Isolation of Dihydroflavonol 4-Reductase cDNA Clones from Angelonia x angustifolia and Heterologous Expression as GST Fusion Protein in Escherichia coli

Blue Angelonia × angustifolia flowers can show spontaneous mutations resulting in white/blue and white flower colourations. In such a white line, a loss of dihydroflavonol 4-reductase (DFR) activity was observed whereas chalcone synthase and flavanone 3-hydroxylase activity remained unchanged. Thus, cloning and characterization of a DFR of Angelonia flowers was carried out for the first time. Two full length DFR cDNA clones, Ang.DFR1 and Ang.DFR2, were obtained from a diploid chimeral white/blue Angelonia × angustifolia which demonstrated a 99% identity in their translated amino acid sequence. In comparison to Ang.DFR2, Ang.DFR1 was shown to contain an extra proline in a proline-rich region at the N-terminus along with two exchanges at the amino acids 12 and 26 in the translated amino acid sequence. The recombinant Ang.DFR2 obtained by heterologous expression in yeast was functionally active catalyzing the NADPH dependent reduction of dihydroquercetin (DHQ) and dihydromyricetin (DHM) to leucocyanidin and leucomyricetin, respectively. Dihydrokaempferol (DHK) in contrast was not accepted as a substrate despite the presence of asparagine in a position assumed to determine DHK acceptance. We show that substrate acceptance testing of DFRs provides biased results for DHM conversion if products are extracted with ethyl acetate. Recombinant Ang.DFR1 was inactive and functional activity could only be restored via exchanges of the amino acids in position 12 and 26 as well as the deletion of the extra proline. E. coli transformation of the pGEX-6P-1 vector harbouring the Ang.DFR2 and heterologous expression in E. coli resulted in functionally active enzymes before and after GST tag removal. Both the GST fusion protein and purified DFR minus the GST tag could be stored at −80°C for several months without loss of enzyme activity and demonstrated identical substrate specificity as the recombinant enzyme obtained from heterologous expression in yeast.     

Identification of Differentially Expressed Proteins in Direct Expressed Prostatic Secretions of Men with Organ-confined Versus Extracapsular Prostate Cancer

