Engineering and mechanistic studies of the Arabidopsis FAE1 beta-ketoacyl-CoA synthase, FAE1 KCS.

The Arabidopsis FAE1 beta-ketoacyl-CoA synthase (FAE1 KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoAs. Sequence analysis of FAE1 KCS predicted that this condensing enzyme is anchored to a membrane by two adjacent N-terminal membrane-spanning domains. In order to characterize the FAE1 KCS and analyze its mechanism, FAE1 KCS and its mutants were engineered with a His6-tag at their N-terminus, and expressed in Saccharomyces cerevisiae. The membrane-bound enzyme was then solubilized and purified to near homogeneity on a metal affinity column. Wild-type recombinant FAE1 KCS was active with several acyl-CoA substrates, with highest activity towards saturated and monounsaturated C16 and C18. In the absence of an acyl-CoA substrate, FAE1 KCS was unable to carry out decarboxylation of [3-(14)C]malonyl-CoA, indicating that it requires binding of the acyl-CoA for decarboxylation activity. Site-directed mutagenesis was carried out on the FAE1 KCS to assess if this condensing enzyme was mechanistically related to the well characterized soluble condensing enzymes of fatty acid and flavonoid syntheses. A C223A mutant enzyme lacking the acylation site was unable to carry out decarboxylation of malonyl-CoA even when 18:1-CoA was present. Mutational analyses of the conserved Asn424 and His391 residues indicated the importance of these residues for FAE1-KCS activity. The results presented here provide the initial analysis of the reaction mechanism for a membrane-bound condensing enzyme from any source and provide evidence for a mechanism similar to the soluble condensing enzymes.     

Monoclonal antibody against K562 cell line accelerates killing of the target cells by large granular lymphocytes

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG_2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG_2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.     

Regional distribution of the enzymes of haem biosynthesis and the inhibition of 5-aminolaevulinate synthase by manganese in the rat brain

The activity of 5-aminolaevulinate synthase, the rate-limiting enzyme of haem biosynthesis, is differentially distributed in various regions of the rat brain. The cerebellum possessed the highest enzyme activity of the eight regions studied. The cerebral cortex and the midbrain also exhibited high 5-aminolaevulinate synthase activity; the septum, hypothalamus, thalamus, amygdala and the hippocampus possessed much lower enzyme activity. However, the total porphyrin and haem contents of the different brain segments did not vary greatly. Mn²⁺, when administered subcutaneously to rats, effectively inhibited the activity of 5-aminolaevulinate synthase in the cerebellum, midbrain and cerebral cortex; however, repeated injections of the metal ion neither decreased the haem and porphyrin contents of the brain nor induced haem oxygenase activity. Mn²⁺ was not an effective inhibitor of 5-aminolaevulinate synthase activity in vitro. On the other hand, studies carried out with the liver in vivo suggested that Mn²⁺ may alter the turnover rate of cellular haem and haemoproteins. In that event, it is likely that the inhibition of 5-aminolaevulinate synthase by Mn²⁺ was in part a result of the inhibition of protein synthesis by the metal ion. It is postulated that the haem and porphyrin contents of the brain are maintained at a steady-state level, due in part to the refractoriness to inducers of the regulatory mechanism for haem catabolic enzymes and in part to the ability of the organ to utilize haem precursors derived from extraneuronal sources.     

Alternative metabolic fates of thymine nucleotides in human cells.

Three types of experiments have been used to study the metabolism of thymine nucleotides by human cells. (1) Cells were labelled continuously with [3H]thymidine and the incorporation of label into DNA compared with the specific radioactivities of pools of individual thymine nucleotides separated by chromatography on polyethylene-imine-cellulose. (2) Cellular thymine nucleotides were labelled with [3H]thymidine at 13 degrees C, followed by incubation at 37 degrees C in unlabelled medium. Incorporation of label into DNA and loss of label from the nucleotide pools were monitored during the 'chase' period at 37 degrees C. (3) The experiments described in (2) above were repeated in the presence of the DNA-synthesis inhibitor cytosine arabinoside, in order to demonstrate more clearly and to quantify degradative pathways for thymine nucleotides. In phytohaemagglutinin-stimulated lymphocytes and in bone-marrow cells, only a proportion (25-60%) of labelled thymine nucleotide was incorporated into DNA, the rest being rapidly degraded and lost from the cell. In contrast, an established cell line (HPB-ALL) from a patient with acute lymphoblastic leukaemia of thymic origin incorporated 100% of its exogenously labelled thymine nucleotides into DNA. These results indicated that alternative metabolic routes are open to thymine nucleotides in human cells. In lymphocytes from patients with megaloblastic anaemia and in normal lymphocytes treated with methotrexate, the utilization of labelled thymine nucleotides for DNA synthesis was more efficient than in controls. These results offer an explanation for the observation of a normal pool of thymidine triphosphate in the cells of patients with untreated megaloblastic anaemia even though the amount of this compound available for DNA synthesis appears to be decreased.     

