Fluorescence investigations of coenzyme and substrate interactions in mannitol-1-phosphate dehydrogenase ofEscherichia coli

Coenzyme and substrate interactions with mannitol-1-phosphate dehydrogenase fromEscherichia coli (a dimer of MW 45,000) have been studied by fluorescence spectroscopy. NAD⁺ quenches the fluorescence emission of the protein tryptophan residues; shifting the excitation wavelength from 280 to 290 nm results in an increase in this quenching and a red shift in the emission maximum. NAD⁺ also quenches the fluorescence of covalently attached pyridoxyl phosphate, and this quenching is accompanied by a spectral broadening above 425 nm. Fructose-6-phosphate increases the binding of NAD⁺, but causes a slight reduction in the quenching of the tryptophan fluorescence observed at saturating levels of coenzyme, and reverses the NAD⁺-induced broadening in the pyridoxyl phosphate emission spectrum. NADH quenches the protein emission much less than NAD⁺; this quenching is not changed by shifting the excitation wavelength and is not affected by the presence of bound mannitol-1-phosphate. Titrations monitoring the quenching by NADH indicate a single class of NADH binding sites, while titrations monitoring NADH fluorescence suggest that coenzyme fluorescence is more enhanced when NADH is bound to less than half of the total enzyme subunits, with the emission per NADH molecule bound decreasing as the number of NADH molecules bound increases. In the absence of coenzyme, neither fructose-6-phosphate nor mannitol-1-phosphate have any effect on the protein tryptophan emission; however, both substrates induce specific changes in the emission spectrum of covalently attached pyridoxyl phosphate. These results suggest that the different coenzymes and substrates cause specific conformational changes in mannitol-1-phosphate dehydrogenase.     

Affinity labeling of the active site of pig liver NADH-cytochrome b5 reductase by 5′-p-fluorosulfonylbenzoyladenosine

Lysosome-solubilized pig liver NADH-cytochrome b₅ reductase is inactivated by 5′-p-fluorosulfonylbenzoyladenosine (5′-FSBA) following pseudo-first-order kinetics. A double reciprocal plot of 1/K _obs versus 1/[5′-FSBA] yields a straight line with a positiveY intercept, indicative of reversible binding of the analogue prior to an irreversible incorporation.K _d or the initial reversible enzyme-analogue complex is estimated at 185 µM withK ₂=0.22 min^?1 (atpH 8.0 and 25°C). A stoichiometry of 1.2 moles of analogue bound/mole of enzyme at 100% inactivation has been determined from incorporation studies using 5′-p-fluorosulfonylbenzoyl-[¹⁴C]adenosine. The irreversible inactivation as well as the covalent incorporation could be completely prevented by the presence of NADH, the substrate of enzyme, during the incubation. Four 5′-FSBA-labeled peptides were isolated by reverse-phase high-performance liquid chromatography of tryptic digest of the modified NADH-cytochrome b₅ reductase and their amino acid sequences were determined. These peptides appear to be related to the NADH binding site of the enzyme.     

Purification and properties of a fructose-1,6-diphosphate activated l-lactate dehydrogenase from Staphylococcus epidermidis

l-(+)-lactate dehydrogenase (LDH) from Staphylococcus epidermidis ATCC 14990 was purified by affinity chromatography. The purified enzyme was specifically activated by fructose-1,6-diphosphate (FDP). The concentration of FDP required for 50% maximal activity was about 0.15 mM. The enzyme activity was inhibited by adenosine diphosphate (ADP) and oxamate. The inhibition by ADP appeared to be competitive with respect to reduced nicotinamide adenine dinucleotide (NADH). The catalytic activity of the LDH for pyruvate reduction exhibited an optimum at pH 5.6. The enzyme is composed of four, probably identical, subunits. Sephadex gel filtration and sedimentation velocity at pH 5.6 yielded molecular weights of about 130000 and 126000 respectively. The molecular weight at pH 6.5 and 7.0 was found to be only about 68000. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and sedimentation velocity at pH 2.0 or 8.5 revealed monomeric subunits with an approximate molecular weight of 36000. The thermostability of the heat labile enzyme was increased in the presence of FDP, NADH and pyruvate. The purified LDH exhibited an anomalous type of kinetic behavior. Plots of initial velocity vs. different concentrations of pyruvate, NADH or FDP led to saturation curves with intermediary plateau regions. As a consequence of these plateau regions the Hill coefficient alternated between lower and higher n-values. Some distinguishing properties of the S. epidermidis LDH and other LDHs activated by FDP are discussed.     