Current protocols for the screening of prostate cancer cannot accurately discriminate clinically indolent tumors from more aggressive ones. One reliable indicator of outcome has been the determination of organ-confined versus nonorgan-confined disease but even this determination is often only made following prostatectomy. This underscores the need to explore alternate avenues to enhance outcome prediction of prostate cancer patients. Fluids that are proximal to the prostate, such as expressed prostatic secretions (EPS), are attractive sources of potential prostate cancer biomarkers as these fluids likely bathe the tumor. Direct-EPS samples from 16 individuals with extracapsular (n = 8) or organ-confined (n = 8) prostate cancer were used as a discovery cohort, and were analyzed in duplicate by a nine-step MudPIT on a LTQ-Orbitrap XL mass spectrometer. A total of 624 unique proteins were identified by at least two unique peptides with a 0.2% false discovery rate. A semiquantitative spectral counting algorithm identified 133 significantly differentially expressed proteins in the discovery cohort. Integrative data mining prioritized 14 candidates, including two known prostate cancer biomarkers: prostate-specific antigen and prostatic acid phosphatase, which were significantly elevated in the direct-EPS from the organ-confined cancer group. These and five other candidates (SFN, MME, PARK7, TIMP1, and TGM4) were verified by Western blotting in an independent set of direct-EPS from patients with biochemically recurrent disease (n = 5) versus patients with no evidence of recurrence upon follow-up (n = 10). Lastly, we performed proof-of-concept SRM-MS-based relative quantification of the five candidates using unpurified heavy isotope-labeled synthetic peptides spiked into pools of EPS-urines from men with extracapsular and organ-confined prostate tumors. This study represents the first efforts to define the direct-EPS proteome from two major subclasses of prostate cancer using shotgun proteomics and verification in EPS-urine by SRM-MS.Prostate cancer is the most common malignancy to affect men in the Western world, but only 15–20% of these men will present with aggressive, lethal disease (1, 2) whereas the majority of patients will die of other causes. Although the implementation of large-scale screening for prostate cancer using serum prostate-specific antigen (PSA) has dramatically improved early detection of disease, unnecessary biopsies and patient overtreatment are becoming increasingly evident (2, 3). Consequently, there has been a shift in emphasis away from detection of prostate cancer and toward identification of lethal disease. Currently, Gleason grading is considered to be one of the best outcome predictors; however, patients with Gleason 7 tumors are in the clinical “gray zone,” whereby the predictive ability of Gleason grading is mixed (4, 5). A recent study constructed a 157-gene signature based on the comparison of Gleason score ≤6 and ≥8 patients, and could show that their panel could predict lethality in the cohort of Gleason 7 patients (5). Nonetheless, the development and large-scale implementation of prognostic markers of prostate cancer has been hampered by numerous factors owing, in part, to the heterogeneous and multifocal nature of the disease (6). Although the widely used Gleason grading system attempts to control for heterogeneity of the glands and multifocality of cancerous lesions by summing the 2–3 most commonly observed histological patterns via inspection of multiple (typically 8–12) core biopsies, cancerous foci are still often missed (2, 6) providing only partial information that can lead to imprecise diagnoses and prognoses. Pathologic staging remains the gold standard for disease staging and risk assessment (7, 8); however, this process lacks timeliness in discriminating organ-confined from extracapsular disease. Indeed, one-third of individuals with nonorgan-confined disease are identified only after surgery (9). Furthermore, ∼35% of men treated with radical prostatectomy with curative intent subsequently develop biochemical recurrence (10–13) and the mean time from surgery to recurrence is 3.5 years (4). Significant risk factors for time to prostate-specific mortality following biochemical recurrence after radical prostatectomy are PSA doubling time, pathological Gleason score, and time from surgery to biochemical recurrence (4). Estimates place the percent of lethal cases at 20–25% of all patients that show biochemical recurrence, suggesting that nearly 75–80% of patients in this group may be overtreated (14).There is an emerging trend toward recruitment of men with perceived low-risk disease to an “active surveillance” monitoring approach. This is based on the supposition that most prostate cancers are slow growing, and that the more aggressive forms can be identified during a period of observation with little increased risk of death. Although a consensus may not exist for defining the disease stage where active surveillance is warranted, there is considerable agreement that men who have a PSA level less than 10 ng/ml, impalpable disease (clinical stage T1c) and only 1 biopsy core out of 12 or more that show Gleason 6 cancer are most likely to harbor indolent disease (15). Even so, these candidates for active surveillance will still contain individuals who will have disease progression and die from their cancer. Thus, despite efforts to recruit individuals to active surveillance protocols, overtreatment of prostate cancer is fueled by the lack of reliable means to accurately discriminate between men with clinically indolent prostate cancer from those with more aggressive disease (16, 17). This inability to accurately predict prostate cancer aggressiveness based solely on standard clinicopathologic features clearly underscores the need to explore the ability of additional biomarkers to enhance outcome prediction for men with prostate cancer. Furthermore, it is important to acknowledge that a single biomarker alone is unlikely to have sufficient prognostic power; rather, the integration of a panel of biomarkers hold the promise for improved prostate cancer detection and prognosis (2).Fluids that are proximal to the prostate are attractive sources of potential prostate cancer biomarkers (2, 18), as they house secreted proteins and sloughed cells that provide a presumably more comprehensive assessment of the organ and extent of disease. Further, fluids such as urine are clinically favorable for their ease of collection, the volume and frequency at which they can be obtained, and their adaptability to routine clinical assays. Prostate-proximal fluids include seminal fluid, semen, and expressed prostatic secretions (EPS)¹. Here, we focus on the analysis of EPS as our biological specimen, using direct-EPS samples for the discovery of candidate prognostic biomarkers and both direct-EPS and pooled EPS-urines derived from independent sets of patients for candidate biomarker verification. Direct-EPS is a prostatic fluid that is collected from patients undergoing prostatectomy by massaging the organ and expelling 0.5–1 ml of the fluid just prior to surgical removal. It was chosen as our discovery fluid as it is expected to house prostate-secreted proteins at a higher concentration and purity, and we have developed a workflow for the in-depth proteomic analysis of this fluid (19). Following discovery proteomics in 16 clinically stratified direct-EPS samples, verification studies were performed using independent sample sets of direct-EPS. Next, we focused our attention on the verification and quantitative analysis of candidate proteins in pooled EPS-urines. Before EPS-urine collection, men undergo digital rectal examination (DRE), often as part of a routine procedure, which causes direct-EPS to be expelled from the prostate and subsequently voided in urine. Because EPS-urine can be collected with substantial ease and in greater volumes and frequencies than direct-EPS, much attention has been paid to this fluid as a valuable resource of prostate cancer biomarkers amenable to routine clinical analysis. Following the recent FDA approval of the EPS-urine assay for prostate cancer gene 3 (PCA3), standardized clinical collection protocols will be widely implemented and easier access to this fluid is expected. Moreover, we have recently identified a number of prostate-enriched proteins in EPS-urine by comparing its proteome to a urine background (20).The present study used multidimensional protein identification technology (MudPIT) coupled with bioinformatics to first catalog and comparatively analyze the direct-EPS proteomes from a small cohort of patients with extracapsular versus organ-confined prostate cancers. A semiquantitative algorithm based on spectral counts (QSpec) (21) and an integrative data mining strategy led to the selection of a number of putative biomarkers that were verified by Western blotting in direct-EPS. Lastly, to demonstrate accurate quantitative measurements of verified candidates in EPS-urine, a pilot study utilizing SRM-MS was undertaken as a proof-of-concept.     

