Carbonic Anhydrase Activities in Pea Thylakoids

Pea thylakoids with high carbonic anhydrase (CA) activity (average rates of 5000 µmol H⁺ (mg Chl)^–1 h^–1 at pH 7.0) were prepared. Western blot analysis using antibodies raised against the soluble stromal -CA from spinach clearly showed that this activity is not a result of contamination of the thylakoids with the stromal CA but is derived from a thylakoid membrane-associated CA. Increase of the CA activity after partial membrane disintegration by detergent treatment, freezing or sonication implies the location of the CA in the thylakoid interior. Salt treatment of thylakoids demonstrated that while one part of the initial enzyme activity is easily soluble, the rest of it appears to be tightly associated with the membrane. CA activity being measured as HCO₃ ^– dehydration (dehydrase activity) in Photosystem II particles (BBY) was variable and usually low. The highest and most reproducible activities (approximately 2000 µmol H⁺ (mg Chl)^–1 h^–1) were observed in the presence of detergents (Triton X-100 or n-octyl--D-glucopyranoside) in low concentrations. The dehydrase CA activity of BBY particles was more sensitive to the lipophilic CA inhibitor, ethoxyzolamide, than to the hydrophilic CA inhibitor, acetazolamide. CA activity was detected in PS II core complexes with average rate of 13,000 µmol H⁺ (mg Chl)^–1 h^–1 which was comparable to CA activity in BBY particles normalized on a PS II reaction center basis.This revised version was published online in October 2005 with corrections to the Cover Date.     

Double-stranded DNA-induced localized unfolding of HCV NS3 helicase subdomain 2

The NS3 helicase of the hepatitis C virus (HCV) unwinds double-stranded (ds) nucleic acid (NA) in an NTP-dependent fashion. Mechanistic details of this process are, however, largely unknown for the HCV helicase. We have studied the binding of dsDNA to an engineered version of subdomain 2 of the HCV helicase (d(2Delta)NS3h) by NMR and circular dichroism. Binding of dsDNA to d(2Delta)NS3h induces a local unfolding of helix (alpha(3)), which includes residues of conserved helicase motif VI (Q(460)RxxRxxR(467)), and strands (beta(1) and beta(8)) from the central beta-sheet. This also occurs upon lowering the pH (4.4) and introducing an R461A point mutation, which disrupt salt bridges with Asp 412 and Asp 427 in the protein structure. NMR studies on d(2Delta)NS3h in the partially unfolded state at low pH map the dsDNA binding site to residues previously shown to be involved in single-stranded DNA binding. Sequence alignment and structural comparison suggest that these Arg-Asp interactions are highly conserved in SF2 DEx(D/H) proteins. Thus, modulation of these interactions by dsNA may allow SF2 helicases to switch between conformations required for helicase function.     

Does a deletion in a virus-like particle protein have pleiotropic effects on the reproductive biology of a parasitoid wasp?

Endoparasitoid insects have evolved mechanisms to counteract host immune defences. At oviposition, the endoparasitoid Venturia canescens injects virus-like particles (VLPs) together with the egg that interfere with the immune system of the host. These prevent encapsulation of the parasitoid egg. It has been shown that the gene coding for one of the VLP proteins exists in two variants (called Repeat-Plus (RP) and Repeat-Minus (RM)). Previous observations suggested that these variants induce pleiotropic effects on the reproductive biology of the female resulting, in an impeded transfer of eggs from the ovarioles to the oviducts in RM females, and in alterations in the egg laying sequence. We show that RM females from another geographical locality do not exhibit any phenotypic alteration of the reproductive biology. By showing a lack of association between VLP alleles and the reproductive phenotypic characteristics in a given strain, our results do not support the hypothesis of pleiotropic effects. Conversely, the results suggest that different genes code for the VLPs and for the reproductive biology characteristics. Different phenomena such as linkage, the action of pathogens, etc. could explain the association between characters that is observed in some strains.     

Structured HCV nucleocapsids composed of P21 core protein assemble primary in the nucleus of Pichia pastoris yeast

The relationship between HCV core protein (HCcAg) processing and the structural composition and morphogenesis of nucleocapsid-like particles (NLPs) produced in Pichia pastoris cells was studied. At early stages of heterologous expression, data suggest that HCcAg (in the P21 form) was transported soon after its synthesis in the cytoplasm into the nucleus. HCcAg assembly into nucleocapsid-like particles with 20-30 nm in diameter took place primary in the cell nucleus. However, at later stages, when P21 and P23 forms were co-detected, data suggest that new assembly of nucleocapsid particles containing P21 possibly occurs at ER membranes and in the cytoplasm. This is the first report showing that structured HCV NLPs composed of P21 core protein assemble primary in the nucleus of P. pastoris yeast.     

Virus-like particle production at low multiplicities of infection with the baculovirus insect cell system

The baculovirus insect cell expression system (BEVS) was used for the production of self-forming Porcine parvovirus-like particles (VLPs) in serum-free medium. A low multiplicity of infection (MOI) strategy was used to overcome an extra virus amplification step, undesirable in industrial production, and to minimize the virus passage effect. It was confirmed that the time of infection (TOI) and MOI are dependent variables. Higher cell densities were obtained at low MOIs, keeping a constant TOI; however, both volumetric and specific productivities were lower. In synchronous infection, at high MOI, the specific productivity decreased when the cells were infected in the late phase of growth. Product degradation due to cell lysis strongly influenced the optimal time of harvest (TOH). Time of harvest was found to be highly dependent on the MOI, and a direct relationship with the cell yield was obtained.Analysis of the culture medium reveals that glutamine depletion occurs in the late phase of the growth. Supplementation of glutamine to uninfected cell cultures resulted in an increased cell yield. Its addition to cultures infected in the middle phase of the growth curve was also able to restore the productivity levels, but addition to cells in their stationary phase caused no observable effect on product expression. The study clearly shows that for a specific TOI it is not obvious what the correct MOI should be to obtain the best volumetric productivity.     

