Total Cellular Protein Content Research Articles

Optimal Protein Allocation Controls the Inhibition of GltA and AcnB in Neisseria gonorrhoeae.

Neisseria gonorrhea (Ngo) is a major concern for global public health due to its severe implications for reproductive health. Understanding its metabolic phenotype is crucial for comprehending its pathogenicity. Despite Ngo's ability to encode TCA cycle proteins, GltA and AcnB, their activities are notably restricted. To investigate this phenomenon, we used the iNgo_557 metabolic model and incorporated a constraint on total cellular protein content. Our results indicate that low cellular protein content severely limits GltA and AcnB activity, leading to a shift towards acetate overflow for ATP production, which is more efficient in terms of protein usage. Surprisingly, increasing cellular protein content alleviates this restriction on GltA and AcnB and delays the onset of acetate overflow, highlighting protein allocation as a critical determinant in understanding Ngo's metabolic phenotype. These findings underscore the significance of Ngo's metabolic adaptation in light of optimal protein allocation, providing a blueprint to understand Ngo's metabolic landscape.

Alkaloid binding to opium poppy major latex proteins triggers structural modification and functional aggregation

Opium poppy accumulates copious amounts of several benzylisoquinoline alkaloids including morphine, noscapine, and papaverine, in the specialized cytoplasm of laticifers, which compose an internal secretory system associated with phloem throughout the plant. The contiguous latex includes an abundance of related proteins belonging to the pathogenesis-related (PR)10 family known collectively as major latex proteins (MLPs) and representing at least 35% of the total cellular protein content. Two latex MLP/PR10 proteins, thebaine synthase and neopione isomerase, have recently been shown to catalyze late steps in morphine biosynthesis previously assigned as spontaneous reactions. Using a combination of sucrose density-gradient fractionation-coupled proteomics, differential scanning fluorimetry, isothermal titration calorimetry, and X-ray crystallography, we show that the major latex proteins are a family of alkaloid-binding proteins that display altered conformation in the presence of certain ligands. Addition of MLP/PR10 proteins to yeast strains engineered with morphine biosynthetic genes from the plant significantly enhanced the conversion of salutaridine to morphinan alkaloids.

Macromolecules Influence Cellular Competence and Expression Level of IGFs Genes in Bovine Oocytes In Vitro.

Simple SummaryThe macromolecule content of culture media can affect the maturation competence of oocytes, which influences the subsequent in vitro development of embryos. This study was designed to determine the effects of macromolecules on cellular competence and the transcript level of insulin-like growth factors (IGF1, IGF2) and their receptors in bovine oocytes. The current study showed that bovine serum albumin (BSA) and fetal calf serum (FCS) improved nuclear maturation and protein biosynthesis (especially FCS). Polyvinyl alcohol did not support the antioxidant defense mechanism due to decreased glutathione peroxidase enzyme activity. The expression of the IGF1 gene could not be detected in all experimental groups, but BSA and FCS increased the transcript level of the IGF2 gene. Moreover, oocyte maturation with BSA increased the transcript level of the IGF1R gene, whereas the transcript level of the IGF2R gene was similar among macromolecule supplementation groups. The BSA and FCS could improve in vitro bovine oocyte development due to supporting cellular characteristics.In vitro maturation (IVM) of mammalian oocytes, which influences subsequent in vitro development of embryos, is affected by the macromolecule content in culture media for the success of oocyte maturation competence, in which the cytoplasmic and nuclear reprogramming events occur. The insulin-like growth factor family (IGFs) promotes the maturation of bovine oocytes and the expansion of cumulus cells and also inhibits apoptosis. This study was, therefore, designed to examine the effects of macromolecules (bovine serum albumin, BSA; fetal calf serum, FCS; and polyvinyl alcohol, PVA) on in vitro nuclear maturation, total cellular protein, glutathione peroxidase (GPx) enzyme activity, and the gene expression level of IGF1, IGF2, and their receptor in bovine oocytes. Oocytes obtained from bovine ovaries were cultured in bicarbonate-buffered medium 199 supplemented with 4 mg/mL BSA, 10% FCS, 1 mg/mL PVA, and without macromolecule supplement (control) during 22 h in the air with a humidified atmosphere and 5% CO2 at 38.5 °C temperature. Supplementation of BSA and FCS increased (χ2 = 9.84; p < 0.05) the percentages of oocytes that reached metaphase II compared to the control and PVA. The amount of protein per ml of cell extracts of oocytes matured in FCS supplemented culture media was higher (p < 0.05) than the oocytes in the PVA and control. The levels of GPx enzyme activity in cell extracts isolated from oocytes in each experimental group did not change over time, but the GPx enzyme activity in oocytes matured in PVA-supplemented culture media was lower (p < 0.05) than in oocytes in the other experimental groups. Transcript for the IGF1 gene was not detected in all experimental groups, but the supplementation of BSA and FCS significantly elevated the transcript level of the IGF2 gene. In addition, the maturation of oocytes with BSA-supplemented media increased the transcript level of the IGF1R gene, whereas the transcript level of the IGF2R gene was similar among macromolecule supplementation groups. The current study concluded that BSA and FCS could improve in vitro bovine oocyte development due to supporting nuclear maturation and increasing the total cellular protein content, GPx enzyme, and transcript activity.

