The Bifunctional Effects of Lactoferrin (LFcinB11) in Inhibiting Neural Cell Adhesive Molecule (NCAM) Polysialylation and the Release of Neutrophil Extracellular Traps (NETs).

The expression of polysialic acid (polySia) on the neuronal cell adhesion molecule (NCAM) is called NCAM-polysialylation, which is strongly related to the migration and invasion of tumor cells and aggressive clinical status. Thus, it is important to select a proper drug to block tumor cell migration during clinical treatment. In this study, we proposed that lactoferrin (LFcinB11) may be a better candidate for inhibiting NCAM polysialylation when compared with CMP and low-molecular-weight heparin (LMWH), which were determined based on our NMR studies. Furthermore, neutrophil extracellular traps (NETs) represent the most dramatic stage in the cell death process, and the release of NETs is related to the pathogenesis of autoimmune and inflammatory disorders, with proposed involvement in glomerulonephritis, chronic lung disease, sepsis, and vascular disorders. In this study, the molecular mechanisms involved in the inhibition of NET release using LFcinB11 as an inhibitor were also determined. Based on these results, LFcinB11 is proposed as being a bifunctional inhibitor for inhibiting both NCAM polysialylation and the release of NETs.

Influence of Calcium Ions on the Thermal Characteristics of α-amylase from Thermophilic Anoxybacillus sp. GXS-BL.

BACKGROUND: α-Amylases are starch-degrading enzymes and used widely, the study on thermostability of α-amylase is a central requirement for its application in life science and biotechnology. OBJECTIVE: In this article, our motivation is to study how the effect of Ca2+ ions on the structure and thermal characterization of α-amylase (AGXA) from thermophilic Anoxybacillus sp.GXS-BL. METHODS: α-Amylase activity was assayed with soluble starch as the substrate, and the amount of sugar released was determined by DNS method. For AGXA with calcium ions and without calcium ions, optimum temperature (Topt), half-inactivation temperature (T50) and thermal inactivation (halflife, t1/2) was evaluated. The thermal denaturation of the enzymes was determined by DSC and CD methods. 3D structure of AGXA was homology modeled with α-amylase (5A2A) as the template. RESULTS: With calcium ions, the values of Topt, T50, t1/2, Tm and ΔH in AGXA were significantly higher than those of AGXA without calcium ions, showing calcium ions had stabilizing effects on α-amylase structure with the increased temperature. Based on DSC measurements AGXA underwent thermal denaturation by adopting two-state irreversible unfolding processes. Based on the CD spectra, AGXA without calcium ions exhibited two transition states upon unfolding, including α- helical contents increasing, and the transition from α-helices to ß-sheet structures, which was obviously different in AGXA with Ca2+ ions, and up to 4 Ca2+ ions were located on the inter-domain or intra-domain regions according to the modeling structure. CONCLUSION: These results reveal that Ca2+ ions have pronounced influences on the thermostability of AGXA structure.

The Inhibition of Polysialyltranseferase ST8SiaIV Through Heparin Binding to Polysialyltransferase Domain (PSTD).

