Guadalupe Zavala

Association of the astrovirus structural protein VP90 with membranes plays a role in virus morphogenesis.

ABSTRACT VP90, the capsid polyprotein precursor of human astrovirus Yuc8, is assembled into viral particles, and its processing at the carboxy terminus by cellular caspases, to yield VP70, has been correlated with the cell release of the virus. Here, we characterized the effect of the VP90-VP70 processing on the properties of these proteins, as well as on their intracellular distribution. VP90 was found in membrane-enriched fractions (mVP90), as well as in fractions enriched in cytosolic proteins (cVP90), while VP70 was found exclusively in the latter fractions. Upon trypsin activation, infectivity was detected in all VP90-containing fractions, confirming that both mVP90 and cVP90 are able to assemble into particles; however, the two forms of VP90 showed differential sensitivities to trypsin, especially at their carboxy termini, which in the case of mVP90 was shown to remain membrane associated after protease digestion. Structural protein oligomers were detected in purified VP70-containing viruses, as well as in membrane-enriched fractions, but they were less evident in cytosolic fractions. Ultrastructural studies of infected cells revealed different types of viral particles, some of which appeared to be associated with membranes. By immunoelectron microscopy, structural proteins were shown to form virus particles in clusters and to associate with the edges of vesicles induced during infection, which also appear to contain subviral particles inside. Nonstructural proteins and viral RNA colocalized with mVP90, but not with cVP90, suggesting that mVP90 might represent the form of the protein that is initially assembled into particles, at the sites where the virus genome is being replicated.

Implications for evolution of nuclear structures of animals, plants, fungi and protoctists

The evolutionary variations of nuclear structure of animals, plants, fungi and protoctists were studied with electron microscopy by using techniques preferentially staining ribonucleoprotein (RNP) particles and chromatin. A remarkable similarity in the general morphological features of the RNP particles and chromatin arrangement is found in animals, plants and fungi. Important variations of these features were found in protoctists. These observations suggest that major evolutionary changes in the nuclear structure predate the acquisition of plastids by the ancestors of green plants. Once evolved, the nuclear structural pattern is conserved in plants and animals. Among protoctists studied, Kinetoplastida, Cryptomonadida and Volvocida have RNP particles and chromatin arrangement resembling those of plants and animals. These similarities may indicate a common ancestor. Important differences in the nuclear structure among Euglenida, Amebida, Cryptomonadida, Volvocida and Kinetoplastida support the view that Sarcomastigophora is a polyphyletic taxon. For the same reason Kinetoplastida and Euglenida must not be grouped in a monophyletic taxon. We propose that the variations of RNP particles may be related to the initial evolution of post-transcriptional processing.

Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli

BackgroundInclusion bodies (IBs) are aggregated proteins that form clusters when protein is overexpressed in heterologous expression systems. IBs have been considered as non-usable proteins, but recently they are being used as functional materials, catalytic particles, drug delivery agents, immunogenic structures, and as a raw material in recombinant therapeutic protein purification. However, few studies have been made to understand how culture conditions affect the protein aggregation and the physicochemical characteristics that lead them to cluster. The objective of our research was to understand how pH affects the physicochemical properties of IBs formed by the recombinant sphingomyelinase-D of tick expressed in E. coli BL21-Gold (DE3) by evaluating two pH culture strategies.ResultsUncontrolled pH culture conditions favored recombinant sphingomyelinase-D aggregation and IB formation. The IBs of sphingomyelinase-D produced under controlled pH at 7.5 and after 24 h were smaller (<500 nm) than those produced under uncontrolled pH conditions (>500 nm). Furthermore, the composition, conformation and β-structure formation of the aggregates were different. Under controlled pH conditions in comparison to uncontrolled conditions, the produced IBs presented higher resistance to denaturants and proteinase-K degradation, presented β-structure, but apparently as time passes the IBs become compacted and less sensitive to amyloid dye binding.ConclusionsThe manipulation of the pH has an impact on IB formation and their physicochemical characteristics. Particularly, uncontrolled pH conditions favored the protein aggregation and sphingomyelinase-D IB formation. The evidence may lead to find methodologies for bioprocesses to obtain biomaterials with particular characteristics, extending the application possibilities of the inclusion bodies.