Administration of Costus igneus Nak leaf extract improves diabetic-induced impairment in hepatorenal functions in male albino rats

ObjectivesThe present study was designed to evaluate the therapeutic efficacy Costus igneus Nak leaf extract against the diabetes-induced hepatorenal injury in rats. MethodsDiabetes was induced in male albino rats by administering single injection of streptozotocin (55 mg/kg) for one day. The diabetic condition was established by estimation of blood glucose level; animals with blood glucose levels above 250 mg were considered as diabetic. Following the confirmation, animals were randomly divided into seven experimental groups, viz group I served as the control, group II-CI-300mg/Kg (per se), group III-Diabetic, group IV-diabetic+CI (100mg/kg), group V-diabetic+CI (200mg/kg), group VI-Diabetic+CI (300 mg/kg) and group VII-diabetic+glibenclamide (0.5mg/kg). Treatment regimens were started 24 h after the confirmation of the successful induction of diabetes and continued for four consecutive weeks at three doses (100–300 mg/kg). After the completion of experimental period animals were sacrificed, blood was collected through retro-orbital puncture for separation of serum for assessment of liver (ALT, AST, ALP, albumin, globulin and bilirubin) and kidney-specific biomarkers (creatinine, urea, and uric acid, Na+ and K+), and inflammatory cytokines (TNF-α, IL-6, IL-1β, TGF-β1, IL-10. Liver and kidneys were harvested for histopathology and biochemical screening. Hepatorenal protective activity was evaluated by estimating different biochemical variables and histopathology by hematoxylin and eosin staining of the liver and kidney tissues. Results and ConclusionsThree different daily doses of the plant extract showed recovery in all the biochemical parameters such as lipid peroxidation, reduced glutathione, superoxide dismutase, catalase, glucose-6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, and glutathione-S-transferase; ALT, AST, and ALP; creatinine, urea, and uric acid; total cellular protein content in tissues, glycogen content, and serum protein content (albumin and globulin); and inflammatory cytokines. However, significantly higher recovery was observed in the 300 mg/kg dose treated group of animals. Considerations of the serological, biochemical and histopathological studies, it may be concluded that that the C. igneus Nak leaf extract abolished various deleterious effects of type 1 diabetes induced hepatorenal functions.

High cytotoxicity of betulin towards fish and murine fibroblasts: Is betulin safe for nonneoplastic cells?

BackgroundBetulin, a natural pentacyclic triterpene with the lupane structure that is present in significant amounts in the outer bark of birch, is known for its broad array of biological and pharmacological properties. Betulin has attracted attention as a potential, natural-origin antimicrobial substance. The literature describes it as selectively toxic to neoplastic cells but safe for normal cells.The research aim was to evaluate the basal cytotoxicity of betulin towards fish (BF-2) and murine (NIH/3T3) fibroblasts. We used four colorimetric tests that provide a preliminary evaluation of possible mechanisms of the cytotoxicity of a compound to assess the degree of the toxicity of betulin after 24, 48 and 72 h of incubation with cells: the MTT assay (mitochondrial activity assessment), the NRU assay (lysosomal membrane integrity assessment), the LDH assay (cellular membrane integrity assessment) and the SRB assay (total cellular protein content determination).ResultsThe results revealed an exceptionally high sensitivity of mitochondria to the effect of betulin, with the other endpoints being less sensitive. Although murine fibroblasts were more vulnerable to the toxic effect of betulin than fish fibroblasts, the betulin CC50 values for both cell lines were comparable with analogous IC50 values determined by other researchers in studies involving cancerous cells.ConclusionsThe results indicate the need to verify the claim about the selective toxicity of betulin towards malignant cells and to conduct safety/toxicity tests before any potential therapeutic use of betulin in veterinary medicine.

