Two types of response property in otolith-optokinetic interaction.

Otolithic influence on optokinetic eye-movements (visual-vestibular interaction) was investigated using oscillation of a lateral linear acceleration-step combined with optokinetic stimulation. According to our preliminary study using a 40-deg/s optokinetic stimulus speed at 0.3 and 0.5 G acceleration-steps, the interaction was characterized by a linear addition during the agonistic stimulus condition, but by suppression of the otolith-ocular reflex during the antagonistic stimulus condition. In the present study, we further examined the interaction using 3 different optokinetic speeds at an acceleration step of 0.3 G. It was revealed an additional type of response property that was characterized by marked elevation in the eye velocity with increase in the optokinetic stimulus speed, probably due to the gain increase in the optokinetic response velocity. In either type of the response property, however, the interaction seemed to be nonlinear in the otolith system, being in contrast to linear interaction postulated for the semicircular canal system.     

Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S₁ of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop.     

Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S1 of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop.     

Growth factors in bone matrix. Isolation of multiple types by affinity chromatography on heparin-Sepharose

The mineralized matrix of osseous tissue harbors abundant mitogenic activity which is extractable by demineralizing solvents. In bovine bone powder free of blood and cartilage contamination, the volume concentration of mitogens is up to 20 times greater than in serum. Growth factor activity in bone extracts was quantitated on quiescent mouse BALB/c/3T3 fibroblasts, where [3H]thymidine incorporation for 48 h was stimulated up to 200-fold in a linear, dose-dependent manner. Six distinct bone-derived growth factors (BDGFs) have been resolved and partially purified (up to 44,000-fold) on heparin-Sepharose using NaCl gradient elution. Provisionally named by the NaCl molarity at which they elute, these BDGFs include BDGF-0.45 (25% of total activity). This factor is heat-stable and sensitive to dithiothreitol, and displaces 125I-labelled bovine platelet-derived growth factor in a radioreceptor assay. BDGF-0.45 (approximately 50 ng/g of bone) is closely related or identical to bovine platelet-derived growth factor. BDGF-1.1 (10%) has a pI of 5.2 and shows a 16,600-dalton doublet on sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blots stained with antiserum to bovine anionic fibroblast growth factor. Two activities with high heparin affinity resemble cationic forms of fibroblast growth factor. BDGF-1.5 is the dominant factor in fetal membranous bone (50%), but is less abundant in adult bone (20%). BDGF-1.7, a 17,500-18,400-dalton triplet, is virtually absent in fetal bone (7%) but abundant (30%) in adult bone and may be related to cartilage derived growth factor. Two minor activities, BDGF-0.1 (10%) and BDGF-2.0 (7%) have not been characterized. Proliferation of bovine capillary endothelial cells was strongly supported by BDGFs 1.1, 1.5, and 1.7, but not by 0.45. These four purified BDGFs and the crude bone extract were also strongly mitogenic for rat osteoblasts while depressing alkaline phosphatase specific activity by 2-3-fold. Bone exhibits the most complex spectrum of growth factor activities of any tissue yet described. Bone cells and other indigenous cell types must be considered as possible sources of the BDGFs, in addition to sequestration from blood. Mechanisms for unmasking or release of BDGFs from the mineralized matrix resulting in local action on target cells are undoubtedly important for the development and maintenance of bone tissue.     

An improved cellular enucleation method with extracellular matrix and colchicine facilitates the study of nucleocytoplasmic interaction

Enucleated mammalian cells (cytoplasts) have been widely used for studying differential roles of the cytoplasm and nucleus in various cellular processes. Here, we reported an improved enucleation protocol, in which cells were seeded in extracellular matrix (ECM)-coated 24-wells and spun at 4600 g and 35 °C for 60 min in the presence of cytochalasin B and colchicine. When glass-bottom wells were used, cellular structures and organelles in cytoplasts could be examined directly by confocal microscopy. Nuclear envelope rupture did not occur probably due to mild centrifugation conditions used in this study. Addition of paclitaxel or doxorubicin completely blocked proliferation of residual nucleated cells; however, to our surprise, paclitaxel dramatically prolonged the survival of cytoplasts. Results from Annexin V and Propidium Iodide staining showed that cytoplasts died predominantly by apoptosis, which was partially inhibited by ECM and further by paclitaxel. Mitochondria were mostly rod-shaped and formed a connected network in paclitaxel-treated cytoplasts, indicating lack of fusion and fission dynamics. Moreover, paclitaxel increased mitochondrial membrane potential, suggesting that perturbation of mitochondria might be critical to the survival of cytoplasts. In conclusion, we had established an efficient and fast procedure for enucleation of adherent animal cells, which could facilitate the investigation of nucleocytoplasmic interaction.     

