Lower Protein Expression Levels Research Articles

Structural and Mechanistic Studies Reveal the Functional Role of Bicovalent Flavinylation in Berberine Bridge Enzyme

Berberine bridge enzyme (BBE) is a member of the recently discovered family of bicovalently flavinylated proteins. In this group of enzymes, the FAD cofactor is linked via its 8alpha-methyl group and the C-6 atom to conserved histidine and cysteine residues, His-104 and Cys-166 for BBE, respectively. 6-S-Cysteinylation has recently been shown to have a significant influence on the redox potential of the flavin cofactor; however, 8alpha-histidylation evaded a closer characterization due to extremely low expression levels upon substitution. Co-overexpression of protein disulfide isomerase improved expression levels and allowed isolation and purification of the H104A protein variant. To gain more insight into the functional role of the unusual dual mode of cofactor attachment, we solved the x-ray crystal structures of two mutant proteins, H104A and C166A BBE, each lacking one of the covalent linkages. Information from a structure of wild type enzyme in complex with the product of the catalyzed reaction is combined with the kinetic and structural characterization of the protein variants to demonstrate the importance of the bicovalent linkage for substrate binding and efficient oxidation. In addition, the redox potential of the flavin cofactor is enhanced additively by the dual mode of cofactor attachment. The reduced level of expression for the H104A mutant protein and the difficulty of isolating even small amounts of the protein variant with both linkages removed (H104A-C166A) also points toward a possible role of covalent flavinylation during protein folding.

Fusion order controls expression level and activity of elastin‐like polypeptide fusion proteins

We have previously developed a method to purify recombinant proteins, termed inverse transition cycling (ITC) that eliminates the need for column chromatography. ITC exploits the inverse solubility phase transition of an elastin-like polypeptide (ELP) that is fused to a protein of interest. In ITC, a recombinant ELP fusion protein is cycled through its phase transition, resulting in separation of the ELP fusion protein from other Escherichia coli contaminants. Herein, we examine the role of the position of the ELP in the fusion protein on the expression levels and yields of purified protein for four recombinant ELP fusion proteins. Placing the ELP at the C-terminus of the target protein (protein-ELP) results in a higher expression level for the four ELP fusion proteins, which also translates to a greater yield of purified protein. The position of the fusion protein also has a significant impact on its specific activity, as ELP-protein constructs have a lower specific activity than protein-ELP constructs for three out of the four proteins. Our results show no difference in mRNA levels between protein-ELP and ELP-protein fusion constructs. Instead, we suggest two possible explanations for these results: first, the translational efficiency of mRNA may differ between the fusion protein in the two orientations and second, the lower level of protein expression and lower specific activity is consistent with a scenario that placement of the ELP at the N-terminus of the fusion protein increases the fraction of misfolded, and less active conformers, which are also preferentially degraded compared to fusion proteins in which the ELP is present at the C-terminal end of the protein.

Boosted expression of the SARS-CoV nucleocapsid protein in tobacco and its immunogenicity in mice

Vaccines produced in plant systems are safe and economical; however, the extensive application of plant-based vaccines is mainly hindered by low expression levels of heterologous proteins in plant systems. Here, we demonstrated that the post-transcriptional gene silencing suppressor p19 protein from tomato bushy stunt virus substantially enhanced the transient expression of recombinant SARS-CoV nucleocapsid (rN) protein in Nicotiana benthamiana. The rN protein in the agrobacteria-infiltrated plant leaf accumulated up to a concentration of 79μg per g fresh leaf weight at 3 days post infiltration. BALB/c mice were intraperitoneally vaccinated with pre-treated plant extract emulsified in Freund's adjuvant. The rN protein-specific IgG in the mouse sera attained a titer about 1:1,800 following three doses of immunization, which suggested effective B-cell maturation and differentiation in mice. Antibodies of the subclasses IgG1 and IgG2a were abundantly present in the mouse sera. During vaccination of rN protein, the expression of IFN-γ and IL-10 was evidently up-regulated in splenocytes at different time points, while the expression of IL-2 and IL-4 was not. Up to now, this is the first study that plant-expressed recombinant SARS-CoV N protein can induce strong humoral and cellular responses in mice.