The Two Genes Encoding Starch-Branching Enzymes IIa and IIb Are Differentially Expressed in Barley

The sbeIIa and sbeIIb genes, encoding starch-branching enzyme (SBE) IIa and SBEIIb in barley (Hordeum vulgare L.), have been isolated. The 5′ portions of the two genes are strongly divergent, primarily due to the 2064-nucleotide-long intron 2 in sbeIIb. The sequence of this intron shows that it contains a retro-transposon-like element. Expression of sbeIIb but not sbeIIa was found to be endosperm specific. The temporal expression patterns for sbeIIa and sbeIIb were similar and peaked around 12 d after pollination. DNA gel-blot analysis demonstrated that sbeIIa and sbeIIb are both single-copy genes in the barley genome. By fluorescence in situ hybridization, the sbeIIa and sbeIIb genes were mapped to chromosomes 2 and 5, respectively. The cDNA clones for SBEIIa and SBEIIb were isolated and sequenced. The amino acid sequences of SBEIIa and SBEIIb were almost 80% identical. The major structural difference between the two enzymes was the presence of a 94-amino acid N-terminal extension in the SBEIIb precursor. The (β/α)₈-barrel topology of the α-amylase superfamily and the catalytic residues implicated in branching enzymes are conserved in both barley enzymes.     

Serum Immune-Related Proteins are Differentially Expressed during Hibernation in the American Black Bear

Hibernation is an adaptation to conserve energy in the face of extreme environmental conditions and low food availability that has risen in several animal phyla. This phenomenon is characterized by reduced metabolic rate (∼25% of the active basal metabolic rate in hibernating bears) and energy demand, while other physiological adjustments are far from clear. The profiling of the serum proteome of the American black bear (Ursus americanus) may reveal specific proteins that are differentially modulated by hibernation, and provide insight into the remarkable physiological adaptations that characterize ursid hibernation. In this study, we used differential gel electrophoresis (DIGE) analysis, liquid chromatography coupled to tandem mass spectrometry, and subsequent MASCOT analysis of the mass spectra to identify candidate proteins that are differentially expressed during hibernation in captive black bears. Seventy serum proteins were identified as changing by ±1.5 fold or more, out of which 34 proteins increased expression during hibernation. The majority of identified proteins are involved in immune system processes. These included α₂-macroglobulin, complement components C1s and C4, immunoglobulin μ and J chains, clusterin, haptoglobin, C4b binding protein, kininogen 1, α₂-HS-glycoprotein, and apoplipoproteins A-I and A-IV. Differential expression of a subset of these proteins identified by proteomic analysis was also confirmed by immunodetection. We propose that the observed serum protein changes contribute to the maintenance of the hibernation phenotype and health, including increased capacities for bone maintenance and wound healing during hibernation in bears.     

An mRNA Encoding a Putative GABA-Gated Chloride Channel Is Expressed in the Human Cardiac Conduction System

Abstract: GABA-gated chloride channels are the main inhibitory neurotransmitter receptors in the CNS. Conserved domains among members of previously described GABA_A receptor subunits were used to design degenerate sense and antisense oligonucleotides. A PCR product from this amplification was used to isolate a full-length cDNA. The predicted protein has many of the features shared by other members of the ligand-gated ion channel family. This channel subunit has significant amino acid identity (25–40%) with members of GABA_A and GABA_C receptor subunits and thus may represent a new subfamily of the GABA receptor channel. Although we cannot rule out that this clone encodes a receptor for an unidentified ligand, it was termed GABA _χ. This gene is mainly expressed in placenta and in heart; however, placenta appears to express only an unspliced mRNA. In situ hybridization reveals that the GABA _χ subunit mRNA is present in the electrical conduction system of the human heart. Our results suggest that novel GABA receptors expressed outside of the CNS may regulate cardiac function.     