Optimization of a T7-RNA polymerase system in Synechococcus sp. PCC 7002 mirrors the protein overproduction phenotype from E. coli BL21(DE3)

With the goal of expanding the diversity of tools available for controlling gene expression in cyanobacteria, the T7-RNA polymerase gene expression system from E. coli BL21(DE3) was adapted and systematically engineered for robust function Synechococcus sp. PCC 7002, a fast-growing saltwater strain. Expression of T7-RNA polymerase was controlled via LacI regulation, while functionality was optimized by both further tuning its expression level along with optimizing the translation initiation region of the expressed gene, in this case an enhanced YFP reporter. Under high CO2 conditions, the resulting system displayed a 60-fold dynamic range in expression levels. Furthermore, when maximally induced, T7-RNA polymerase-dependent protein production constituted up to two-thirds of total cellular protein content in Synechococcus sp. PCC 7002. Ultimately, however, this came at the cost of 40% reductions in both biomass and pigmentation levels. Taken together, the developed T7-RNA polymerase gene expression system is effective for controlling and achieving high-level expression of heterologous genes in Synechococcus sp. PCC 7002, making it a valuable tool for cyanobacterial research. KEY POINTS: • Promoter driving T7-RNA polymerase was optimized. • Up to 60-fold dynamic range in expression, depending on CO2 conditions. • Two-thirds of total protein is T7-RNA polymerase dependent.

Morphological plasticity of hyperelongated cells caused by overexpression of translation elongation factor P in Synechococcus elongatus PCC7942.

Translation elongation factors (EFs) are proteins that play important roles during the elongation stage of protein synthesis. In prokaryotes, at least four EFs function in repetitive reactions (EF-Tu, EF-Ts, EF-G, and EF-P). EF-P plays a vital role in the specialized translation of consecutive proline amino acid motifs. It was also recently recognized that EF-P acts throughout translation elongation. Here, we demonstrated for the first time that cell division and morphology are intimately linked to the control of EF-P in the model cyanobacterium Synechococcus elongatus PCC7942. We constructed the overexpression of a wild-type gene product for EF-P (Synpcc7942_2565) as a tool to identify EF-P functionality. The overexpression of EF-P resulted in the morphological plasticity of hyperelongated cells. During the stationary phase, EF-P overexpressors displayed cell lengths of 150μm or longer, approximately 35 times longer than the control. Total cellular protein and amino acid content were also increased in overexpressors. To explore the molecular mechanisms underlying hyperelongation, gene expression analysis was performed. The results revealed that cell division genes, including ftn6, minD, mreB, mreC, and ftsZ, were modulated in overexpressors. Strikingly, ftn6 was severely down-regulated. Little is known regarding EF-P in prokaryotic photosynthetic organisms. Our results suggest that cyanobacterial EF-P participates in the acceleration of protein synthesis and also regulates cell division processes. These findings suggest new ways to modify translation and metabolism in cyanobacteria. Phenotypic and metabolic alterations caused by overexpressing EF-P may also be beneficial for applications such as low-cost, green molecular factories. KEY POINTS: • Cell division and cell morphology in the cyanobacterium Synechococcus elongatus PCC7942 are closely linked with the control of translation elongation factor P (EF-P). • Overexpression of EF-P leads to morphological plasticity in hyperelongated cells. • Cyanobacterial EF-P is involved in the acceleration of protein synthesis and the regulation of cell division processes.