Expression of Nucleotide-Binding Oligomerization Domain 2 in Normal Human Dental Pulp Cells and Dental Pulp Tissues

Introduction The nucleotide-binding oligomerization domain (NOD) proteins belong to a distinct family of proteins that are implicated in the intracellular recognition of bacterial components. NOD2 appears to be a sensor of bacterial peptidoglycans because it recognizes a minimal motif present in all peptidoglycans. The interaction of NOD2 with downstream signaling molecules ultimately results in the activation of NF-κB and production of inflammatory mediators in innate immunity. As such, NOD2 may play an important role in the detection of bacterial pathogens and the initiation of inflammation within the dental pulp. This study was designed to evaluate the expression of NOD2 in normal human dental pulp cells (HDPCs) and human pulp tissues. Methods Human pulp tissue samples were collected from freshly extracted human wisdom teeth, and HDPCs were prepared from the explants of normal human dental pulp tissues. Nested reverse-transcription polymerase chain reaction (Nested RT-PCR) and Western blotting were performed to detect the expression of NOD2 messenger RNA and protein, respectively. Immunohistochemical staining was used to determine the distribution of NOD2 in the pulp tissues. Results The NOD2 messenger RNA and protein were present in normal human dental pulp tissues, with most NOD2 protein expression being localized to odontoblasts and some pulp vascular endothelial cells. In contrast, HDPCs only showed a low level of NOD2 protein expression. Conclusions Our results suggest that NOD2 protein expressed in HDPCs and pulp tissues may play an important role in dental immune defense.

HBZ is an immunogenic protein, but not a target antigen for human T-cell leukemia virus type 1-specific cytotoxic T lymphocytes

Recently, HBZ has been reported to play an important role in the proliferation of adult T-cell leukaemia (ATL) cells and might be a target of novel therapy for ATL. To develop a novel immunotherapy for ATL, we verified the feasibility of cellular immunotherapy targeting HBZ. We established an HBZ-specific and HLA-A*0201-restricted cytotoxic T lymphocyte (CTL) clone. Detailed study using this CTL clone clearly showed that HBZ is certainly an immunogenic protein recognizable by human CTLs; however, HBZ-specific CTLs could not lyse ATL cells. Failure of HBZ-specific CTLs to recognize human T-cell leukemia virus type 1 (HTLV-1)-infected cells might be due to a low level of HBZ protein expression in ATL cells and resistance of HTLV-1-infected cells to CTL-mediated cytotoxicity. Although HBZ plays an important role in the proliferation of HTLV-1-infected cells, it may also provide a novel mechanism that allows them to evade immune recognition.

An allelic series of CD45 dosages uncouples basal and inducible TCR signaling during thymic development (84.13)

Abstract CD45 is a receptor-like tyrosine phosphatase expressed at very high levels on all nucleated hematopoietic cells. CD45 serves as a critical positive regulator of TCR signal transduction by dephosphorylating the inhibitory tyrosine of the Src family kinases, Lck and Fyn. Previously, CD45 deficient animals have revealed a critical positive regulatory role in beta-selection and positive selection during thymic development. Here we describe a novel allele of CD45, lightning, in which a single residue substitution in the extracellular domain produces low protein expression levels but normal isoform splicing. An allelic series of CD45 dosages reveals differential requirements for CD45 in basal and inducible TCR signaling during thymic development. While low levels of CD45 are sufficient to rescue T cell development and TCR inducible signaling, high doses of CD45 are required to fully reconstitute beta-selection and basal signaling. Furthermore, a negative regulatory role for CD45 is identified during inducible but not basal signaling. Hyperphosphorylation of the activation loop tyrosine of Lck but not Fyn at intermediate doses of CD45 correlates with hyper-responsive inducible signaling and demonstrates differential regulation of the src family kinases. Further, distinct thymic subsets exhibit differential requirements for CD45, Fyn and co-receptor-associated Lck in TCR signaling.

