High resolution 3D perspective of Plasmodium biology: advancing into a new era

Apicomplexan parasites exhibit a great variety of complex life cycles that require adaptation to different niches of parasitism. They invade different host cells and highjack their biological functions. Plasmodium falciparum, responsible for the deadliest form of human malaria, causes disease while completely remodeling the erythrocytes of its human host through mechanisms that are only partly understood. Recent developments in ultrastructural technologies offer new opportunities to investigate fundamental aspects in the biology of the parasite in a three-dimensional (3D) perspective. Here we bring together recent work on host cell invasion, hemoglobin uptake, protein export and nuclear dynamics. A comprehensive 3D view of the ultrastructural biology of the parasite may shed new light on cellular mechanisms that underlie the pathogenicity of P. falciparum.     

Galectin-8 Promotes Cytoskeletal Rearrangement in Trabecular Meshwork Cells through Activation of Rho Signaling

Purpose

The trabecular meshwork (TM) cell-matrix interactions and factors that influence Rho signaling in TM cells are thought to play a pivotal role in the regulation of aqueous outflow. The current study was designed to evaluate the role of a carbohydrate-binding protein, galectin-8 (Gal8), in TM cell adhesion and Rho signaling.

Methods

Normal human TM cells were assayed for Gal8 expression by immunohistochemistry and Western blot analysis. To assess the role of Gal8 in TM cell adhesion and Rho signaling, the cell adhesion and spreading assays were performed on Gal8-coated culture plates in the presence and the absence of anti-β₁ integrin antibody and Rho and Rho-kinase inhibitors. In addition, the effect of Gal8-mediated cell-matrix interactions on TM cell cytoskeleton arrangement and myosin light chain 2 (MLC2) phosphorylation was examined.

Principal Findings

We demonstrate here that Gal8 is expressed in the TM and a function-blocking anti-β₁ integrin antibody inhibits the adhesion and spreading of TM cells to Gal8-coated wells. Cell spreading on Gal8 substratum was associated with the accumulation of phosphorylated myosin light chain and the formation of stress fibers that was inhibited by the Rho inhibitor, C3 transferase, as well as by the Rho-kinase inhibitor, Y27632.

Conclusions/Significance

The above findings present a novel function for Gal8 in activating Rho signaling in TM cells. This function may allow Gal8 to participate in the regulation of aqueous outflow.     

ZnT-1 enhances the activity and surface expression of T-type calcium channels through activation of Ras-ERK signaling

Zinc transporter-1 (ZnT-1) is a putative zinc transporter that confers cellular resistance from zinc toxicity. In addition, ZnT-1 has important regulatory functions, including inhibition of L-type calcium channels and activation of Raf-1 kinase. Here we studied the effects of ZnT-1 on the expression and function of T-type calcium channels. In Xenopus oocytes expressing voltage-gated calcium channel (CaV) 3.1 or CaV3.2, ZnT-1 enhanced the low-threshold calcium currents (I(caT)) to 182 ± 15 and 167.95 ± 9.27% of control, respectively (P < 0.005 for both channels). As expected, ZnT-1 also enhanced ERK phosphorylation. Coexpression of ZnT-1 and nonactive Raf-1 blocked the ZnT-1-mediated ERK phosphorylation and abolished the ZnT-1-induced augmentation of I(caT). In mammalian cells (Chinese hamster ovary), coexpression of CaV3.1 and ZnT-1 increased the I(caT) to 166.37 ± 6.37% compared with cells expressing CaV3.1 alone (P < 0.01). Interestingly, surface expression measurements using biotinylation or total internal reflection fluorescence microscopy indicated marked ZnT-1-induced enhancement of CaV3.1 surface expression. The MEK inhibitor PD-98059 abolished the ZnT-1-induced augmentation of surface expression of CaV3.1. In cultured murine cardiomyocytes (HL-1 cells), transient exposure to zinc, leading to enhanced ZnT-1 expression, also enhanced the surface expression of endogenous CaV3.1 channels. Consistently, in these cells, endothelin-1, a potent activator of Ras-ERK signaling, enhanced the surface expression of CaV3.1 channels in a PD-98059-sensitive manner. Our findings indicate that ZnT-1 enhances the activity of CaV3.1 and CaV3.2 through activation of Ras-ERK signaling. The augmentation of CaV3.1 currents by Ras-ERK activation is associated with enhanced trafficking of the channel to the plasma membrane.     

