Investigation of factors responsible for cell line cytoplasmic expression differences

Background  

Previous work has described a novel cytoplasmic expression system that results in a 20-fold increase in the levels of gene expression over a standard CMV-based nuclear expression system, as compared with a 2–3 fold increase seen with previous similar systems. While this increase was seen with BHK and Neuro-2a cells, further studies revealed that some cell lines, such as COS-7, demonstrated relatively poor levels of cytoplasmic expression. The objective of this study was to determine what factors were responsible for the different expression levels between BHK (a high expressing cell line) and COS-7 (a low expressing cell line).     

Molecular factors responsible for host cell recognition and invasion in Plasmodium falciparum.

In Plasmodium falciparum, the rhoptries involved in the invasion process are a pair of flask-shaped organelles located at the apical tip of invading stages. They, along with the more numerous micronemes and dense granules, constitute the apical complex in Plasmodium and other members of the phylum Apicomplexa. Several proteins of varying molecular weight have been identified in P. falciparum rhoptries. These include the 225-, 140/130/110-, 80/60/40-, RAP-1 80-, AMA-1 80-, QF3 80-, and 55-kDa proteins. Some of these proteins are lost during schizont rupture and release of merozoites. Others such as the 140/130/110-kDa complex are transferred to the erythrocyte membrane during invasion. The ring-infected surface antigen (RESA), a 155-kDa polypeptide located in dense granules also associates with the erythrocyte membrane during invasion. Erythrocyte-binding studies have demonstrated that both the 140/130/110-kDa rhoptry complex and RESA bind to inside-out-vesicles (IOVs) prepared from human erythrocytes. The 140/130/110-kDa complex also binds to erythrocyte membranes prepared by hypotonic lysis. These proteins, however, do not bind to intact human erythrocytes. In a heterologous erythrocyte model, both the 140/130/110-kDa complex and RESA are shown to bind directly to mouse erythrocytes. Other studies have shown that RESA associates with spectrin in the erythrocyte cytoskeleton. We have recently developed a liposome-binding assay to demonstrate the lipophilic binding properties of the P. falciparum rhoptry complex of 140/130/110 kDa. The rhoptry complex binds to liposomes containing neutrally, positively, and negatively charged phospholipids. However, liposomes containing phosphatidylethanolamine compete effectively for rhoptry protein binding to mouse erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological factors in Peromyscus speciation

Reciprocal interspecific F1 hybrids of deermice (Peromyscus maniculatus) and oldfield mice (P. polionotus) differ significantly and substantially in fetal and placental, as well as adult, size and weight. Hybrid fetal mortality is associated with large conceptus size. Skin grafts were exchanged between and within the two species to ascertain whether any relationship exists between mean graft retention time and body size of fetuses and adults. P. maniculatus skin grafted to P. polionotus rejected significantly earlier than the reciprocal xenograft. All interspecific graft combinations rejected significantly earlier than intraspecific grafts. Pre-immunization of female P. maniculatus with con- and trans-specific paternal spleen cell antigens reduced fetal, placental, neonatal, and ten-day size compared with controls. Size, weight, fertility, and graft rejection data were compared with several theoretical models. The data were consistent with the hypothesis that immunological disparity between the species could produce marked size variations in reciprocal hybrids. Multiple minor histocompatibility factors can account for large placental size and fetal mortality in Peromyscus hybrids. Physiological reproductive isolation may result from immunological differences between closely allied species.     

Separation of factors responsible for lysozyme catalysis

The four major factors contributing to lysozyme facilitation of acetal hydrolysis are general acid catalysis, distortion, electrostatic stabilization of the transition state and entropy effects. The importance of general acid catalysis is assessed from the Brønsted relationship using an a coefficient of 0·5 based on model compounds. The degree of electrostatic stabilization is assessed from the electric field interactions of Asp 52 and Glu 35. Interaction of lysozyme with transition state analogs is used to calculate the contribution of distortion to catalysis. The remaining acceleration factor is assigned to entropy effects. The value of the entropy effect suggests that the transition state has some freedom of motion on the enzyme.     