Nitrate reductase of Dunaliella parva

Nitrate reductase of the salt tolerant alga Dunaliella parva, in contrast to that of most green algae, can use NADPH as well as NADH as electron donor. Extracts of cells contained various amounts of latent nitrate reductase. The latent enzyme could be activated at 45°C but only in the presence of flavine adenine dinucleotide. The heat activated enzyme did not require flavine adenine dinucleotide for activity and was fully active with NADH, NADPH or reduced flavine mononucleotide as electron donors.     

Apparent Reduction of ADAM10 in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents

It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP^C) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP^C. Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP^C. Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.     

ATP-driven succinate oxidation in the catabolism of Desulfuromonas acetoxidans

The oxidation of succinate with elemental sulphur in Desulfuromonas acetoxidans was investigated using a membrane preparation of this bacterium. The following results were obtained:

1. The preparation catalyzed the oxidation of succinate with sulphur and NAD. These reactions were dependent on ATP and were abolished by the presence of protonophores or dicyclohexylcarbodiimide (DCCD).
2. The membrane preparation also catalyzed the reduction of fumarate with H₂S or with NADH. These activities were not dependent on ATP and were not affected by protonophores or DCCD.
3. By extraction-reincorporation experiments it could be shown that menaquinone is involved in electron transport between H₂S and fumarate and between NADH and fumarate.
4. The membrane fraction catalyzed the reduction of the water-soluble menaquinone-analogue dimethylnaphthoquinone (DMN) by succinate, H₂S, or NADH, and the oxidation of DMNH₂ by fumarate. These activities were not dependent on the presence of menaquinone and were not influenced by ATP.
5. The activities involving succinate oxidation or fumarate reduction were similarly sensitive to 2(n-nonyl)-4-hydroxyquinoline-N-oxide, while H₂S and NADH oxidation by DMN were not affected by the inhibitor.

It is concluded that the catabolism of D. acetoxidans involves the energy-driven oxidation of succinate with elemental sulphur or NAD as electron acceptors and that menaquinone is a component of the electron transport chain catalyzing these reactions.     

Polymorphism of DNA Repair Gene xrcc1 and Lung Cancer Risk

The current large-scale meta-analysis was performed to reach a reliable conclusion on the association between X-ray repair cross-complementing 1 (xrcc1) rs1799782 and the development of lung cancer. Studies that investigated the association between rs1799782 and lung cancer risk were identified by searching PubMed. We calculated odds ratio (OR) with corresponding 95 % confidence interval (CI) for Trp/Trp vs Arg/Arg, Trp/Trp + Arg/Trp vs Arg/Arg, and Trp/Trp vs Arg/Trp + Arg/Arg contrast models. Combining all 25 studies, we yielded three summary ORs: 1.07 (95 % CI 0.92–1.23) for Trp/Trp vs Arg/Arg, 0.93 (95 % CI 0.87–1.00) for Trp/Trp + Arg/Trp vs Arg/Arg, and 1.08 (95 % CI 0.94–1.25) for Trp/Trp vs Arg/Trp + Arg/Arg, suggesting rs1799782 was not associated with overall risk of lung cancer. Strikingly, a significantly deceased risk was found among Caucasian populations (Trp/Trp + Arg/Trp vs Arg/Arg, OR = 0.86, 95 % CI 0.76–0.97). This study confirms that xrcc1 rs1799782 may lower the risk of lung cancer among Caucasians.     