Diversity of Ehrlichia ruminantium Major Antigenic Protein 1-2 in Field Isolates and Infected Sheep

Proteins expressed from the map1 multigene family of Ehrlichia ruminantium are strongly recognized by immune T and B cells from infected animals or from animals that were infected and have recovered from heartwater disease (although still remaining infected carriers). Analogous multigene clusters also encode the immunodominant outer membrane proteins (OMPs) in other ehrlichial species. Recombinant protein analogs of the expressed genes and DNA vaccines based on the multigene clusters have been shown to induce protective immunity, although this was less effective in heterologous challenge situations, where the challenge strain major antigenic protein 1 (MAP1) sequence differed from the vaccine strain MAP1. Recent data for several ehrlichial species show differential expression of the OMPs in mammalian versus tick cell cultures and dominant expression of individual family members in each type of culture system. However, many genes in the clusters appear to be complete and functional and to generate mRNA transcripts. Recent data also suggest that there may be a low level of protein expression from many members of the multigene family, despite primary high-level expression from an individual member. A continuing puzzle, therefore, is the biological roles of the different members of these OMP multigene families. Complete genome sequences are now available for two geographically divergent strains of E. ruminantium (Caribbean and South Africa strains). Comparison of these sequences revealed amino acid sequence diversity in MAP1 (89% identity), which is known to confer protection in a mouse model and to be the multigene family member primarily expressed in mammalian cells. Surprisingly, however, the greatest sequence diversity (79% identity) was in the less-studied map1-2 gene. We investigated here whether this map1-2 diversity was a general feature of E. ruminantium in different cultured African strains and in organisms from infected sheep. Comparison of MAP1-2s revealed amino acid identities of 75 to 100% (mean of 86%), compared to 84 to 100% (mean of 89%) for MAP1s. Interestingly, MAP1-2s varied independently of MAP1s such that E. ruminantium strains with similar MAP1s had diverse MAP1-2s and vice versa. Different MAP1-2s were found in individual infected sheep. Different regions of a protein may be subjected to different evolutionary forces because of recombination and/or selection, which results in those regions not agreeing with a phylogeny deduced from the whole molecule. This appears to be true for both MAP1 and MAP1-2, where statistical likelihood methods detect heterogeneous evolutionary rates for segments of both molecules. Sera from infected cattle recognized a MAP1-2 variable-region peptide in enzyme-linked immunosorbent assay, but less strongly and consistently than a MAP1 peptide (MAP1B). Heterologous protective immunity may depend on recognition of a complex set of varying OMP epitopes.

HER2 protein expression predicts response to trastuzumab in FISH-positive patients.

Abstract Abstract #32 BACKGROUND: Fluorescent in Situ Hybridization (FISH) is currently employed to select patients for trastuzumab or lapatinib therapy. However, only a portion of patients will respond to these HER2-directed therapies. Using a novel assay to quantitate total HER2 (H2T) and HER2 homodimer (H2D) levels, we examined the relationship between these measurements and clinical outcomes in a cohort of trastuzumab-treated metastatic breast cancer patients previously assessed as FISH positive by central testing.
 METHODS: The VeraTag assay was used to measure H2T and H2D. A cutoff previously defined by positional scanning was used to classify patients as H2T high or low (Leitzel et al. ASCO abstract # 1002, 2008). FISH status was determined using the Vysis assay and was performed by a single pathologist under standardized conditions (central testing). FISH + was defined as HER2/CEP17 ratio greater than 2.2. Kaplan-Meier and Cox proportional hazards analyses (TTP and OS) were conducted.
 RESULTS: 99 MBC patients previously scored as IHC 3+ or 2+, FISH+ and treated with first line trastuzumab-containing regimens were re-tested by central FISH. 22 patients were found to be FISH- and 77 were FISH+. 67 FISH+ patients had high H2T levels, and 19 FISH- patients were H2T low. 14% (3/22) of FISH- patients were H2T high, and 13% (10/77) of FISH+ patients were H2T low (overall discordance rate = 13%). In Kaplan Meier analyses, patients assessed as FISH+, H2T high had a significantly longer TTP (median TTP=11.3 mos.) compared to those who were FISH-, H2T low (median TTP=4.5 mos. HR=0.42, p=0.0006), and also compared to those who were FISH+, H2T low (median TTP=3.7 mos. HR=0.43, p=0.01). Those patients who had discordant results (FISH+, but H2T low) behaved similarly compared with FISH-, H2T low (HR=1, p=0.99). Similar results were observed using H2D. In analyses restricted to central FISH+ patients only (N=77), Cox proportional hazards multivariate regression identified H2T as an independent predictor of TTP (HR=0.29, p=0.0015) and OS (HR=0.37, p=0.02).
 CONCLUSION: In metastatic breast cancer patients previously assessed as FISH-positive by central testing, we observed an approximate 55-fold distribution of HER2 protein expression. Patients with HER2 gene amplification by FISH but low HER2 total protein expression or homodimer levels as measured by the VeraTag assay respond poorly to trastuzumab-containing therapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 32.