Purification of biomolecules by the method of the expanded bed adsorption chromatography. III. Method optimization. Aplications

This review introduces the principles of the expanded bed adsorption (EBA) and serves as a practical guide to the use STREAMLINE adsorbent and columns available on the market. Critical operational parameters will be discussed as well as the principles for the method design and optimization that will ensure maximum operation of this unique unit. The review is illustrated with the examples of different types of biological molecules which have been purified when using the expanded bed adsorption.     

The isolation of nuclei and basic nucleoproteins from the cellular slime mold Dictyostelium discoideum.

A method is described for the isolation of nuclei from an axenic strain of Dictyostelium discoideum using a sorbitol/Ficoll solution and low concentration of Triton X-100. Basic proteins have been extracted from the nuclei and on polyacrylamide gel electrophoresis yield a consistent pattern in which five major groups or bands predominate. Four of these five fractions comigrate with calf thymus histones and one fraction seems to be unique to D. discoideum. The slowest moving of the five fractions is soluble in 0.5 M perchloric acid and comigrates with calf thymus histone F1. After recovery from the perchloric acid solution by precipitation with acetone this fraction yielded one major band on electrophoresis.     

Evaluation of modified chitosan as matrix for hydrophobic interaction chromatography

Chitosan beads were modified with glutaraldehyde and modified chitosan was investigated as matrix for hydrophobic interaction chromatography. The influence of temperature, type of salt and its ionic strength on the adsorption of -galactosidase was studied. -Galactosidase was found to bind in presence of high concentration of ammonium sulphate (3 M, w/v) and 90% of the bound enzyme was eluted with decreasing salt concentration in presence of 10% ethylene glycol. Attempt was made to purify -galactosidase from modified chitosan, -galactosidase showed 1.7-fold purification with 43.96% recovery of enzyme activity. The SDS–PAGE analysis of enzyme showed considerable purification and its molecular weight was found to be 63–64 kDa. Unlike other chromatographic matrices, the prepared chitosan beads were used five times. The results showed that purification and recovery of the enzyme did not change even when column size was increased.     

Analysis of human alpha-thrombin by hydrophobic interaction high-performance liquid chromatography

The major active form of human thrombin, alpha-thrombin, was analyzed by hydrophobic interaction high-performance liquid chromatography (HIC-HPLC). The chromatographic system included a polymeric phenyl column and elution was performed by a gradient, 2-0M sodium chloride (5-20 min). Total analysis time was 30 min per injection. By this method, a good resolution between alpha-thrombin and the proteolytically modified thrombin forms, beta- and gamma-thrombin, was obtained. In addition, the thrombin preforms, prothrombin, prethrombin 1, and prethrombin 2, were also resolved from alpha-thrombin in the system. The results from the HIC method were compared to those obtained from non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By this high-resolution chromatographic method, the rapid analysis of purified alpha-thrombin is possible.     

Efficient method for preparation of highly purified lipopolysaccharides by hydrophobic interaction chromatography

A method for the efficient preparation of highly purified lipopolysaccharides (LPSs) by hydrophobic interaction chromatography (HIC) has been developed. The procedure can be used for the purification of cell wall bound LPSs after hot phenol–water extraction and for the isolation of extracellular LPSs from the supernatant, respectively. The method described has been tested with artificial mixtures containing LPSs, polysaccharide, protein and RNA and subsequently employed for the preparative purification of two LPSs of different origin, namely the extracellular LPS secreted by Escherichia coli E49 into the culture medium, and the cell wall bound LPS from Pseudomonas aeruginosa VA11465/1. Compared to currently used methods for LPS purification such as enzymatic digestion and ultracentrifugation, the chromatographic separation reported here combines superior purity with minimal loss of LPS, high reproducibility and simple handling. The removal of contaminants such as protein, RNA and polysaccharides and the recovery of LPSs were monitored by appropriate assays.     

Ribosomal and informational ribonucleoprotein complexes of animal cells. Study on rat liver ribonucleic acids as constituents of ribonucleoprotein complexes by chromatography on nucleoprotein-celite columns.

A novel method of RNA fractionation has been developed. Nuclear and cytoplasmic rat liver RNA species were fractionated as constituents of corresponding ribonucleoprotein particles, which were previously adsorbed on a Celite-column by their protein component. The fractionation is based on a dissociation of the particles (linear concentration gradient of LiCl and urea with subsequent temperature gradient), which results in a gradual release of the RNA molecules from ribonucleoprotein complexes. Thus the fractionation is in accordance with the tightness of the RNA-protein bonds. A gradient elution of RNA from a nucleoprotein-Celite column permitted fractionation of both ribosomal and rapidly labelled non-ribosomal RNA. The latter, both nuclear and cytoplasmic, could be separated by chromatography on nucleoprotein-Celite columns into two main fractions (components I and V). In cytoplasmic RNA components I and V presumably correspond to mlRNA (messenger-like RNA of free cytoplasmic particles) and mRNA (template RNA associated with ribosomes) respectively.     

