum above cells) resulted in a reorientation of internal structure which preceded pseudopod formation toward the activated serum and migration off the filter. Coordinated orientation of the entire neutrophil population did not occur in buffer (random migration) or in a uniform concentration of activated serum (activated random migration). Conditions of activated random migration resulted in increased numbers of cells with locomotory morphology, i.e. cellular asymmetry with linear alignment of nucleus, centriole, microtubule array, and pseudopods. Thus, activated serum increased the number of neutrophils exhibiting locomotory morphology, and a gradient of activated serum induced the alignment of neutrophils such that this locomotory morphology was uniform in the observed neutrophil populayion. In related studies, cytochalasin B and colchicines were used to explore the role of microfilaments and microtubules in the neutrophil orientation and migration response to activated serum. Cytochalasin B (3.0 μg/ml) prevented migration and decreased the microfilaments seen, but allowed normal orientation of neutrophil structures. In an activated serum gradient, colchicines, but not lumicolchicine, decreased the orientation of nuclei and centrioles, and caused a decrease in centriole-associated microtubules in concentrations as low as 10(-8) to 10(-7) M. These colchicines effects were associated with the rounding of cells and impairment of pseudopod formation. The impaired pseudopod formation was characterized by an inability to form pseudopods in the absence of a solid substrate, a formation of narrow pseudopods within a substrate, and a defect in pseudopod orientation in an activated serum gradient. Functional studies of migration showed that colchicines, but not lumicolchicine, minimally decreased activated random migration and markedly inhibited directed migration, but had not effect on random migration. These studies show that, although functioning microfilaments are probably necessary for neutrophil migration, intact microtubules are essential for normal pseudopod formation and orientation, and maximal unidirectional migration during chemotaxis.     

Mesozooplankton community structure changes in the Mfolozi–Msunduzi estuarine system,South Africa,during contrasting river flow conditions

The Mfolozi–Msunduzi estuarine system is subject to periodic dry and wet cycles, with subsequent changes in the abiotic and biotic characteristics of the system. The aim of the current study was to compare its mesozooplankton composition during relatively dry and wet periods. Mesozooplankton samples were collected between 2007 and 2010 in both the Mfolozi and the Msunduzi, covering a dry period between 2007 and 2008 and a period of relatively high freshwater inputs during 2009 and 2010. High flows during the wet period reduced the densities of most of the dominant estuarine mesozooplankton taxa in the Mfolozi Estuary, such as estuarine calanoids Pseudodiaptomus stuhlmanni (Poppe & Mrázek, 1895) and Acartiella natalensis (Connell & Grindley, 1974). The Msunduzi Estuary functioned as a reservoir from which recolonisation by estuarine taxa would quickly take place after the Mfolozi was scoured by floodwaters. Densities of dominant meroplankton taxa, such as zoeae of the crab Paratylodiplax blephariskios and Macrobrachium spp., were not noticeably different in the Mfolozi–Msunduzi system between the low- and high-flow periods.