BACKGROUND: The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface Of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. Heparin has been found to be effective in inhibiting the ST8Sia IV activity, but no clear molecular rationale. It has been found that polysialyltransferase domain (PSTD) in polyST plays a significant role in influencing polyST activity, and thus it is critical for NCAM polysialylation based on the previous studies. OBJECTIVE: To determine whether the three different types of heparin (unfractionated hepain (UFH), low molecular heparin (LMWH) and heparin tetrasaccharide (DP4)) is bound to the PSTD; and if so, what are the critical residues of the PSTD for these binding complexes? METHODS: Fluorescence quenching analysis, the Circular Dichroism (CD) spectroscopy, and NMR spectroscopy were used to determine and analyze interactions of PSTD-UFH, PSTD-LMWH, and PSTD-DP4. RESULTS: The fluorescence quenching analysis indicates that the PSTD-UFH binding is the strongest and the PSTD-DP4 binding is the weakest among these three types of the binding; the CD spectra showed that mainly the PSTD-heparin interactions caused a reduction in signal intensity but not marked decrease in α-helix content; the NMR data of the PSTD-DP4 and the PSTDLMWH interactions showed that the different types of heparin shared 12 common binding sites at N247, V251, R252, T253, S257, R265, Y267, W268, L269, V273, I275, and K276, which were mainly distributed in the long α-helix of the PSTD and the short 3-residue loop of the C-terminal PSTD. In addition, three residues K246, K250 and A254 were bound to the LMWH, but not to DP4. This suggests that the PSTD-LMWH binding is stronger than the PSTD-DP4 binding, and the LMWH is a more effective inhibitor than DP4. CONCLUSION: The findings in the present study demonstrate that PSTD domain is a potential target of heparin and may provide new insights into the molecular rationale of heparin-inhibiting NCAM polysialylation.

Inhibition of α-amylase Activity by Zn2+: Insights from Spectroscopy and Molecular Dynamics Simulations.

BACKGROUND: Inhibition of α-amylase activity is an important strategy in the treatment of diabetes mellitus. An important treatment for diabetes mellitus is to reduce the digestion of carbohydrates and blood glucose concentrations. Inhibiting the activity of carbohydrate-degrading enzymes such as α-amylase and glucosidase significantly decreases the blood glucose level. Most inhibitors of α-amylase have serious adverse effects, and the α-amylase inactivation mechanisms for the design of safer inhibitors are yet to be revealed. OBJECTIVE: In this study, we focused on the inhibitory effect of Zn2+ on the structure and dynamic characteristics of α-amylase from Anoxybacillus sp. GXS-BL (AGXA), which shares the same catalytic residues and similar structures as human pancreatic and salivary α-amylase (HPA and HSA, respectively). METHODS: Circular dichroism (CD) spectra of the protein (AGXA) in the absence and presence of Zn2+ were recorded on a Chirascan instrument. The content of different secondary structures of AGXA in the absence and presence of Zn2+ was analyzed using the online SELCON3 program. An AGXA amino acid sequence similarity search was performed on the BLAST online server to find the most similar protein sequence to use as a template for homology modeling. The pocket volume measurer (POVME) program 3.0 was applied to calculate the active site pocket shape and volume, and molecular dynamics simulations were performed with the Amber14 software package. RESULTS: According to circular dichroism experiments, upon Zn2+ binding, the protein secondary structure changed obviously, with the α-helix content decreasing and ß-sheet, ß-turn and randomcoil content increasing. The structural model of AGXA showed that His217 was near the active site pocket and that Phe178 was at the outer rim of the pocket. Based on the molecular dynamics trajectories, in the free AGXA model, the dihedral angle of C-CA-CB-CG displayed both acute and planar orientations, which corresponded to the open and closed states of the active site pocket, respectively. In the AGXA-Zn model, the dihedral angle of C-CA-CB-CG only showed the planar orientation. As Zn2+ was introduced, the metal center formed a coordination interaction with H217, a cation-π interaction with W244, a coordination interaction with E242 and a cation-π interaction with F178, which prevented F178 from easily rotating to the open state and inhibited the activity of the enzyme. CONCLUSION: This research may have uncovered a subtle mechanism for inhibiting the activity of α-amylase with transition metal ions, and this finding will help to design more potent and specific inhibitors of α-amylases.

[Molecular evolution of the sulphite efflux gene SSU1 in Saccharomyces cerevisiae].