Nitrogen limitation reveals large reserves in metabolic and translational capacities of yeast

Cells maintain reserves in their metabolic and translational capacities as a strategy to quickly respond to changing environments. Here we quantify these reserves by stepwise reducing nitrogen availability in yeast steady-state chemostat cultures, imposing severe restrictions on total cellular protein and transcript content. Combining multi-omics analysis with metabolic modeling, we find that seven metabolic superpathways maintain >50% metabolic capacity in reserve, with glucose metabolism maintaining >80% reserve capacity. Cells maintain >50% reserve in translational capacity for 2490 out of 3361 expressed genes (74%), with a disproportionately large reserve dedicated to translating metabolic proteins. Finally, ribosome reserves contain up to 30% sub-stoichiometric ribosomal proteins, with activation of reserve translational capacity associated with selective upregulation of 17 ribosomal proteins. Together, our dataset provides a quantitative link between yeast physiology and cellular economics, which could be leveraged in future cell engineering through targeted proteome streamlining.

Differential Effects of Yeast NADH Dehydrogenase (Ndi1) Expression on Mitochondrial Function and Inclusion Formation in a Cell Culture Model of Sporadic Parkinson's Disease.

Parkinson’s disease (PD) is a neurodegenerative disorder that exhibits aberrant protein aggregation and mitochondrial dysfunction. Ndi1, the yeast mitochondrial NADH dehydrogenase (complex I) enzyme, is a single subunit, internal matrix-facing protein. Previous studies have shown that Ndi1 expression leads to improved mitochondrial function in models of complex I-mediated mitochondrial dysfunction. The trans-mitochondrial cybrid cell model of PD was created by fusing mitochondrial DNA-depleted SH-SY5Y cells with platelets from a sporadic PD patient. PD cybrid cells reproduce the mitochondrial dysfunction observed in a patient’s brain and periphery and form intracellular, cybrid Lewy bodies comparable to Lewy bodies in PD brain. To improve mitochondrial function and alter the formation of protein aggregates, Ndi1 was expressed in PD cybrid cells and parent SH-SY5Y cells. We observed a dramatic increase in mitochondrial respiration, increased mitochondrial gene expression, and increased PGC-1α gene expression in PD cybrid cells expressing Ndi1. Total cellular aggregated protein content was decreased but Ndi1 expression was insufficient to prevent cybrid Lewy body formation. Ndi1 expression leads to improved mitochondrial function and biogenesis signaling, both processes that could improve neuron survival during disease. However, other aspects of PD pathology such as cybrid Lewy body formation were not reduced. Consequently, resolution of mitochondrial dysfunction alone may not be sufficient to overcome other aspects of PD-related cellular pathology.

Biotin Identification Proteomics in Three-Dimensional Organotypic Human Skin Cultures.

Biotin identification (BioID) proteomics facilitates the unbiased detection of protein interaction neighborhoods in live cells. The BioID technique relies on the covalent biotin alteration of vicinal proteins by a modified bacterial biotin ligase. The biotin ligase is fused to a protein of interest to identify putative protein-protein interactions. Here, we describe the adaptation of this technique for use in three-dimensional epidermal cultures. Due to the covalent biotin modification of proteins, our protocol allows for the complete solubilization of the total cellular protein content in differentiated keratinocytes. Thus, a comprehensive network of potential interactors of a protein of interest can be mapped.

α-Cedrene, a Newly Identified Ligand of MOR23, Increases Skeletal Muscle Mass and Strength.

Skeletal muscle atrophy is a common and debilitating condition that lacks an effective therapy. In this study, the effects of α-cedrene are tested, a natural ligand of mouse olfactory receptor 23 (MOR23) whose ectopic function regulating myogenesis on skeletal muscle growth was reported recently. α-Cedrene not only stimulated hypertrophy but also attenuated free fatty acid-induced atrophy of cultured skeletal myotubes, as evidenced by an increased myotube diameter, fusion index, and total cellular protein content. These hypertrophic and antiatrophic properties of α-cedrene in cultured myotubes were confirmed in corresponding mouse models. The skeletal muscle mass, total muscle protein content, average cross-sectional area of myofibers, and muscle strength were significantly greater in α-cedrene-treated mice compared with untreated animals during either a regular chow diet or high-fat diet. Receptor knockdown experiments using RNA interference in cultured skeletal myotubes revealed that the hypertrophic and antiatrophic properties of α-cedrene may be mediated by MOR23. Furthermore, α-cedrene induced the expression of MOR23 and enhanced its downstream cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cyclic AMP-responsive element-binding protein (CREB) signaling in the skeletal muscle of mice fed chow or high-fat diet. α-Cedrene is a promising agent that may be applied to enhance the mass and strength of skeletal muscle.