Sonic hedgehog mutations identified in holoprosencephaly patients can act in a dominant negative manner

Sonic hedgehog (SHH) plays an important instructional role in vertebrate development, as exemplified by the numerous developmental disorders that occur when the SHH pathway is disrupted. Mutations in the SHH gene are the most common cause of sporadic and inherited holoprosencephaly (HPE), a developmental disorder that is characterized by defective prosencephalon development. SHH HPE mutations provide a unique opportunity to better understand SHH biogenesis and signaling, and to decipher its role in the development of HPE. Here, we analyzed a panel of SHH HPE missense mutations that encode changes in the amino-terminal active domain of SHH. Our results show that SHH HPE mutations affect SHH biogenesis and signaling at multiple steps, which broadly results in low levels of protein expression, defective processing of SHH into its active form and protein with reduced activity. Additionally, we found that some inactive SHH proteins were able to modulate the activity of wt SHH in a dominant negative manner, both in vitro and in vivo. These findings show for the first time the susceptibility of SHH driven developmental processes to perturbations by low-activity forms of SHH. In conclusion, we demonstrate that SHH mutations found in HPE patients affect distinct steps of SHH biogenesis to attenuate SHH activity to different levels, and suggest that these variable levels of SHH activity might contribute to some of the phenotypic variation found in HPE patients.

Heme Oxygenase-1 Prevents Airway Mucus Hypersecretion Induced by Cigarette Smoke in Rodents and Humans

We investigated the role of heme oxygenase-1 (HO-1), a powerful anti-inflammatory and anti-oxidant enzyme, in modulating cigarette smoke (CS)-induced mucus secretion. In both rats and mice, 5-day CS exposure increased HO-1 expression and activity, mucus secretion, MUCIN 5AC (MUC5AC) gene and protein expression, and local inflammation, along with up-regulation of dual oxidase 1 gene expression and both the activity and phosphorylation of the epidermal growth factor receptor, which is involved in MUC5AC induction. Pharmacological induction of HO-1 prevented these actions and inhibition of HO-1 expression by a specific siRNA potentiated them. In French participants to the European Community Respiratory Health Survey II (n = 210, 30 to 53 years of age, 50% males) exposed to CS, a significant increase in the percentage of participants with chronic sputum was observed in those harboring at least one allele with a long (GT)(n) in the HO-1 promoter gene (>33 repeats), which is associated with a low level of HO-1 protein expression, compared with those with a short number of (GT)n repeats (21.7% versus 8.6%, P = 0.047). No such results were observed in those who had never smoked (n = 297). We conclude that HO-1 has a significant protective effect against airway mucus hypersecretion in animals and humans exposed to CS.

Signaling-mediated Functional Activation of Inducible Nitric-oxide Synthase and Its Role in Stimulating Platelet Activation