Structural Studies of ZnS Nanoparticles by High Resolution Transmission Electron Microscopy

Zinc sulfide (ZnS), a representative of wide band gap semiconductor nanocrystals, has an excitonic Bohr radius (aBZnS ) of 2.5 nm. It makes ZnS nanoparticles (ZnS NP) having such size very interesting as small biomolecular probes for fluorescence and laser scanning microscopy. To date, ZnS NP of diameters larger than aBZnS has been subject of extensive experimental and theoretical studies. However many questions remain open concerning the synthesis of undoped and uncapped ZnS NP of diameters less than 2.5 nm. To further probe into the physical properties of undoped and uncapped ZnS NP, in this work we report on studies of uncapped ZnS nanoparticles synthesized by a wet chemical process at room temperature. Three colloidal suspensions (named A, B and C, respectively) were obtained from 9:1, 1:1 and 1:9 volume mixtures of 1mM ZnSO4 and 0.85mM Na2S aqueous solutions. Qualitative differences in UV-Vis absorption spectra are discussed in the context of Z-contrast scanning transmission electron microscopy (Z-contrast), low and high resolution transmission electron microscopy (TEM) results. Distribution of particle size is dependent on different volumes of source solutions. For the intermediate mixture, it has been found that about 78% of ZnS nanoparticles have a diameter smaller than the excitonic Bohr Radius of 2.5 nm. HRTEM studies have revealed that nanoparticles grow preferentially with hexagonal structure.

Relations between nucleolar morphometric parameters and pre-rRNA synthesis in animal and plant cells.

In order to study if there are differences between cells of the same tissue with one and two nucleoli, nuclear and nucleolar volume, density of tritiated uridine incorporation, amount of DNA per nucleus and intensity of cytoplasmic basophilia were measured in mononucleolated and binucleolated rat epithelial endometrial cells, in onion root meristematic cells and in chick embryo matrix cells of the central nervous system, neuroblasts and neurons. No significant differences in nuclear volume, density of tritiated uridine incorporation and amount of DNA per nucleus were found between cells of the same type with diverse numbers of nucleoli. Binucleolated endometrial cells, matrix cells, and root meristematic cells have biphasic distributions of nucleolar volumes. One peak of this distribution roughly coincides with the nucleolar volume of mononucleolated cells, the other peak corresponds almost to double the volume. As the density of uridine incorporation is the same irrespective of the nucleolar number and volume, the cells with larger nucleolar volumes have higher pre-rRNA synthesis. These cells also have higher amounts of ribosomes in the cytoplasm, as revealed by the photometric study of basophilia. It is concluded that in this population of cells the ribosomal production is regulated to a higher steady equilibrium than in the general population. This difference is not due to polyploidism or to the increased DNA content of G2 phase cells. Binucleolated neuroblasts and neurons have nucleolar volumes similar to those of mononucleolated ones.

Bio-synthesis of gold nanoparticles by human epithelial cells, in vivo

Healthy epithelial cells, in vivo, have the ability to synthesize gold nanoparticles when aqueous tetrachloroauric acid is made to react with human skin. Neither a reducing agent nor a protecting chemical is needed for this bio-synthesis method. The first indication of gold nanoparticle formation is the staining of the skin, which turns deep purple. Stereoscopic optical micrographs of human skin tissue in contact with aqueous tetrachloroauric acid clearly show the staining of the epithelial cells. The UV-Vis spectrum of these epithelial cells shows an absorption band with a maximum at 553 nm. This absorption peak is within the wavelength region where the surface plasmon resonance (SPR) band of aqueous colloidal gold exhibits a maximum. Transmission electron micrographs show that gold nanoparticles synthesized by epithelial cells have sizes between 1 and 100 nm. The electron diffraction pattern of these nanoparticles reveals a crystalline structure whose interplanar distances correspond to fcc metallic gold. Transmission electron micrographs of ultra-thin (70 nm thick) slices of epithelial cells clearly and undoubtedly demonstrate that gold nanoparticles are inside the cell. According to high resolution transmission electron micrographs of intracellular single gold nanoparticles, they have the shape of a polyhedron.