Selenium as a modulator of membrane stability parameters and surface changes during the initiation phase of 1,2-dimethylhydrazine induced colorectal carcinogenesis

Epithelial ovarian cancer (EOC) represents the most challenging of gynecological malignancies. Defective apoptosis is a major causative factor in the development and progression of cancer. The two important pathways of apoptosis are extrinsic death receptor pathway (Fas family) and intrinsic mitochondrial pathway (Bcl-2 family). In this study, differential protein expression of the major Fas family members (Fas, FasL, and FAP-1) and Bcl-2 family members (Bax, Bcl-2, and Bcl-X(L)) in benign versus malignant surface epithelial ovarian tumors was evaluated at the protein level by immunohistochemistry. The expression of these molecules was compared in 30 benign versus 35 malignant surface epithelial ovarian tumors. The findings of the present study showed that there was no significant difference in the expression of the Fas family members in benign and malignant ovarian tumors. However, benign tumors showed higher levels of anti-apoptotic Bcl-2 protein levels (p < 0.009), whereas malignant tumors showed higher levels of pro-apoptotic Bax (p < 0.001). In general, there was no significant difference in Bcl-X(L) protein levels. The observations made in the present study suggest that alterations in expression of the Fas family and the Bcl-2 family members occur and play a key role in the deregulated growth of epithelial ovarian cancer.     

Intracellular trafficking of FXYD1 (phospholemman) and FXYD7 proteins in Xenopus oocytes and mammalian cells

FXYD proteins are a group of short single-span transmembrane proteins that interact with the Na(+)/K(+) ATPase and modulate its kinetic properties. This study characterizes intracellular trafficking of two FXYD family members, FXYD1 (phospholemman (PLM)) and FXYD7. Surface expression of PLM in Xenopus oocytes requires coexpression with the Na(+)/K(+) ATPase. On the other hand, the Na(+)/Ca(2+) exchanger, another PLM-interacting protein could not drive it to the cell surface. The Na(+)/K(+) ATPase-dependent surface expression of PLM could be facilitated by either a phosphorylation-mimicking mutation at Thr-69 or a truncation of three terminal arginine residues. Unlike PLM, FXYD7 could translocate to the cell surface of Xenopus oocytes independently of the coexpression of α1β1 Na(+)/K(+) ATPase. The Na(+)/K(+) ATPase-independent membrane translocation of FXYD7 requires O-glycosylation of at least two of three conserved threonines in its ectodomain. Subsequent experiments in mammalian cells confirmed the role of conserved extracellular threonine residues and demonstrated that FXYD7 protein, in which these have been mutated to alanine, is trapped in the endoplasmic reticulum and Golgi apparatus.     

Treatment of hyperfunctioning thyroid nodules by percutaneous ethanol injection

BACKGROUND: Autonomous thyroid nodules can be treated by a variety of methods. We assessed the efficacy of percutaneous ethanol injection in treating autonomous thyroid nodules. METHODS: 35 patients diagnosed by technetium-99 scanning with hyperfunctioning nodules and suppressed sensitive TSH (sTSH) were given sterile ethanol injections under ultrasound guidance. 29 patients had clinical and biochemical hyperthyroidism. The other 6 had sub-clinical hyperthyroidism with suppressed sTSH levels (<0.24 &mgr;IU/ml) and normal thyroid hormone levels. Ethanol injections were performed once every 1-4 weeks. Ethanol injections were stopped when serum T3, T4 and sTSH levels had returned to normal, or else injections could no longer be performed because significant side effects. Patients were followed up at 3, 6 and, in 15 patients, 24 months after the last injection. RESULTS: Average pre-treatment nodule volume [18.2 PlusMinus; 12.7 ml] decreased to 5.7 PlusMinus; 4.6 ml at 6 months follow-up [P < 0.001]. All patients had normal thyroid hormone levels at 3 and 6 months follow-up [P < 0.001 relative to baseline]. sTSH levels increased from 0.09 PlusMinus; 0.02 &mgr;IU/ml to 0.65 PlusMinus; 0.8 &mgr;IU/ml at the end of therapy [P < 0.05]. Only 3 patients had persistent sTSH suppression at 6 months post-therapy. T4 and sTSH did not change significantly between 6 months and 2 years [P > 0.05]. Ethanol injections were well tolerated by the patients, with only 2 cases of transient dysphonia. CONCLUSION: Our findings indicate that ethanol injection is an alternative to surgery or radioactive iodine in the treatment of autonomous thyroid nodules.     