Molecular dynamics of FMRP and other RNA-binding proteins in MEG-01 differentiation: the role of mRNP complexes in non-neuronal development.

Asymmetrically differentiating cells are formed with the aid of RNA-binding proteins (RBPs), which can bind, stabilize, regulate, and transport target mRNAs. The loss of RBPs in neurons may lead to severe neurodevelopmental diseases such as the Fragile X Syndrome with the absence of the Fragile X Mental Retardation Protein (FMRP). Because the latter is ubiquitous and shares many similarities with other RBPs involved in the development of peripheral cells, we suggest that FMRP would have a role in the differentiation of all tissues where it is expressed. A MEG-01 differentiation model was, therefore, established to study the global developmental functions of FMRP. PMA induction of MEG-01 cells causes important morphological changes driven by cytoskeletal dynamics. Cytoskeleton change and colocalization analyses were performed by confocal microscopy and sucrose gradient fractionation. Total cellular protein content and de novo synthesis were also analyzed. Microtubular transport mediates the displacement of FMRP and other RBP-containing mRNP complexes towards regions of the cell in development. De novo protein synthesis decreases significantly upon differentiation and total protein content composition is altered. Because those results are comparable with those obtained in neurons, the absence of FMRP would have significant consequences in cells everywhere in the body. The latter should be further investigated to give a better understanding of the systemic implications of imbalances of FMRP and other functionally similar RBPs.

Immunosuppressive and cytotoxic potential of aqueous leaves extract of Thlaspi arvense

Medicinal plants are traditionally used in Asian countries for treatment of various intracellular as well as extracellular infections. The present study was undertaken to investigate Thlaspi arvense for determining its immunosuppressive potential and cytotoxic activity in infected lysed human whole blood samples. For these studies, virally infected human whole blood samples (n = 5; symptoms such as sneezing, cough etc.) were collected and analysing its cytotoxicity of variable doses of aqueous leaves extract of Thlaspi arvense in the form of proliferation using Concanavalin (Con A; 2.5 µg/ml). In addition, immunosuppressive activity was also determined using lysed human whole blood samples were incubated with variable doses of aqueous extract in presence or absence of incomplete freunds adjuvant (IFA) pertaining to determine the total cellular (protein) content. In contrast, in vitro studies of aqueous extract of Thlaspi arvense were also determined using lactoferrin (100 µg/well) and ovalbumin (OVA, 100 µg/well) as coating antigen pertaining to antibody production using Elisa. The results showed that aqueous leaves extract of Thlaspi arvense showed markedly decline in Con A proliferation at higher doses. In addition, this aqueous extract also showed decline in total cellular content in presence or absence of IFA but it showed enhancement in antibody production in vitro against lactoferrin and OVA at higher doses. Overall the data showed that aqueous leaves extract of Thlaspi arvense displayed immunosuppressive and cytotoxic effect in virally infected human whole blood samples.

Immunological Exploration of Primary Metabolite Extracted From Aqueous Stem Extract of Caralluma Fimbriata

Objective: The objective of our study is to determined its immunological property of primary (protein) metabolite extracted from aqueous stem extract of Caralluma fimbriata against specific protein antigens (lactoferrin and Ovalbumin, OVA) pertaining to determine B cell production in vitro by ELISA (enzyme linked immunosorbent assay) and also measured T cell production in vitro using immature bone marrow cells of mice exposed with Incomplete freunds adjuvant (IFA) and Concanavalin (Con) A. Methods: For these studies, estimation of protein from stem extract of Caralluma fimbriata were determined and also determining the total cellular (protein) content in immature bone marrow cells along with variable concentration of Caralluma fimbriata in presence or absence of IFA. In addition, lactoferrin (100 µg/well) and weak antigen i.e. ovalbumin (OVA; 100 µg/well) pertaining to antibody (IgG) production were determined in vitro through Elisa and also measured its proliferative response using Con A (2.5 µg/ml). Results: The results showed that aqueous stem extract of Caralluma fimbriata showed the presence of protein (0.698 mg/ml; 10 µl) content and also showed the enhancement in total cellular (protein) content in presence or absence of IFA which is determined through NanoDrop method. In addition, this aqueous stem extract showed enhancement in anti-lactoferrin and anti-OVA IgG titre in vitro at higher doses as compared to control but there is sudden decline in bone marrow cell proliferation containing Con A at higher doses. Conclusion: Our data suggest that aqueous stem extract of Caralluma fimbriata may help to raise antibodies in vitro against lactoferrin and OVA but sudden decline in Con A proliferative response in bone marrow cells. In other words, aqueous stem extract containing primary metabolite of Caralluma fimbriata could be a potent immune enhancer of B cells in vitro but inhibitor of T cells at higher doses.