Analysis of Nuclear Ribonucleoproteic Structures During Notochordal Cell Differentiation and Maturation in Chick Embryos

The ultrastructure of notochordal cells and the quantitative changes of nuclear mRNA‐containing particles were studied in several stages of the development of the chick embryo. The modifications in the frequency of perichromatin granules (PCG) were analyzed in embryos at 24 hr to 10 days of incubation (stages 6–36 of Hamburger and Hamilton). The ultrastructural and morphometric data show that notochordal cells undergo changes that can be systematized in four periods. Very early notochordal cells (stages 6–11), are characterized by the presence of large nucleoli and abundant PCG, traits probably related to the frequent mitotic division and the expression of inductive signals reported in numerous papers. During the second period (stages 16–21) the number of PCG and the size of the nucleolus decrease. These changes are coincident with the beginning of vacuolization. In the third period (stages 21–30), the notochordal cells undergo a second cytodifferentiation characterized by a large increase of cytoplasmic vacuolization and secretion of materials that thicken the perichordal sheath. During this period, the nucleolus becomes smaller and the number of PCG increases. Similar features were previously described during functional maturation of embryonic neurons and striated fibers at synaptogenesis, and epidermal cells. The fourth period, beginning at stage 30, is characterized by the decrease of the density of PCG and of the nucleolar volume and corresponds to cessation of mitosis and cell degeneration. Anat Rec 259:113–123, 2000.

Changes in the ribonucleoprotein constituents of the nucleus during the differentiation of muscle cells in the chick embryo.

Summary— The ribonucleoprotein components of the nucleus of chick embryo muscle cells in different stages of development were studied by electron microscopic quantitative stereology. The changes of the constituents were related with the appearance of the innervation by means of silver impregnation for light microscope. The numerical density of the perichromatin granules (PCG) is low in mononuclear cells and myotubes. It is noteworthy that the frequency of the PCG does not change during the transition of the cells in mitotic cycle to postmitotic myoblasts and during myofibril differentiation. However, there is an important increment in this parameter when the motor nerve fibers arrive at the muscle and the synaptic contacts are established. This change is correlated with appearance, or at least with a great increasé, of the importance of posttranscriptional controls of the expression of some genes. The augmentation in the frequency of PCG is not accompanied by alterations of the abundance of total RNP particles, in close resemblance with the phenomena occurring in neuroblast during the differentiation of synaptic endings. The variations of the nucleolar volume coincide with the changes in rRNA synthesis.

Changes of ribonucleoproteic structures of embryonic epidermal cell nuclei during differentiation and maturation

The nuclear structure of the epiblast and skin cells was studied during the development of the chick embryo (Gallus domesticus). Ribonucleoproteic structures (RNPs) and chromatin were characterized with preferential staining techniques. The changes of the frequency of a type of RNP particle, the perichromatin granule, from gastrulating embryos to newly hatched chick, were analyzed by electron microscopic quantitative stereology. The changes of nuclear and nucleolar volume were estimated using the light microscope. The differentiation of ectoderm from epiblast is characterized by a significant increase in the nucleolar volume and in the frequency of perichromatin granules. These features suggest that ectoderm differentiation involves a general increment of mRNA and rRNA transcription. At the beginning the synthesis of a sulfhydryl-rich protein, which is coincident with the thickening of the bilayered epithelium to a multilayered epithelium, a large increase of the numerical density of perichromatin granules not accompanied by any modification of the nucleolar volume was found. The same changes characterize the acquisition of epidermal mature cell features immediately after hatching. These findings as well as previous results concerning the differentiation of central nervous system and striated muscle suggest that the increases of the number of perichromatin granules take place during the acquisition of new cytophysiological states related to functional maturation and not to early stages of cell differentiation.

Recombinant-phospholipase A2 production and architecture of inclusion bodies are affected by pH in Escherichia coli

Aggregation of recombinant proteins into inclusion bodies (IBs) is the major drawback of heterologous expression in Escherichia coli. Here, we evaluated the effects of a pH shift after expression induction on recombinant phospholipase A2 production and its aggregation in IBs in E. coli Origami™, as compared to cultures with pH maintained at 7.5 or uncontrolled pH. Cultures shifted from 7.5 to pH 6.5 or 8.5 produced ∼15-25% less biomass as compared with those kept at 7.5 or without pH control. The cultures shifted to pH 8.5 showed a ∼50% higher yield of acetate per biomass, and the rPLA2 yield was improved 2.4-fold. Purified IBs formed at pH 8.5 containing ∼50% of rPLA2, were more susceptible to proteinase-K cleavage and bound less thioflavin-T, indicating lower amyloid content, with the concomitant enrichment of α-helical and random-coil secondary structures, as demonstrated by FTIR. Moreover, only one IB per cell was formed at pH 8.5; instead, more than two were observed under the other culture pH conditions. Nevertheless, under uncontrolled pH conditions, ∼300nm larger IBs were observed. Our work presents evidence of the usefulness of recombinant protein expression cultivated at pH 8.5 allowing the reduction of amyloid content in IBs.