A nicotinamide adenine dinucleotide phosphate phosphatase fromChlorella

A phosphatase enzyme hydrolysing NADP⁺ and NADPH to NAD⁺ and NADH was found to be present in extracts ofChlorella pyrenoidosa     

Mechanism of bFGF-binding Peptide Reversing Adriamycin Resistance in Human Gastric Cancer Cells

Development of drug resistance is a challenging problem in cancer chemotherapy. It has been shown that basic fibroblast growth factor (bFGF) plays an important role in an epigenetic mechanism of drug resistance. We have isolated a bFGF binding peptide P7 with inhibitory activity against bFGF-induced proliferation of human gastric cancer cells by screening a phage display library. In this study, we found that P7 peptide also has efficacy of reversing bFGF-induced resistance to Adriamycin (ADM) in human gastric cancer cells. Further investigations with SGC-7901 cells revealed that inhibition of Akt activation triggered by bFGF, and reversal of bFGF-induced up-regulation of Bcl-2 and XIAP and down-regulation of Bax, contribute to P7 peptide counteracting the anti-apoptotic effect of bFGF, and further reversing bFGF-induced resistance to ADM. The results suggested that the bFGF-binding peptide may have therapeutic potential of drug resistance in gastric cancer.     

EGFR-AKT-mTOR activation mediates epiregulin-induced pleiotropic functions in cultured osteoblasts

Epidermal growth factor (EGF) receptor (EGFR) emerges as an essential molecule for the regulating of osteoblast cellular functions. In the current study, we explored the effect of epiregulin, a new EGFR ligand, on osteoblast functions in vitro, and studied the underlying mechanisms. We found that epiregulin-induced EGFR activation in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, epiregulin activated AKT-mammalian target of rapamycin (mTOR) and Erk-mitogen-activated protein kinase (MAPK) signalings in cultured osteoblasts, which were blocked by EGFR inhibitor AG1478 or monoclonal antibody against EGFR (anti-EGFR). Further, in primary and MC3T3-E1 osteoblasts, epiregulin promoted cell proliferation and increased alkaline phosphatase activity, while inhibiting dexamethasone (Dex)-induced cell death. Such effects by epiregulin were largely inhibited by AG1478 or anti-EGFR. Notably, AKT-mTOR inhibitors, but not Erk inhibitors, alleviated epiregulin-induced above pleiotropic functions in osteoblasts. Meanwhile, siRNA depletion of Sin1, a key component of mTOR complex 2 (mTORC2), also suppressed epiregulin-exerted effects in MC3T3-E1 cells. Together, these results suggest that epiregulin-induced pleiotropic functions in cultured osteoblasts are mediated through EGFR-AKT-mTOR signalings.     

Expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the Spinal Tuberculous Focus and Its Impact on the Disease

To investigate the expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the spinal tuberculous focus and its relationship with the lesions type, severity, and bone destruction. The pathological samples of patients with spinal tuberculosis (TB) were divided into hyperplasia group and necrosis group according to their intra-operative and post-operative pathological findings. Normal bone tissues were taken as the control group. Pathology and expression of TNF-α, IFN-γ, TGF-β, and IL-4 in different tissues were compared among these three groups using immunohistochemical staining, quantitative image analysis, and measurement of bone tissue. 286 granulomas observed in the 14 samples in the hyperplasia group, which included 84 necrotizing and 202 non-necrotizing granulomas. As for the 20 samples in the necrosis group, there were 356 necrotizing and 186 non-necrotizing granulomas among all the 542 granulomas. The proportion of necrotizing granulomas in the necrosis group was significantly higher than that of the hyperplasia group. By inter-group comparison, expression of TNF-α, IFN-γ of granulomas in the hyperplasia group was significantly higher than that of the necrosis group, while the expression of TGF-β, IL-4 of granulomas in the necrosis group was significantly higher than that of the hyperplasia group. Also, expression of IFN-γ of non-necrotizing granulomas was significantly higher than that of necrotizing granulomas in the hyperplasia group, and expression of TGF-β in necrotizing granulomas was significantly higher than that of non-necrotizing granulomas in the necrosis group. The lesions were mainly bone resorption in the hyperplasia group, whereas mostly necrotic bones accompanied by local fibrosis in the necrosis group. Expression levels of TNF-α, IFN-γ in the hyperplasia group have a positive correlation to bone loss, whereas expression levels of TGF-β, IL-4 in the necrosis group have a positive correlation to the bone formation. The high expressions of TNF-α, IFN-γ in the spinal tuberculous focus were associated with protective immune cells. TGF-β and IL-4 were related to allergic lesions, fibrosis and osteogenesis. Expression imbalance of TNF-α, IFN-γ, TGF-β, and IL-4 might aggravate the allergy of TB.     

