Growth distribution and surface pH patterns along maize roots

The distribution of elongation and surface pH patterns along the primary roots of maize (cv. LG 11), maintained vertically in humid air (darkness, 22°C), have been analysed quantitatively. A new technique employing Sephadex G 25 beads containing a pH indicator dye (bromocresol purple), was used for measuring both the growth gradient of the roots (Sephadex beads as markers) and at the same time, the surface pH changes (referring to a standard scale). The optimal axial growth was located between 2 and 4 mm from the tip. This coincides with the optimal decrease in surface pH.     

Solid-phase synthesis of ragweed allergen Ra5

A two-stage strategy was used to synthesize the 45-residue, four-disulfide-bonded ragweed protein allergen Ra5: (i) the protected linear chain was prepared by solid-phase, stepwise synthesis according to the sequence of the fully reduced protein; (ii) after removal from the resin and deprotection by hydrogen fluoride, the reduced chain was purified by gel filtration on Sephadex G-50 and allowed to fold “spontaneously” in the presence of oxidized dithiothreitol to form the disulfide links. The product (synthetic Ra5) was purified to disc-electrophoretic homogeneity by cycling through Sephadex G-50 and CM-Sephadex (overall yield: 6% theoretical). Synthetic Ra5 was closely similar in structure to the natural protein by amino acid analysis. polyacrylamide gel electrophoresis, tryptic peptide mapping, and circular dichroism spectra, as well asin vitro andin vivo immunoassays of antigenic/allergenic activities.     

Purification and amino acid analysis of cytochrome c fromUstilago violacea

Cytochrome c fromUstilago violacea was further analyzed in order to characterize the pink phenotype. NaOH-extracted cytochrome c was purified in three steps, which included ammonium sulfate precipitation, CM-Sephadex ion-exchange chromatography with 0.5 N NaCl elution, and CM-Sephadex ion-exchange chromatography with a 0.0–0.6 N NaCl gradient elution. Polyacrylamide gel electrophoresis of the purified protein yielded a single red band, which was the only band detected upon Coumassie brilliant blue staining. HCl-hydrolysates of the protein were examined for their amino acid composition, which indicated that the cytochrome c fromU. violacea contains approximately 104 residues, with high levels of alanine, histidine, serine, and low levels of phenylalanine and arginine.     

Biochemical characterization of a metallothionein-like protein in rat brain

Recent investigations from this laboratory have identified a metallothionein-like protein in the rat brain with an elution volume (ve/vo) of 2.08 and a molecular weight of smaller than 10,000. The synthesis of this protein was stimulated following intracerebroventricular (icv, 0.20 μmol zinc/μL/h, 48 h), but not intraperitoneal (ip) administration of ZnSO₄. Furthermore, chronic ip administration of ZnSO₄ (5.0 mg/kg/d/10 d) did not alter the level of the metallothionein-like protein in the brain. However, the hepatic metallothionein was induced following icv administration of ZnSO₄. The chromatofocusing of metallothionein-like protein isolated by gel permeation chromatography on Sephadex G-75 exhibits three zinc-binding peaks, which focus on pH 6.8, 6.2, and 5.3, respectively. It is expected that the protein peak focusing at 5.3 is a metallothionein-like protein. Purification of the zinc stimulated metallothionein-like protein on ion exchange chromatography on DEAE-Sephadex A-25 columns, using a linear gradient elution procedure produced two isoforms, eluting, respectively, at 75 and 137 mM of Tris-acetate buffer, pH 7.5. The comparative high-performance liquid chromatographic (HPLC) profiles of the zinc-induced hepatic metallothionein isoforms I and II (retention times 17.39 and 18.73 min) and that of the zinc-stimulated metallothionein-like protein isoforms I and II (retention times 17.32 and 18.64 min) are very similar. The function(s) of the metallothionein-like protein isoforms in the brain remains to be elucidated.     

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.     

The problem of isolation of the X virus inhibitor from potato leaf sap

Using moving boundary electrophoresis in a veronal buffer, pH 8·5, of 0·05 ionic strength and at 4·3 mA, we succeeded in separating the inhibitor, which was obtained by fractionation on a Sephadex G-50 column, into four components. The two most substantial components represent 25% and 65% respectively from the separated proteins as a whole. The heterogeneity of the inhibitor was proved by analytical ultracentrifugation, too. The ionex chromatography was applied for the quantitative separation of the inhibitor. We used ionex chromatography on DEAE Cellulose the concentration gradient being 0–1m NaCl in a 0·01m phosphate buffer, pH 7·3, and on DEAE Sephadex A-50 using the same concentration gradient in a phosphate buffer, of 0·1 ionic strength and pH 7·3. In both cases four components were obtained. The most substantial component, representing 65% of the whole analysed material, was eluted at the concentration 0·15–0·3m NaCl, and was electrophoretically homogenous and showed the most effective inhibitory ability.     