Nitric oxide (NO) is a short lived secondary messenger, synthesized by nitric-oxide synthases (NOS). It is believed that the activity of inducible NOS (iNOS) is regulated primarily at the transcription level by inducing expression of iNOS mRNA and protein, which then continuously produces NO, until its degradation. Platelets do not have the nuclear transcriptional regulatory mechanisms of the iNOS gene and are believed to generate NO in response to agonist stimulation via endothelial NOS (eNOS). However, here we show that agonist-induced NO production is only partially eNOS-dependent and is also mediated by iNOS. Platelet agonist-induced NO production is significantly reduced in iNOS-knockout platelets. Platelet NO production occurs within seconds after agonist addition and is not accompanied by changes in iNOS protein levels, indicating a signaling-mediated functional activation mechanism of iNOS. Importantly, iNOS knock-out and iNOS inhibitors reduce agonist-induced platelet secretion and aggregation and cGMP levels, indicating that iNOS activation is important in stimulating platelets via the newly identified NO-cGMP-dependent platelet secretion pathway. Furthermore, iNOS knock-out mice have prolonged bleeding time, suggesting that this novel mode of regulation of iNOS activity plays a physiologically relevant role in hemostasis.

Protein Quaternary Structure and Expression Levels Contribute to Peroxisomal-Targeting-Sequence-1-Mediated Peroxisomal Import of Human Soluble Epoxide Hydrolase

The peroxisomal targeting sequence 1 (PTS1) is a consensus tripeptide 1 (S/C/A)(K/R/H)(L/M) that is found at the C-terminus of most peroxisomal proteins. However, the only known mammalian protein containing a terminal methionine PTS1 (SKM), human soluble epoxide hydrolase (hsEH), shows both peroxisomal and cytosolic localizations in vivo. Mechanisms regulating the subcellular localization of hsEH thus remain unclear. Here we utilized green fluorescent protein–hsEH fusion constructs to study the peroxisomal targeting of hsEH in transiently and stably transfected Chinese hamster ovary cells. Our results suggest that the peroxisomal import of hsEH is regulated by three factors. First, we show that SKM is required, but not sufficient, for peroxisomal import. Second, by manipulating protein expression levels, we show that SKM mediates peroxisomal import of wild-type hsEH only when expression levels are high. Third, we show that amino acid modifications that decrease subunit oligomerization and presumably enhance accessibility of the SKM motif confer peroxisomal targeting even at low protein expression levels. We conclude that, in hsEH, SKM is a necessary but inefficient and context-dependent PTS1. Peroxisomal import occurs when expression levels are high or when the SKM motif is accessible. These results provide a mechanistic basis for understanding the cell-specific and tissue-specific localization of hsEH in vivo.

Expression and promoter methylation of the RASSF1A gene in sporadic breast cancers in Chinese women

The novel tumor suppressor RASSF1A is frequently inactivated during human tumorigenesis by promoter methylation. In this study, we detected the RASSF1A promoter methylation by methylated-specific PCR and investigated RASSF1A gene expression by semi-quantitative RT-PCR and immunohistochemical staining in 36 cases of breast cancer and their adjacent normal tissues in Chinese women. The promoter methylation of the RASSF1A gene was found to be a frequent event in the breast cancers (61.1%). RASSF1A methylation was not found in the matched adjacent normal tissues. The loss frequency of RASSF1A mRNA was 33.3% and that of the RASSF1A protein was 44.4% in breast cancers. RASSF1A mRNA and protein were all expressed in adjacent normal tissues. The mRNA and protein expression level of RASSF1A was significantly lower in breast cancer than in adjacent normal tissue. However, the promoter methylation of the RASSF1A gene in breast cancers were not correlated with clinical parameters, such as ages, histological types, TNM stages and lymph node metastases. Thus, the promoter methylation of RASSF1A was one reason for the low level of RASSF1A mRNA and protein expression and was a frequent event in primary sporadic breast tumorigenesis in Chinese women.

The Aspergillus nidulans FcyB cytosine‐purine scavenger is highly expressed during germination and in reproductive compartments and is downregulated by endocytosis