Diffusible factors are responsible for differences in nuclease sensitivity among chromatins originating from different cell types

We have examined the kinetics of nuclease digestion of chromatin from committed and uncommitted cells in experiments where the nuclei are mixed and co-digested. Cultures of the sea urchin, Arbacia punctulata, were grown to the 16-cell stage in either [3H]thymidine or [14C]thymidine and the macromere, mesomere, and micromere cell types separated. After isolation, sets of nuclei with two different blastomere types (each having different radionucleotide tagging) were mixed and co-digested with micrococcal nuclease or DNase. I. The extent of digestion was monitored by solubility in 5% perchloric acid (PCA). We find no significant differences in initial digestion rates or limit digests among the different cell types when co-digested with either nuclease. Differences in nuclease sensitivity observed when nuclei are digested separately are abolished when nuclei are probed in a mixing experiment. The results support the hypothesis that phenotypic differences in digestibility among different cell types in vitro reflect differences in chromatin-condensing factors which can diffuse between nuclei.     

Identification of factors responsible for insecticide resistance in Helicoverpa armigera

Moth larvae (Helicoverpa armigera Hübner) collected from field crops were tested for resistance to cypermethrin, fenvalerate, endosulfan, monocrotophos and quinolphos. Larvae were treated with a dose of the pesticide that would kill 99% of the susceptible insects. The percent survival of the resistant strains was determined. Highest seasonal average percentage survival was recorded by fenvalerate (65.0%) followed by cypermethrin (62.4%). Acetylcholinesterase of resistant larvae was less sensitive to monocrotophos and methyl paraoxon. Resistant larvae showed higher activities of esterases, phosphatases and methyl paraoxon hydrolase compared with susceptible larvae. The presence of high activity of esterases was attributed to appearance of extra bands of esterases in native PAGE. The presence of P-glycoprotein expression was detected in resistant larvae using P-gp antibodies; this was not detected in the susceptible larvae. Our results indicate that the high level of resistance detected in the field pests could be because of a combined effect of decreased sensitivity to AChE, higher levels of esterases, phosphatases and the expression of P-gp.     

Genes responsible for etiopathogenesis in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma is a very important cancer type, not only because of its frequent cases around the world but also because of its high mortality rate. Despite of the fact that this is a very rare cancer in Poland (around 2% of patients), we need to know more about this disease in order to find better ways to fight it. Epidemiological factors: smoking and use of psychoactive substances like alcohol and drugs, poor diet as well as changes in expression of genes may have influence on this cancer. During last years there has been a lot of researche on the role of GEAC1, which seems to play a very important role in normal and cancer cells. Perhaps the protein coded by this gene is an important part in cancer development, also being an important element in processes in normal tissues.     

Separation of factors responsible for change in breathing pattern induced by instrumentation

Employment of mouthpiece and noseclips (MP + NC) has repeatedly been shown to increase tidal volume (VT), but its effect on respiratory frequency (f) and its subsets is controversial. The mechanisms accounting for this alteration in breathing pattern are poorly understood and may include stimulation of oral or nasal sensory receptors or alteration in the route of breathing. In this study we demonstrated that use of a MP + NC, compared with nonobtrusive measurement with a calibrated respiratory inductive plethysmograph, alters the majority of the volume and time indexes of breathing pattern, with increases in minute ventilation (P less than 0.01), VT (P less than 0.001), inspiratory time (TI, P less than 0.05), expiratory time (TE, P less than 0.05), mean inspiratory flow (P less than 0.05), and mean expiratory flow (P less than 0.05) and a decrease in f(P less than 0.05). Separating the potential mechanisms we found that when the respiratory route was not altered, independent oral stimulation (using an occluded MP) or nasal stimulation (by applying paper clips to the alae nasi) did not change the breathing pattern. In contrast, obligatory oral breathing without additional stimulation of the oral or nasal sensory receptors caused increases in VT (P less than 0.05), TI (P less than 0.05), and TE (P less than 0.01) and a fall in f(P less than 0.05). Heating and humidifying the inspired air did not prevent the alteration in breathing pattern with a MP. Thus change in the respiratory route is the major determinant of the alteration in breathing pattern with a MP + NC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological aspects of human amniotic fluid cells: implication for normal pregnancy