The SSU1 gene encoding a membrane sulfite pump is a main facilitator invovled in sulfite efflux. In Saccharomyce cerevisiae, various range of resistance to sulfite was observed among strains. To explore the evolution traits of SSU1 gene, the population data of S. cerevisiae were collected and analyzed. The phylogenetic analysis indicated that S. cerevisiae population can be classified into three sub-populations, and the positive selection was detected in population by McDonald-Kreitman test. The anaylsis of Ka/Ks ratios further showed that S. cerevisiae sub-population was undergoing positive selection. This finding was also supported by PAML branch model. Nine potential positive selection sites were predicted by branch-site model, and four sites exclusively belong to the sub-population under positive seletion. The data from ssulp protein structure demonstrated that three sites are substitutions between polar and hydrophobic amino acids, and only one site of substitutaion from basic amino acid to basic amino acid (345R/K). Because amino acid pKa values are crucial for sulfite pump to maintain their routine function, positive selection of these amino acid substitutions might affect sulfite efflux efficient.

[Using Raman spectroscopy to analyze apoptosis of gastric cancer cells induced by cisplatin].

Apoptosis of gastric cancer cells induced by cisplatin was investigated using laser Raman spectroscopy. Gastric cancer cells (SGC7901) were treated with 10 microg x mL(-1) cisplatin for 24, 48 and 72 hours, then were divided into two parts, one for fluorescence staining, the other for collection of Raman spectra by means of scanning. The acquired spectra were then preprocessed by background elimination, smoothing, normalization, baseline correction, and peak fitting. Fluorescence staining result showed that the nucleuses from untreated group were uniformly stained, while those from the group treated for 72 hours were densely stained and broken. The spectra results revealed that the intensity of peaks associated with nucleic acid and protein decreased after the cells were incubated with cisplatin for 24, 48 and 72 hours. The intensity of peaks at 783, 1002 and 1343 cm(-1) respectively fell to 52, 64 and 76 percent of the original value after 72 h of treatment, which indicated that cisplatin could induce apoptosis of gastric cancer cells and reduce the amount of nucleic acid and protein in the cells. The above results suggest that Raman spectra can provide abundant information about the changes in materials in cells and serve as an effective method for real time measurement of apoptosis.

[The effect of abnormal cell shape on the spectral distinguishing of erythrocytes using laser tweezers Raman spectroscopy].

Erythrocyte is a mature blood cell that contains hemoglobin to carry oxygen to the bodily tissues. Erythrocyte, which takes on a biconcave disc that has no nucleus, is flexible and changeful. Erythrocyte is so sensitive to the environment that the shape of cell goes crimpy, even acanthoid. A laser tweezers Raman spectroscopy (LTRS) setup was used to trap single erythrocyte from healthy donors and patients with thalassemia and to collect the Raman scattering of trapping cell. Normal shape, crimpy erythrocytes and acanthoid erythrocytes were tested, and the averaged spectra, and principal component analysis (PCA) which detailed the spectral difference and the change of hemoglobin, were used to evaluate the effects of different cell shape on the spectral distinguishing of erythrocyte. The results reveal that in normal physiological environment the change in cell shape does not effect the spectral distinguishing of abnormal erythrocyte.

[FTIR-HATR to identify beta-thalassemia and its mechanism study].

Fourier transform infrared spectroscopy (FTIR) associated with horizontal attenuated total reflectance (HATR) was firstly used to diagnose beta-thalassemia patients. With excellent linearity (r = 0.997) and reproducibility (RSD < 4%), FTIR-HATR shows an order-of-magnitude increase in IR absorption bands over the single-path transmission FTIR Based on above, spectra from 37 patients' and 68 health samples indicated several observable differences in IR vibrational spectra of the Hb lysates between the beta-thalassemia major patients and control: (1)Because of decreasing hemoglobin, the peak intensities are obviously lower in beta-thalassemia group that is consistent with index from routine hemoglobin diagnosis. (2) In 1750-1500 cm(-1) region, slight decrease at 1652 cm(-1) (alpha-helix), 1638 and 1 628 cm(-1) bands but mild increase at 1682 cm(-1) all demonstrate structure changes by both Fourier self-deconvolution and second derivative spectra. (3) More importantly, difference spectra substantially demonstrate decreased intensities at 1440, 1453, 1479 cm(-1) bands arising from CH2/CH3 deformation vibration of phospholipids but increased intensities at 1150 cm(-1) band originating from C--O stretching vibration of carbohydrate and at 1081 and 1053 cm(-1) bands that are attributed to 2,3-diphosphoglycerate (DPG) in beta-thalassemia major group. Statistical analysis demonstrates significant difference of DPG/phospholipids ratio between two groups. All the samples can be 100% correctly classified into groups on the basis of this ratio. These finding could help understand possible mechanism for diagnosing thalassemia. It makes large-scale screening of thalassemia by FTIR a possibility.