Synthesis,anticancer activity,and β‐lactoglobulin binding interactions of multitargeted kinase inhibitor sorafenib tosylate (SORt) using spectroscopic and molecular modelling approaches

Sorafenib tosylate (SORt) is an oral multikinase inhibitor used for treatment of advanced renal cell, liver, and thyroid cancers. In this study, this drug was synthesized and its antiproliferative activities against HCT116 and CT26 cells were assessed. The interaction of SORt with β‐lactoglobulin (BLG) was studied using different fluorescence techniques, circular dichroism (CD), zeta potential measurements, and docking simulation. The results of infrared (IR), mass, ^HNMR, and ^CNMR spectra demonstrated that the drug was produced with high quality, purity, and efficiency. SORt showed potent cytotoxicity against HCT116 and CT26 cells with IC₅₀ of 8.12 and 5.42 μM, respectively. For BLG binding of SORt, the results showed that static quenching was the cause of the high affinity drug–protein interaction. Three‐dimensional fluorescence and synchronous spectra indicated that SORt conformation was changed at different levels. CD suggested that the α‐helix content remained almost constant in the BLG–SORt complex, whereas random coil content decreased. Zeta potential values of BLG were more positive after binding with SORt, due to electrostatic interactions between BLG and SORt. Thermodynamic parameters confirmed van der Waals and hydrogen bond interactions in the complex formation. Molecular modelling predicted the presence of hydrogen bonds and electrostatic forces in the BLG–SORt system, which was consistent with the experimental results.     

Characteristics of Quantum Plasmonic Waves Guided by a Symmetric Metal–Gap–Dielectric Nano-system

Plasmonics - The guiding properties of a symmetric conductor–gap–dielectric system consists of a metal film symmetrically surrounded by media of two dielectrics, and is theoretically...     

Comparing the frequency of CD33+ pSTAT3+ myeloid-derived suppressor cells and IL-17+ lymphocytes in patients with prostate cancer and benign prostatic hyperplasia

Prostate cancer (PCa) is one of the most epidemic types of cancer in men. The tumor microenvironment (TME) of PCa is involved in the emergence of immunosuppressive factors such as myeloid-derived suppressor cells (MDSC), which regulate the immune system by several mechanisms, including interleukin (IL)-10 production. On the other hand, IL-17⁺ helper T cells (Th17) induce MDSCs and chronic inflammation in TME by producing IL-17. This study demonstrated that the frequency of CD33⁺ pSTAT3⁺ MDSC and IL-17⁺ lymphocyte as well as IL-10 messenger RNA (mRNA) expression were significantly higher in the PCa patients than in the benign prostatic hyperplasia (BPH) group. Moreover, there was no significant relationship between the frequency of CD33⁺ pSTAT3⁺ MDSC, and IL-17⁺ lymphocyte with Gleason scores in the PCa group. We suggested that the higher frequency of CD33⁺ pSTAT3⁺ MDSC and IL-17⁺ lymphocyte and the more frequent expression of IL-10 mRNA in PCa patients may play roles in tumor progression from BPH to PCa.     

Competition and overlap of Oryzaephilus surinamensis and Plodia interpunctella populations under condition of stored date fruits

Plodia interpunctella and Oryzaephilus surinamensis are found in food storehouses including dates and palm storages. The current study aimed to determine competition and overlap potentials of the two pests of date fruits. Time series models were used to study two species populations and logistic growth model to estimate the effect of density of the species. The results revealed the environmental capacities of O. surinamensis and P. interpunctella were 433 and 1610 (maximum number per 20 g), respectively, and the population growth rates (r) were 1.2 and 1.3, respectively. Ecological balances of the two species were close to each other from the first to the third week. The population of O. surinamensis decreased in the fourth week of the competition. The highest population balance of the two species was in the 14th week. The potential of exploitable ecological niches (e_ij) and the amount of non-exploited ecological niches by any species (z_ij) for O. surinamensis was higher than for P. interpunctella from the 8th week untill the end of sampling period. The overlap of ecological niches in the two species (D) ranged from 0.94 to 1, indicating a complete overlap of temporal activity in the two populations on date palm. The current results of this study can be used by integrated pest management specialists. Information over the effects of species competition on population dynamics and their coexistence can be used to predict population status and to adopt simple pest control methods.     

Genome-wide analysis of key salinity-tolerance transporter (HKT1;5) in wheat and wild wheat relatives (A and D genomes)

Exclusion of sodium ions from cells is one of the key salinity tolerance mechanisms in plants. The high-affinity cation transporter (HKT1;5) is located in the plasma membrane of the xylem, excluding Na⁺ from the parenchyma cells to reduce Na⁺ concentration. The regulatory mechanism and exact functions of HKT genes from different genotypic backgrounds are relatively obscure. In this study, the expression patterns of HKT1;5 in A and D genomes of wheat were investigated in root and leaf tissues of wild and domesticated genotypes using real-time PCR. In parallel, the K⁺/Na⁺ ratio was measured in salt-tolerant and salt-sensitive cultivars. Promoter analysis were applied to shed light on underlying regulatory mechanism of the HKT1;5 expression. Gene isolation and qPCR confirmed the expression of HKT1;5 in the A and D genomes of wheat ancestors (Triticum boeoticum, AbAb and Aegilops crassa, MMDD, respectively). Interestingly, earlier expression of HKT1;5 was detected in leaves compared with roots in response to salt stress. In addition, the salt-tolerant genotypes expressed HKT1;5 before salt-sensitive genotypes. Our results suggest that HKT1;5 expression follows a tissue- and genotype-specific pattern. The highest level of HKT1;5 expression was observed in the leaves of Aegilops, 6 h after being subjected to high salt stress (200 mM). Overall, the D genome allele (HKT1;5-D) showed higher expression than the A genome (HKT1;5-A) allele when subjected to a high NaCl level. We suggest that the D genome is more effective regarding Na⁺ exclusion. Furthermore, in silico promoter analysis showed that TaHKT1;5 genes harbor jasmonic acid response elements.