A Novel Quality Control Compartment Derived from the Endoplasmic Reticulum

Degradation of proteins that, because of improper or suboptimal processing, are retained in the endoplasmic reticulum (ER) involves retrotranslocation to reach the cytosolic ubiquitin-proteasome machinery. We found that substrates of this pathway, the precursor of human asialoglycoprotein receptor H2a and free heavy chains of murine class I major histocompatibility complex (MHC), accumulate in a novel preGolgi compartment that is adjacent to but not overlapping with the centrosome, the Golgi complex, and the ER-to-Golgi intermediate compartment (ERGIC). On its way to degradation, H2a associated increasingly after synthesis with the ER translocon Sec61. Nevertheless, it remained in the secretory pathway upon proteasomal inhibition, suggesting that its retrotranslocation must be tightly coupled to the degradation process. In the presence of proteasomal inhibitors, the ER chaperones calreticulin and calnexin, but not BiP, PDI, or glycoprotein glucosyltransferase, concentrate in the subcellular region of the novel compartment. The "quality control" compartment is possibly a subcompartment of the ER. It depends on microtubules but is insensitive to brefeldin A. We discuss the possibility that it is also the site for concentration and retrotranslocation of proteins that, like the mutant cystic fibrosis transmembrane conductance regulator, are transported to the cytosol, where they form large aggregates, the "aggresomes."     

The complement of enzymatic sets in different species

We present here a comprehensive analysis of the complement of enzymes in a large variety of species. As enzymes are a relatively conserved group there are several classification systems available that are common to all species and link a protein sequence to an enzymatic function. Enzymes are therefore an ideal functional group to study the relationship between sequence expansion, functional divergence and phenotypic changes. By using information retrieved from the well annotated SWISS-PROT database together with sequence information from a variety of fully sequenced genomes and information from the EC functional scheme we have aimed here to estimate the fraction of enzymes in genomes, to determine the extent of their functional redundancy in different domains of life and to identify functional innovations and lineage specific expansions in the metazoa lineage. We found that prokaryote and eukaryote species differ both in the fraction of enzymes in their genomes and in the pattern of expansion of their enzymatic sets. We observe an increase in functional redundancy accompanying an increase in species complexity. A quantitative assessment was performed in order to determine the degree of functional redundancy in different species. Finally, we report a massive expansion in the number of mammalian enzymes involved in signalling and degradation.     

Detection of Helicobacter pylori-specific genes in the oral yeast

BACKGROUNDS: Until today, human stomach is the only recognized habitat of Helicobacter pylori. However, recruitment of DNA-based methods has made possible the detection of H. pylori in water and oral cavity, thus suggesting fecal-oral and oral-oral routes for transmission of H. pylori, respectively. In this study, yeast has been proposed as a common vector for transmission of H. pylori. Thus designed primers were recruited to target 16S rDNA and cagA genes in the oral yeasts by PCR. MATERIALS AND METHODS: Eighteen yeasts were examined microscopically for the presence of bacterial-like bodies. DNAs were extracted from oral yeasts using phenol-chloroform method. Amplification conditions were optimized as 33 cycles and annealing temperatures of 63 degrees C for 16S rDNA and 51 degrees C and 52 degrees C for cagA gene which was targeted in two steps. DNAs of H. pylori and Saccharomyces cerevisiae were used as controls. Polymerase chain reaction (PCR) products of two genes from one yeast and from H. pylori were cloned in pCAP and subsequently subcloned in pSK+ and were sequenced. RESULTS: Bacterial-like bodies were observed in all oral yeasts. The amplified products of 16S rDNA from all oral yeasts were homologous in size with those of H. pylori. Fifteen out of eighteen (83%) yeasts contained cagA gene, homologous to H. pylori. CagA was not amplified from three yeasts and S. cerevisiae. Analysis of the sequenced products of 16S rDNA and cagA from one oral yeast showed 98% homology with those of H. pylori. CONCLUSIONS: The presence of H. pylori inside the yeast was indicated by light microscopy and PCR. It appears that yeasts, which are abundant in nature and thrive the mucosal surfaces of human, might serve as reservoirs and vehicles of H. pylori.     

Relationship between the tissue-specificity of mouse gene expression and the evolutionary origin and function of the proteins

Background  

The combination of complete genome sequence information with expression data enables us to characterize the relationship between a protein's evolutionary origin or functional category and its expression pattern. In this study, mouse proteins were assigned into functional and phyletic groups and the gene expression patterns of the different protein groupings were examined by microarray analysis in various mouse tissues.