A Systems Biology Approach to Study the Biology Characteristics of Esophageal Squamous Cell Carcinoma by Integrating microRNA and Messenger RNA Expression Profiling

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant tumors. The aim of this study was to investigate the biology characteristics of ESCC by analyzing microRNA and mRNA expression profile. We used BRB-array tools to analyze the deregulated microRNA and mRNA between esophageal squamous cell carcinomas and paired normal adjacent tissues. We used miRTrail and protein–protein interaction methods to explore the related pathways and networks of deregulated microRNA and mRNA. By combining the results of pathways and networks, we found that the deregulated microRNA and their deregulated target mRNA are enriched in the following pathways: DNA replication, cell cycle, ECM-receptor interaction, focal adhesion, mismatch repair, and pathways in cancer. The results showed that many deregulated microRNAs and mRNAs may play a vital role in the pathogenesis of ESCC, and the systems biology approach is very helpful to explore molecular mechanism of ESCC.     

Separation of zinc ligands in human seminal plasma

Zinc is an essential trace element for male fertility, especially in the capacitation process of spermatozoa. In the present work, after taking into account the contamination and absorption phenomena that occur during chromatography, we found four different zinc ligands in seminal plasma. Citrate is the main (79%) one, then an unknown low-molecular-weight ligand (15%), albumin (4%), and transferrin (2%). Semen samples were successively fractionated on immobilized Cibacron blue, Sephadex G 75 and G 15, and immobilized concanavalin A.     

Studies on choline acetyltransferase isolated from human brain

The purified choline acetyltransferase from human striatal tissue was found to have aK _m value of 8 μM for acetyl-coenzyme A and 250 μM for choline. The predominant enzyme component has a molecular weight of about 67,000 daltons, measured by molecular filtration through Sephadex G-100. In a sucrose-density gradient, the enzyme cosedimented with bovine serum albumin with an estimatedS-value of 4.5. The enzyme activity was enhanced 2- to 3-fold by KCl, NaCl, (NH₄)₂SO₄, and chelating agents like EDTA or EGTA. Cupric sulfate (0.1 mM) inhibited the enzyme activity almost completely. This inhibition was circumvented by increasing concentrations of enzyme protein, dithiothreitol, and EDTA, but not by the substrates, histidine, or imidazole.     

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.     

Relationship between the composition of the regulatory complex of esterases and the age ofMycobacterium phlei culture

During submerged cultivation ofMycobacterium phlei a mixture of macromolecular compounds ia released into the medium. Concentrated filtrates of cultures of different ages were separated on Sephadex G-25 fine and polyanions were found to predominate in the young culture. During further days of fermentation the proportion of polycations significantly increases. The results are discussed with respect to the regulatory complex of esterases.     

Feasibility study of gel filtration as a separation method for blood serum proteins in combination with PIXE analysis

The combination between a protein separation technique and the PIXE method has a great potential for large surveys, including thousands of samples, in which multielemental analysis is required. Gel filtration with a Sephadex G-200 gel and a TRIS-acetate buffer was used for separating proteins in human serum. The fractions were doped with an yttrium/vanadium standard and then concentrated and pipeted onto Kimfol^? backing foils. Using the PIXE technique, the distributions of Fe, Cu, Zn, and Se, with respect to molecular size, were found, indicating binding to specific proteins. Sulfur and phosphorus were found to correlate well with the protein content measured by UV-absorption at 280 nm. Further developments and tests on the protein separation technique is required, taking restrictions imposed by the PIXE method into consideration.     

Progression of O6-methylguanine-DNA methyltransferase and temozolomide resistance in cancer research

Temozolomide (TMZ) is an alkylating agent that is widely used in chemotherapy for cancer. A key mechanism of resistance to TMZ is the overexpression of O⁶-methylguanine-DNA methyltransferase (MGMT). MGMT specifically repairs the DNA O⁶-methylation damage induced by TMZ and irreversibly inactivates TMZ. Regulation of MGMT expression and research regarding the mechanism of TMZ resistance will help rationalize the clinical use of TMZ. In this review, we provide an overview of recent advances in the field, with particular emphasis on MGMT structure, function, expression regulation, and the association between MGMT and resistance to TMZ.     

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.     

Chemical composition and immunological properties of glycoproteins of Sporothrix species

Glycoproteins of 11Sporothrix species were purified from their respective culture filtrates by use of DEAE-Sephadex A-50 and QAE-Sephadex A-25 column chromatography and investigated for their chemical and immunological properties. On the basis of sugar composition, the glycoproteins of the 11Sporothrix species could be divided into two groups, i.e., rhamnose containing (i.e., Rha⁺), and non rhamnose containing (i.e., Rha^?) groups. The species in the former group wereS. curviconia, S. inflata, S. schenckii andS. schenckii var. luriei, and those in the latter group wereS. cyanescens, S. foliorum, S. fungorum, S. ghanensis, S. imectorum, S. luteoalba andS. ramosissima. The glycoproteins of four of the (Rha⁺) species were relatively similar in elution patterns of DEAE-Sephadex A-50 chromatograms, sugar and amino acid compositions, serological reactivity with rabbit andS. schenckii serum and rabbit antiKlebsiella pneumoniae K47 serum, and cutaneous delayed hypersensitivity. In the case of the (Rha^?) species, the glycoproteins of five species cross-reacted with rabbit antiS. schenckii serum and all, but theS. cyanescens, glycoprotein were reactive to some degree in skin tests in sporotrichotic patients. These results strongly suggest that the chemical and immunological properties of these glycoproteins correspond with the morphological observations amongSporothrix species.     

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.