Butyrate stimulates ApoA-IV-containing lipoprotein secretion in differentiated Caco-2 cells: role in cholesterol efflux

The aim of this study was to determine: (1) whether the Short Chain Fatty Acids (SCFA) Acetate, Propionate, and Butyrate enhance the synthesis and secretion of intestinal apolipoprotein A-IV-containing lipoproteins and (2) if so, whether these particles are able to promote cholesterol efflux in vitro. For this purpose Caco-2 cells were used for their functional properties of differentiated enterocytes. They were incubated with the three SCFA (2, 4, and 8 mM) for 48 h. Only butyrate stimulated apoA-IV gene expression and this was associated with an increase in apoA-IV secretion. A nondenaturing 2D-PAGE (agarose gel was followed by PAGE) was used to identify apoA-IV-containing lipoproteins in various media, and showed that butyrate stimulated the secretion of two small HDL sized particles. The influence of these secreted particles on cholesterol efflux was investigated using incubation of media with (3)H-cholesterol-labeled Fu5AH cells. The data indicate that conditioned media from Caco-2 cells treated with butyrate resulted in an increase of 20-30% in cholesterol efflux. We conclude that butyrate may regulate apoA-IV secretion and, therefore, modulate reverse cholesterol transport.     

A technique for isolation of rubella virus-like particles by sucrose gradient ultracentrifugation using Coomassie brilliant blue G crystals

An improved method for the isolation of rubella virus-like particles (RVLP) from cell culture supernatant of transfected Chinese hamster ovary (CHO24S) cells is described. It employs a combination of membrane filtration with sucrose gradient ultracentrifugation. It was found that staining the RVLP band with Coomassie brilliant blue G (CBB) resulted in the CBB crystals adsorbing RVLP. After ultracentrifugation (25,000 rpm, 3h, 4 degrees C) a sharp blue band with crystals (diameter 30-40 microm) was observed (at a density of 1.250 g/ml at 25 degrees C) in a 30-60% sucrose gradient. Using a combination of SDS-PAGE and Western blotting techniques, E1 rubella virus structural protein was detected only in the solutions derived from the sharp blue band. A decrease in crystal concentration a few millimeters above or below the main band was associated with a decrease in protein concentration. By dilution with a saturated ice-cold 30% sucrose solution it was possible to pellet the crystals by centrifugation (15,000 rpm, 10 min). SDS-PAGE showed a much higher concentration of RVLP structural protein in the pellet than in the supernatant. This RVLP-containing material is especially suitable for the preparation of rubella virus immunoblot stripes.     

Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt, 1925: differences in their cell surfaces and in the bacteria-containing vacuoles

Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt. 1925 are two of eight species of Entamoeba that sometimes inhabit the human colon. The former is an invasive organism capable of causing life-threatening intestinal and extra-intestinal disease: the latter appears not to be invasive. Because the two species, when viewed by light microscopy appear morphologically similar, they were long regarded as a single species. However, recent biochemical. immunological, and genetic studies provided convincing evidence that they belong to separate species. Our ultrastructural studies revealed distinct differences in at least two features of the trophozoites. 1) The cell surfaces of the trophozoites of each species differ with regard to structures exposed on the surface, and the distribution and arrangement of intra-membranous proteins. 2) The phagocytosis of bacteria differs in respect to the formation of the phagocytic vacuoles. Loose vacuoles containing several bacteria were seen in E. histolytica whereas tight vacuoles containing a single bacterium were observed in E. dispar. Furthermore, bacteria were found only within vacuoles in E. histolytica; in E. dispar, bacteria were found within vacuoles and some were found free in the cytoplasm.     

Human eukaryotic initiation factor 4AII associates with hepatitis C virus NS5B protein in vitro

Hepatitis C virus (HCV) NS5B protein has been shown to have RNA-dependent RNA polymerase (RdRp) activity by itself and is a key enzyme involved in viral replication. Using analyses with the yeast two-hybrid system and in vitro binding assay, we found that human eukaryotic initiation factor 4AII (heIF4AII), which is a component of the eIF4F complex and RNA-dependent ATPase/helicase, interacted with NS5B protein. These two proteins were shown to be partially colocalized in the perinuclear region. The binding site in HCV NS5B protein was localized within amino acid residues 495 to 537 near the C terminus. Since eIF4A has a helicase activity and functions in a bidirectional manner, the binding of HCV NS5B protein to heIF4AII raises the possibility that heIF4AII facilitates the genomic RNA synthesis of NS5B protein by unwinding the secondary structure of the HCV genome and is a host component of viral replication complex.     

In vitro self-assembled HCV core virus-like particles induce a strong antibody immune response in sheep.

The in vitro self-assembly properties of the entire hepatitis C virus core protein (HCcAg) obtained from Pichia pastoris cells and the induction of specific antibody immune response were studied. HCcAg was purified as a low-molecular-weight species by electroelution under denaturing conditions for confirmation of its self-assembly properties. After renaturalization, electron microscopy showed that HCcAg assembled into spherical particles of 30 nm. HCcAg also showed homogeneity and was specifically recognized by serum from a chronic HCV carrier patient. The data indicated that in vitro assembly of HCcAg, into virus-like particles resembling HCV nucleocapsid particles at a mature stage, is an intrinsic quality of this protein. Finally, HCcAg generated a strong antibody immune response in sheep, suggesting its usefulness for stimulating the host immune response against HCV.