On the existence of two GABA pools associated with newly synthesized GABA and with newly taken up GABA in nerve terminals

[¹⁴C]Glutamic acid and [³H]GABA were injected into the lateral ventricle of mouse and then [¹⁴C]GABA and [³H]GABA in synaptosomes isolated from the animals were analysed. The [¹⁴C]GABA was interpreted to be newly synthesized GABA from [¹⁴C]glutamic acid while the [³H]GABA to be newly taken up GABA. We have obtained the following results: (1) when the animals were pretreated with aminooxyacetic acid and thus the GABA content in synaptosomes increased to about 2 times of the control level, only the [³H]GABA was enhanced to 3 times of the control level without any change of [¹⁴C]GABA, (2) the release of [¹⁴C]GABA from synaptosomes by high K⁺ depolarization was 1.5 times greater than that of [³H]GABA, (3) the releases of both [¹⁴C]GABA and [³H]GABA were increased in the presence of cold GABA,l-2,4-diaminobutyric acid or γ-amino-β-hydroxybutyric acid, but only slightly increased in the presence of β-alanine. These results would suggest that newly synthesized GABA and newly taken up GABA localized individually in different pools, which might localize either in different nerve terminals or separately in the same nerve terminal.     

The selective roles of chaperone systems on over-expression of human-like collagen in recombinant Escherichia coli

Human-like collagen (HLC) is a novel biomedical material with promising applications. Usually, insoluble HLC was formed due to over-expression. In order to improve the production of soluble HLC, the effective chaperone proteins and their mediation roles on HLC were clarified. Trigger factor (TF) pathway with low specificity and high binding affinity to nascent chains could increase soluble HLC expression; GroEL-GroES could increase the expression level of HLC by assisting the correct folding of HLC and increase mRNA level of the gene coding for HLC by enhancing mRNA stability. DnaK chaperone system did not work positively on soluble HLC due to the unbalanced ratio of DnaK:DnaJ:GrpE, especially too high GrpE significantly inhibited DnaK-mediated refolding. The production of soluble HLC with co-expression of exogenous TF and GroEL-GroES was increased by 35.3 % in comparison with the highest value 0.26 g/L reported previously.     

Ribosomal RNA contents of maize genotypes with different ribosomal RNA gene numbers

Ribosomal RNA (rRNA) contents were determined in 16 maize genotypes whose individual rRNA gene numbers varied from 5000 to 23,000 per 2C nucleus. Analytical polyacrylamide gel electrophoresis of total RNA showed that no obvious relation existed between rRNA gene number and rRNA content. Only two of nine common inbred lines contained more rRNA than W-23, the inbred with the lowest rRNA gene number. Two of four lines with altered protein content (due to long-term experimental selection) had rRNA contents significantly reduced from those of W-23. A line with an apparent duplication of the nucleolus organizer region of chromosome 6 (called 2-NOR) was expected to possess an elevated quantity of rRNA because it possesses a larger nucleolus; however, we produced a 2-NOR isogenic version and found no difference in rRNA content. The rRNA genes in maize are distributed throughout the NOR-heterochromatin and the NOR-secondary constriction portions of the NOR. The absence of an obvious correlation between rRNA gene number and cellular rRNA content may reflect the presence of a large number of rRNA genes in an inactive state, at least during the stage of growth examined in these experiments.     

The amino-terminal sequence ofStreptomyces griseus carboxypeptidase

The amino-terminal sequence of carboxypeptidase fromStreptomyces griseus was determined using a new protocol for automatic Edman degradation that reduced background noise. The sequence of the first 48 residues is: Asp-Phe-Pro-Pro-Ala-Asp-Ser-Arg-Tyr-His-Asn-Tyr-Ala-Glu-Met-Asn-Ala-Ala-Ile-Asp-Ala-Arg-Ile-Ala-Ala-Asn-Pro-Ser-Ile-Met-Ser-Lys-Arg-Val-Ile-Gly-Lys-Thr-Tyr-Gln-Gly-(Arg)-Asp-Val-Ile-Ala-Val-Lys, which is homologous to that of other zinc-containing carboxypeptidase from vertebrate and invertebrate sources.