We cloned and characterized an Aspergillus nidulans gene, called fcyB, encoding the closest homologue to the yeast Fcy2p/Fcy21p permeases. Deletion of fcyB (DeltafcyB) does not affect growth, development, reproduction or bulk purine uptake, but eliminates the leaky growth on purines of DeltaazgADeltauapCDeltauapA strains, lacking all known purine transporters, and confers resistance to the antifungal 5-fluorocytosine. Kinetic analyses showed FcyB is a low-capacity, high-affinity, cytosine-purine transporter sharing similar molecular interactions for substrate recognition with the yeast Fcy2p/Fcy21p carriers. fcyB transcription is highly activated during germination but drops at low constitutive levels throughout vegetative development. UaY-mediated purine induction of fcyB transcription is only moderate, while ammonium represses transcription through an AreA-dependent mechanism. A strain expressing FcyB-GFP confirms a low protein expression level in the plasma membrane of vegetative mycelia, but reveals an abundant expression in sexual and asexual compartments. FcyB-GFP was also shown to be downregulated by endocytosis in response to ammonia or the presence of cytosine. The expression profile of FcyB supports that its main physiological role is cytosine-purine scavenging.

Relationship between the expression of RASSF1A protein and promoter hypermethylation of RASSF1A gene in bladder tumor

To investigate the relationship between the expression of RASSF1A protein and promoter hypermethylation of RASSF1A gene, RASSF1A protein expression was measured by Western blotting in 10 specimens of normal bladder tissues and 23 specimens of bladder transitional cell carcinoma (BTCC). The promoter methylation in BTCC and normal bladder tissues was detected by methylation-specific PCR (MSP). The results showed that the expression level of RASSF1A protein was significantly lower in BTCC tissues than that in normal bladder tissues. However, it was not correlated with its clinical stages and pathological grades. The frequency of promoter methylation of RASSF1A gene was higher in BTCC tissues than that in normal bladder tissues. In 14 patients with the aberrant promoter methylation, 13 showed loss or low expression of RASSF1A protein. It is concluded that RASSF1A gene promoter methylation may contribute to the low level or loss of RASSF1A protein expression, the inactivation of RASSF1A gene and the genesis of BTCC. But, it may bear no correlation with its clinical stages and pathological grades.

Purification of a human immunoglobulin G1 monoclonal antibody from transgenic tobacco using membrane chromatographic processes

Efficient purification of protein biopharmaceuticals from transgenic plants is a major challenge, primarily due to low target protein expression levels, and high impurity content in the feed streams. These challenges may be addressed by using membrane chromatography. This paper discusses the use of cation-exchange and Protein A affinity-based membrane chromatographic techniques, singly and in combination for the purification of an anti- Pseudomonas aerugenosa O6ad human IgG1 monoclonal antibody from transgenic tobacco. Protein A membrane chromatography on its own was unable to provide a pure product, mainly due to extensive non-specific binding of impurities. Moreover, the Protein A membrane showed severe fouling tendency and generated high back-pressure. With cation-exchange membrane chromatography, minimal membrane fouling and high permeability were observed but high purity could not be achieved using one-step. Therefore, by using a combination of the cation-exchange and Protein A membrane chromatography, in that order, both high purity and recovery were achieved with high permeability. The antibody purification method was first systematically optimized using a simulated feed solution. Anti- P. aeruginosa human IgG1 type monoclonal antibody was then purified from transgenic tobacco juice using this optimized method.

Age Related Changes from Youth to Adulthood in Rat Brain Cortex: Nitric Oxide Synthase and Mitochondrial Respiratory Function

Age related changes in brain cortex NO metabolism were investigated in mitochondria and cytosolic extracts from youth to adulthood. Decreases of 19%, 40% and 71% in NO production were observed in mitochondrial fractions from 3, 7, and 14 months old rats, respectively, as compared with 1-month-old rats. Decreased nNOS protein expression in 14 months old rats was also observed in mitochondria as compared with the nNOS protein expression in 1-month-old rats. Low levels of eNOS protein expression close to the detection limits and no iNOS protein expression were significantly detected in mitochondrial fraction for both groups of age. NO production in the cytosolic extracts also showed a marked decreasing tendency, showing higher levels than those observed in mitochondrial fractions for all groups of age. In the cytosolic extracts, however, the levels were stabilized in adult animals from 7 to 14 months. nNOS protein expression showed a similar age-pattern in cytosolic extracts for both groups of age, while the protein expression pattern for eNOS was higher expressed in adult rats (14 months) than in young animals. As well as in mitochondrial extracts iNOS protein expression was not significantly detected in cytosolic extracts at any age. RT-PCR assays indicated increased levels of nNOS mRNA in 1-month-old rats as compared with 14 months old rats, showing a similar pattern to that one observed for protein nNOS expression. A different aged pattern was observed for eNOS mRNA expression, being lower in 1-month-old rats as compared with 14 months old animals. iNOS mRNA was very low expressed in both groups of age, showing a residual iNOS mRNA that was not significantly detected. State 3 respiration rates were 78% and 85% higher when succinate and malate-glutamate were used as substrates, respectively, in 14 months rats as compared with 1-month-old rats. No changes were observed in state 4 respiration rates. These results could indicate 1 that nNOS and eNOS mRNA and protein expression can be age-dependent, and confirmed the nNOS origin for the mitochondrial NOS. During rat growth, the respiratory function seems to be modulated by NO produced by the different NOS enzymes: nNOS, eNOS and mtNOS present in the cytosol and in the mitochondria.