Amniotic fluid cells (AFCs) are routinely obtained and expanded in vitro for prenatal diagnosis; nevertheless current knowledge about their properties is limited. The detailed mechanisms underlying normal pregnancies are yet to be discovered. The goal of this study was to identify the immunological aspects of AFCs including cytokine production and human leukocyte antigen (HLA) expression, and to discuss its implication for pregnancy. Eighty-six samples of AFCs were determined for HLA expression before and after culture. Cytokine production was measured with flow cytometry in AFC culture supernatants. Treatment of interferon (IFN)-gamma on induction of HLA-DR expression in cultured AFCs was also investigated. Data indicated that both fresh and cultured AFCs express HLA-I, HLA-G, but not HLA-DR, and the cultured AFCs predominately produce the cytokine interleukin (IL)-6. Importantly, we observed that IFN-gamma could induce HLA-DR expression on cultured AFCs in a dose-dependent manner. Taken together, our results indicated that AFCs are functionally active cells and are significant in pregnancy.     

Three determinants in ezrin are responsible for cell extension activity.

The ERM proteins--ezrin, radixin, and moesin--are key players in membrane-cytoskeleton interactions. In insect cells infected with recombinant baculoviruses, amino acids 1-115 of ezrin were shown to inhibit an actin- and tubulin-dependent cell-extension activity located in ezrin C-terminal domain (ezrin310-586), whereas full-length ezrin1-586 did not induce any morphological change. To refine the mapping of functional domains of ezrin, 30 additional constructs were overexpressed in Sf9 cells, and the resulting effect of each was qualitatively and semiquantitatively compared. The removal of amino acids 13-30 was sufficient to release a cell-extension phenotype. This effect was abrogated if the 21 distal-most C-terminal amino acids were subsequently deleted (ezrin31-565), confirming the existence of a head-to-tail regulation in the whole molecule. Surprisingly, the deletion in full-length ezrin of the same 21 amino acids provided strong cell-extension competence to ezrin1-565, and this property was recovered in N-terminal constructs as short as ezrin1-310. Within ezrin1-310, amino acid sequences 13-30 and 281-310 were important determinants and acted in cooperation to induce cytoskeleton mobilization. In addition, these same residues are part of a new actin-binding site characterized in vitro in ezrin N-terminal domain.     

Large plasmid in Shigella boydii is responsible for epithelial cell penetration

A large plasmid in a virulent Shigella boydii 5 strain was transferred to plasmid-cured avirulent strains of S. boydii 5, S. boydii 12, S. sonnei form II, and Escherichia coli K12. The transconjugants acquired the ability to invade tissue culture cells, which indicated that the large plasmid in S. boydii is responsible for epithelial cell invasiveness.     

Ultrastructural evidence for adrenergic nerve degeneration in the guinea pig uterus during pregnancy

Summary In the guinea pig myometrium, the adrenergic nerves selectively demonstrated at the ultrastructural level after treatment with 5-OH-DA, show varying degree of degeneration during pregnancy. The changes are more extensive in a late gestational stage (40–45 days) than in an early one (20–25 days), and are particularly evident in the uterus overlying the conceptus as compared to the regions between the fetuses. Scattered degenerative changes were also observed in myometrial specimens from virgin animals, but probably reflect the normal continuous turnover of axons.     

Involvement of mitochondria in cell degeneration

Human WI-38 fibroblasts were microinjected with isolated mitochondria, and survival of the injected cells was followed. More than 95% of the cells were alive and able to divide when they were injected with fresh mitochondrial preparations having a high respiratory control ratio (RCR). The presence of lysosomes was found to be toxic to the cells, and hence mitochondria had to be isolated without being contaminated by lysosomes. The microinjection of isolated mitochondria from old rats induced a 20% degeneration of the injected cells. The proportion of dead cells was also found to be dependent on the metabolic control of the injected mitochondria. Moreover, an easily metabolized energy substrate such as D(-)-beta-hydroxybutyrate sodium salt was able to inhibit, in a dose-dependent manner, cell degeneration induced by microinjection of uncoupled mitochondria. These results suggest that modifications of mitochondria leading to their uncoupling are harmful to the cells, and this can explain some of the degenerative processes observed in natural or externally induced cell death.