Genetic Manipulation of Alcohol Dehydrogenase Levels in Ripening Tomato Fruit Affects the Balance of Some Flavor Aldehydes and Alcohols

Tomato (Lycopersicon esculentum) plants were transformed with gene constructs containing a tomato alcohol dehydrogenase (ADH) cDNA (ADH 2) coupled in a sense orientation with either the constitutive cauliflower mosaic virus 35S promoter or the fruit-specific tomato polygalacturonase promoter. Ripening fruit from plants transformed with the constitutively expressed transgene(s) had a range of ADH activities; some plants had no detectable activity, whereas others had significantly higher ADH activity, up to twice that of controls. Transformed plants with fruit-specific expression of the transgene(s) also displayed a range of enhanced ADH activities in the ripening fruit, but no suppression was observed. Modified ADH levels in the ripening fruit influenced the balance between some of the aldehydes and the corresponding alcohols associated with flavor production. Hexanol and Z-3-hexenol levels were increased in fruit with increased ADH activity and reduced in fruit with low ADH activity. Concentrations of the respective aldehydes were generally unaltered. The phenotypes of modified fruit ADH activity and volatile abundance were transmitted to second-generation plants in accordance with the patterns of inheritance of the transgenes. In a preliminary taste trial, fruit with elevated ADH activity and higher levels of alcohols were identified as having a more intense “ripe fruit” flavor.     

Osteopontin Is Expressed in the Mouse Uterus during Early Pregnancy and Promotes Mouse Blastocyst Attachment and Invasion In Vitro

Embryo implantation into the maternal uterus is a decisive step for successful mammalian pregnancy. Osteopontin (OPN) is a member of the small integrin-binding ligand N-linked glycoprotein family and participates in cell adhesion and invasion. In this study, we showed that Opn mRNA levels are up-regulated in the mouse uterus on day 4 and at the implantation sites on days 5 and 8 of pregnancy. Immunohistochemistry localized the OPN protein to the glandular epithelium on day 4 and to the decidual zone on day 8 of pregnancy. OPN mRNA and proteins are induced by in vivo and in vitro decidualization. OPN expression in the endometrial stromal cells is regulated by progesterone, a key regulator during decidualization. As a secreted protein, the protein level of OPN in the uterine cavity is enriched on day 4, and in vitro embryo culturing has indicated that OPN can facilitate blastocyst hatching and adhesion. Knockdown of OPN attenuates the adhesion and invasion of blastocysts in mouse endometrial stromal cells by suppressing the expression and enzymatic activity of matrix metalloproteinase-9 in the trophoblast. Our data indicated that OPN expression in the mouse uterus during early pregnancy is essential for blastocyst hatching and adhesion and that the knockdown of OPN in mouse endometrial stroma cells could lead to a restrained in vitro trophoblast invasion.     

Identification of a Saccharomyces cerevisiae Gene that Is Required for G1 Arrest in Response to the Lipid Oxidation Product Linoleic Acid Hydroperoxide*

Reactive oxygen species cause damage to all of the major cellular constituents, including peroxidation of lipids. Previous studies have revealed that oxidative stress, including exposure to oxidation products, affects the progression of cells through the cell division cycle. This study examined the effect of linoleic acid hydroperoxide, a lipid peroxidation product, on the yeast cell cycle. Treatment with this peroxide led to accumulation of unbudded cells in asynchronous populations, together with a budding and replication delay in synchronous ones. This observed modulation of G1 progression could be distinguished from the lethal effects of the treatment and may have been due to a checkpoint mechanism, analogous to that known to be involved in effecting cell cycle arrest in response to DNA damage. By examining several mutants sensitive to linoleic acid hydroperoxide, the YNL099c open reading frame was found to be required for the arrest. This gene (designated OCA1) encodes a putative protein tyrosine phosphatase of previously unknown function. Cells lacking OCA1 did not accumulate in G1 on treatment with linoleic acid hydroperoxide, nor did they show a budding, replication, or Start delay in synchronous cultures. Although not essential for adaptation or immediate cellular survival, OCA1 was required for growth in the presence of linoleic acid hydroperoxide, thus indicating that it may function in linking growth, stress responses, and the cell cycle. Identification of OCA1 establishes cell cycle arrest as an actively regulated response to oxidative stress and will enable further elucidation of oxidative stress-responsive signaling pathways in yeast.