Biochemical Methods to Analyze Wnt Protein Secretion.

Wnt proteins act as potent morphogens in various aspects of embryonic development and adult tissue homeostasis. However, in addition to its physiological importance, aberrant Wnt signaling has been linked to the onset and progression of different types of cancer. On the cellular level, the secretion of Wnt proteins involves trafficking of lipid-modified Wnts from the endoplasmic reticulum (ER) to Golgi and further compartments via the Wnt cargo receptor evenness interrupted. Others and we have recently shown that Wnt proteins are secreted on extracellular vesicles (EVs) such as microvesicles and exosomes. Although more details about specific regulation of Wnt secretion steps are emerging, it remains largely unknown how Wnt proteins are channeled into different release pathways such as lipoprotein particles, EVs and cytonemes. Here, we describe protocols to purify and quantify Wnts from the supernatant of cells by either assessing total Wnt proteins in the supernatant or monitoring Wnt proteins on EVs. Purified Wnts from the supernatant as well as total cellular protein content can be investigated by immunoblotting. Additionally, the relative activity of canonical Wnts in the supernatant can be assessed by a dual-luciferase Wnt reporter assay. Quantifying the amount of secreted Wnt proteins and their activity in the supernatant of cells allows the investigation of intracellular trafficking events that regulate Wnt secretion and the role of extracellular modulators of Wnt spreading.

Cytotoxic and genotoxic effects of abamectin, chlorfenapyr, and imidacloprid on CHOK1 cells.

The cytotoxicity and genotoxicity of abamectin, chlorfenapyr, and imidacloprid have been evaluated on the Chinese hamster ovary (CHOK1) cells. Neutral red incorporation (NRI), total cellular protein content (TCP), and methyl tetrazolium (MTT) assays were followed to estimate the mid-point cytotoxicity values, NRI50, TCP50, and MTT50, respectively. The effects of the sublethal concentration (NRI25) on glutathione S-transferase (GST), glutathione reductase (GRD), glutathione peroxidase (GPX), and total glutathione content have been evaluated in the presence and absence of reduced glutathione (GSH), vitamin C, and vitamin E. The genotoxicity was evaluated using chromosomal aberrations (CA), micronucleus (MN) formation, and DNA fragmentation techniques in the presence and absence of the metabolic activation system, S9 mix. Abamectin was the most cytotoxic pesticide followed by chlorfenapyr, while imidacloprid was the least cytotoxic one. The glutathione redox cycle components were altered by the tested pesticides in the absence and presence of the tested antioxidants. The results of genotoxicity indicate that abamectin, chlorfenapyr, and imidacloprid have potential genotoxic effects on CHOK1 cells under the experimental conditions.

Study of Protein Phosphatase 2A (PP2A) Activity in LPS-Induced Tolerance Using Fluorescence-Based and Immunoprecipitation-Aided Methodology.