Activity of matrix metalloproteinase-9 against native collagen types I and III

Interstitial collagen types I, II and III are highly resistant to proteolytic attack, due to their triple helical structure, but can be cleaved by matrix metalloproteinase (MMP) collagenases at a specific site, approximately three-quarters of the length from the N-terminus of each chain. MMP-2 and -9 are closely related at the structural level, but MMP-2, and not MMP-9, has been previously described as a collagenase. This report investigates the ability of purified recombinant human MMP-9 produced in insect cells to degrade native collagen types I and III. Purified MMP-9 was able to cleave the soluble, monomeric forms of native collagen types I and III at 37 degrees C and 25 degrees C, respectively. Activity against collagens I and III was abolished by metalloproteinase inhibitors and was not present in the concentrated crude medium of mock-transfected cells, demonstrating that it was MMP-9-derived. Mutated, collagenase-resistant type I collagen was not digested by MMP-9, indicating that the three-quarters/one-quarter locus was the site of initial attack. Digestion of type III collagen generated a three-quarter fragment, as shown by comparison with MMP-1-mediated cleavage. These data demonstrate that MMP-9, like MMP-2, is able to cleave collagens I and III in their native form and in a manner that is characteristic of the unique collagenolytic activity of MMP collagenases.     

生物功能化色谱技术研究胰岛素与其受体间的相互作用

生物功能化色谱技术是通过模拟细胞膜表面环境,在色谱系统内实现研究生物分子间相互作用过程的新技术。利用生物功能化色谱方法研究了胰岛素与其受体间的相互作用。将提取出的胰岛素受体混合物固定化在人工膜(Immobilized artificial membrane, IAM)的表面,并确定了固定化的最佳pH值为7.2,最佳离子强度为20 mmol,最佳有机溶剂浓度为2% (v/v)。通过区带洗脱法确定了胰岛素受体的存在及其与胰岛素间存在相互作用;通过前沿分析法确定了胰岛素受体与胰岛素间相互作用的结合常数大小约为0.701 nmol/L。     

Fractionation of avian erythrocyte histones by hydrophobic interaction chromatography.

The interactions of H1 (H1A, H1B), H2A, H2B, H3, H4, and H5 with phenyl cross-linked agarose were studied. Procedures are described whereby all six histones can be bound, released, and fractionated by using appropriate salt concentrations or pH. The binding can be totally abolished by inclusion of hydrophobic disrupting agents. Control experiments with nonderivated cross-linked agarose ruled out a passive aggregation-disaggregation phenomenon governing the binding patterns. The absorption sequence based on the identification and quantitation of individual histones from either unfractionated (whole) histone or separate histone classes is as follows: H3 greater than or equal to H4 greater than H2B greater than or equal to H5 greater than or equal to H2A greater than H1A greater than or equal to H1B. The order differs only slightly from the reverse of the desorption sequence, H1B less than or equal to H1A less than or equal to H5 less than H2A less than or equal to H3. Preferential interaction of H2A-H2B, H3-H4, and H2A-H2B-H4 occur; these interactions can modify the original relative affinity of each individual component for the matrix. The variability in matrix affinity appears to involve simple stoichiometry of the histone components.     

Two types of P neurons in the medullary dorsal respiratory cellular group in cats

Electrophysiological properties of P neurons localized in the medullary dorsal respiratory cellular group and of vagal afferent fibers innervating these neurons were studied in acute experiments on nembutal-anesthetized cats with preserved spontaneous respiration. P neurons were shown to form a non-homogeneous cellular population. They generated phasic discharges during the whole inspiration period, but differed in their responses to lung inflation. These findings allowed us to classify P neurons as slowly adapting and rapidly adapting units, probably activated by slowly and rapidly adapting pulmonary receptors, respectively. Sensitivity of the slowly adapting P neurons to activation by the corresponding receptors and the mechanisms underlying the participation of the two types of P neurons in the reflex feedback between the respiratory center and lungs are discussed.Neirofiziologiya/Neurophysiology, Vol. 26, No. 3, pp. 211–217, May–June, 1994.     

An analytical method for the separation of dopamine metabolites in cellular extracts by high-pressure liquid chromatography

An analytical scheme using high-performance liquid chromatography (HPLC) has been developed to separate radiolabeled catecholamines in cell extracts derived from mammalian cells grown in tissue culture. Four different types of chromatographic systems have been employed to effect separations of groups of metabolites that possess similar organic functional groups. Data obtained by thin-layer chromatography are also presented and it is demonstrated that HPLC is the system of choice for the separation and quantitative analysis of metabolites of dopamine in physiological fluids.