[Study of Raman spectra of single carcinoma of nasopharynx cell].

The Raman spectra from carcinoma of nasopharynx cell lines (CNE2) and normal airway epithelial cell lines (HBE) were investigated using a laser tweezers Raman spectroscopy (LTRS). The Raman scattering measurements were obtained from three different places in every single cell. Visual inspection of the spectra shows that the differences observed in spectra of the cancer cells and normal cells are obvious. The peak ratio I1 304/I1 336 is 1.05 for the normal cell and 1.22 for the cancer cell. Using a combination of principal component analysis (PCA) and discriminant function analysis (DFA), the authors are able to predict cancer cells, and normal cells and the DFA is better for single Raman spectrum. The sampling locations did not seriously affect the result of PCA and DFA. PCA and DFA also show that the uniformity of normal cells is better than that of cancer cells. The results indicate that the Raman spectra may offer the experimental basis for colorectal cancer diagnosis.

[Raman micro-spectroscopy of single blood platelets].

The authors collected the Raman spectra of single blood platelets of human, pig, rat and rabbit suspended in saline so-lution by using a laser tweezers Raman spectroscopy (LTRS) setup. A single platelet cell was trapped in the focus of a near-infrared laser beam at 785 nm and the excited Raman spectrum was acquired. For each species, the Raman spectra of up to 20 platelet cells were acquired and were used to perform a principal components analysis (PCA) or a discriminate function analysis (DFA). The average Raman spectra indicate that the vibration bands at 1 524 and 1 157 cm(-1) of human platelets are obviously different from those of the platelets from pig, rat and rabbit. The Raman intensities at 1 157 and 1 524 cm(-1) bands are significantly high for human platelets. The ratio I1 157 / I1 003 of human platelets was 0.795, but those of pig, rat and rabbit were 0.532, 0.502 and 0.485, respectively. In addition, the platelets from four different species can be discriminated with multivariate analysis. These findings demonstrate that the LTRS, combined with multivariate analysis, could be used to rapidly discriminate platelets from various species and may find valuable application in rapid sensing of biochemical changes in a single cell.

Expression of MaMAPK gene in seedlings of Malus L. under water stress.

Seedlings of three species of Malus were used to study the expression of mitogen-activated protein kinase (MAPK) in response to water stress: Malus hupehensis, a drought-sensitive species; Malus sieversii, a drought-tolerant species; and Malus micromalus, a middle type. Results showed that Malus MAPK (MaMAPK, GenBank accession No. AF435805) was expressed in both roots and leaves of seedlings of the three Malus species treated with 20% polyethylene glycol for different time periods. Expression levels peaked at 1.5 h after treatment with polyethylene glycol, then decreased to their lowest levels. Liquid kinase assays indicated that the dynamic changes of MAPK activity were very similar to those of the relative expression of MaMAPK mRNA. However, the peak of the former occurred slightly behind the latter. It was noticed that, although the kinase activity decreased after the peak, it was still higher than that of the control during the whole time period. These results suggested that MaMAPK was regulated not only by water stress at the transcription level, but also by phosphorylation and dephosphorylation at the protein level. In addition, of these three apple species, the highest MAPK activity and MaMAPK expression level was found in M. sieversii, followed by M. micromalus and M. hupehensis, suggesting that MAPK might be correlated with drought tolerance in these three species. The different expression levels might be one of the molecular mechanisms of the different drought tolerances in Malus.