Protein phosphatase 2A (PP2A) is one of the most abundant intracellular serine/threonine (Ser/Thr) phosphatases accounting for 1% of the total cellular protein content. PP2A is comprised of a heterodimeric core enzyme and a substrate-specific regulatory subunit. Potentially, at least seventy different compositions of PP2A exist because of variable regulatory subunit binding that accounts for various activity modulating numerous cell functions. Due to the constitutive phosphatase activity present inside cells, a sensitive assay is required to detect the changes of PP2A activity under various experimental conditions. We optimized a fluorescence assay (DIFMU assay) by combining it with prior anti-PP2A immunoprecipitation to quantify PP2A-specific phosphatase activity. It is also known that prior exposure to lipopolysaccharides (LPS) induces “immune tolerance” of the cells to subsequent stimulation. Herein we report that PP2A activity is upregulated in tolerized peritoneal macrophages, corresponding to decreased TNF-α secretion upon second LPS stimulation. We further examined the role of PP2A in the tolerance effect by using PP2ACαlox/lox;lyM-Cre conditional knockout macrophages. We found that PP2A phosphatase activity cannot be further increased by tolerance. TNF-α secretion from tolerized PP2ACαlox/lox;lyM-Cre macrophages is higher than tolerized control macrophages. Furthermore, we showed that the increased TNF-α secretion may be due to an epigenetic transcriptionally active signature on the promoter of TNF-α gene rather than regulation of the NFκB/IκB signaling pathway. These results suggest a role for increased PP2A activity in the regulation of immune tolerance.

Decrease in Ribosomal RNA in Candida albicans Induced by Serum Exposure.

Candida albicans is an important polymorphic human pathogen. It can switch from a unicellular yeast form to germinating hypha, which may play a role in making it the successful pathogen it is. This hyphal transformation can be triggered by various extracellular stimuli, the most potent one being serum from any source. We have previously reported that Candida albicans transiently polyadenylates portions of both the large and small subunits of ribosomal RNA, shortly after serum exposure. Northern blots at the same time suggested that serum might induce a decrease in total ribosomal RNA. We have carried out a number of experiments to carefully assess this possibility and now report that serum significantly reduces ribosomal RNA in Candida albicans. Fluorometric measurements, Northern blotting and quantitative RT-PCR, have all confirmed this decrease. Timed experiments show that serum induces this decrease rapidly, as it was seen in as early as five minutes. Cell mass is not decreased as total cellular protein content remains the same and metabolic activity does not appear to slow, as assessed by XTT assay, and by the observation that cells form hyphal structures robustly. Another hyphal inducer, N-acetylglucosamine, also caused RNA decrease, but to a lesser extent. We also observed it in non-germinating yeast, such as Candida glabrata. The reason for this decrease is unknown and overall our data suggests that decrease in rRNA does not play a causal role in hyphal transformation. Rapid and significant decrease in a molecule so central to the yeast’s biology is of some importance, and further studies, such as its effect on protein metabolism, will be required to better understand its purpose.

Cytotoxic effects of acephate, ethoprophos, and monocrotophos in CHO-K1 cells

This work evaluates the cytotoxicity of acephate, ethoprophos, and monocrotophos in the Chinese hamster ovary cell line. The neutral red incorporation (NRI), total cellular protein (TCP) content, and methyl tetrazolium (MTT) assays were followed to estimate the midpoint cytotoxicity values, NRI50, TCP50, and MTT50, respectively. The targeted cells were exposed to the tested pesticides in the presence and absence of fetal calf serum. The effects of the sublethal concentration (NRI25) on glutathione S-transferase, glutathione reductase, glutathione peroxidase, and total glutathione content have been evaluated. In addition, the ameliorative effects of extracellular reduced glutathione (GSH, 1 mM), vitamin C (70 µM), and vitamin E (30 µM) were investigated. The exposure period and protein content in the culture medium affected the cytotoxicity of the tested pesticides. Unexpectedly, acephate showed the least MTT50 value, 56.6 µM. The tested antioxidants caused different and significant effects on the components of the glutathione redox cycle.

Inhibition of hepatitis E virus replication by proteasome inhibitor is nonspecific.

The ubiquitin proteasome system plays important role in virus infection. A previous study showed that the proteasome inhibitor MG132 could potentially affect hepatitis E virus (HEV) replication. In this study, we found that MG132 could inhibit HEV and hepatitis C virus (HCV) replication-related luciferase activity in subgenomic models. Furthermore, treatment with MG132 in a HEV infectious model resulted in a dramatic reduction in the intracellular level of HEV RNA. Surprisingly, MG132 concurrently inhibited the expression of a luciferase gene used as a control as well as a wide range of host genes. Consistently, the total cellular RNA and protein content was concurrently reduced by MG132 treatment, suggesting a nonspecific antiviral effect.