Glucose up‐regulates HIF‐1α expression in primary cortical neurons in response to hypoxia through maintaining cellular redox status

It has been suggested that hypoxia-inducible factor 1 (HIF-1), a key regulator in cell's adaptation to hypoxia, plays an important role in the fate of neurons during ischemia. However, the mechanism of HIF-1 regulation is still not fully understood in neurons subjected to ischemia. In this study, we demonstrated that glucose up-regulated the expression of HIF-1alpha, the oxygen-dependent subunit of HIF-1, in rat primary cortical neurons exposed to hypoxia. To understand the mechanism of glucose-regulated HIF-1alpha expression, we investigated the relationships between HIF-1alpha expression, reactive oxygen species (ROS), and redox status. Low levels of HIF-1alpha protein expression were observed in the neurons exposed to in vitro ischemic conditions that had high levels of ROS (oxidizing environments), and vice versa. The glutathione (GSH) precursor, N-acetyl cysteine, induced HIF-1alpha protein expression in hypoxic neurons while the GSH synthesis inhibitor, l-buthionine sulfoximine, inhibited the expression. Moreover, (-)-epicatechin gallate, a ROS scavenger, elevated HIF-1alpha expression in the neurons subjected to in vitro ischemia. Furthermore, results from a systemic hypoxia model showed that a reducing environment increased HIF-1alpha expression in rat brains. Taken together, these data presented the first evidence that glucose promoted HIF-1alpha stabilization through regulating redox status in primary neurons exposed to hypoxia. The results imply that hypoxia only may not be sufficient to stabilize HIF-1alpha and that a reducing environment is required to stabilize HIF-1alpha in neurons exposed to hypoxia.

Development and Characterization of Pectinate Micro/Nanoparticles for Gene Delivery

The aim of this study was to investigate the possibility of using pectinate micro/nanoparticles as gene delivery systems. Pectinate micro/nanoparticles were produced by ionotropic gelation. Various factors were studied for their effects on the preparation of pectinate micro/nanoparticles: the pH of the pectin solution, the ratio of pectin to the cation, the concentration of pectin and the cation, and the type of cation (calcium ions, magnesium ions and manganese ions). After the preparation, the size and charge of the pectin micro/nanoparticles and their DNA incorporation efficiency were evaluated. The results showed that the particle sizes decreased with the decreased concentrations of pectin and cation. The type of cations affected the particle size. Sizes of calcium pectinate particles were larger than those of magnesium pectinate and manganese pectinate particles. The DNA loading efficiency showed that Ca-pectinate nanoparticles could entrap DNA up to 0.05 mg when the weight ratio of pectin:CaCl(2):DNA was 0.2:1:0.05. However, Mg-pectinate could entrap only 0.01 mg DNA when the weight ratio of pectin:MgCl(2):DNA was 1:100:0.01 The transfection efficiency of both Ca-pectinate and Mg-pectinate nanoparticles yielded relatively low levels of green fluorescent protein expression and low cytotoxicity in Huh7 cells. Given the negligible cytotoxic effects, these pectinate micro/nanoparticles can be considered as potential candidates for use as safe gene delivery carriers.

Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate

Background:Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD) genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate – basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial – and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes.Results:Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63). Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50) but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance.Conclusion:Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers.