Type analysis of the oligosaccharide chains on microheterogeneous components of bovine pancreatic DNAase by the lectin-nitrocellulose sheet method.

The oligosaccharide chains of microheterogeneous bovine pancreatic DNAases were characterized by the lectin-nitrocellulose sheet method. The active fractions of the DNAases from column chromatography showed four major and several minor spots on a two-dimensional polyacrylamide gel. They were transferred on to nitrocellulose sheets and treated with glycosidases (neuraminidase, endo-beta-N-acetyl glucosaminidase H or F, or peptide N-glycosidase F) and treated with peroxidase-coupled lectins (concanavalin A, Ricinus communis agglutinin or wheat-germ agglutinin). From the results, the most probable oligosaccharide types were proposed to be as follows: the four major spots contained components which had high-mannose type or hybrid-type oligosaccharides, such as those susceptible to endo-beta-N-acetylglucosaminidase H. In addition, spot 1 contained a complex-type biantennary oligosaccharide without sialic acid and spot 3 contained a tri- or tetra-antennary complex-type oligosaccharide with sialic acid. The component corresponding to spot 2 had a hybrid-type oligosaccharide chain with a 'bisecting' acetylglucosamine, linked 1-4 to the beta-mannose residue of the trimannosyl core, and the component corresponding to spot 4 had a high-mannose-type oligosaccharide chain.     

Biological half time of deposited nickel oxide aerosol in rat lung by inhalation

Wistar male rats were exposed to nickel oxide (NiO) aerosols (mass median aerodynamic diameter, 1.2 and 4.0 μm). The average exposure concentration was controlled from a low level of 0.6 mg/m³ to a high level of 70 mg/m³ and total exposure time was 140 h. Some rats were sacrificed just after the exposure, whereas others were exposed for 1 mo and kept for 12 and 20 mo clearance periods before sacrifice. There were no differences in body weight gain between NiO exposure groups and controls. Nickel concentrations in lungs of exposure groups were much higher than those of controls and decreased with the passing of the clearance time. No apparent deposition of nickel was observed in the liver, kidney, spleen, and blood immediately after the exposure, but in the case of the high exposure groups, the nickel concentration in the liver, spleen, and blood slightly increased with the increasing time of clearance. The biological half time of NiO deposited in the lungs was estimated by the assumption that the amount of the clearance is proportional to the amount of the NiO deposited. This resulted in a biological half time of 11.5 and 21 mo for 1.2 and 4.0 μm, respectively.     

Special smooth muscle cells along the submucosal surface of the guinea pig colon with reference to its spontaneous contractions

Antiperistalses occur from the flexure region of the guinea pig colon. We previously demonstrated that the circular muscle at the mesenteric border of the flexure region produced spontaneous regular contractions and found special smooth muscle cells believed to be pacemakers along the submucosal surface of the circular muscle layer. In this study, we revealed bipolar- and multipolar-type special smooth muscle cells along the submucosal surface of the muscle layer. Their slender cell processes contacted each other and formed a cellular network. Caveolae, filament structures expressing smooth muscle actin, vimentin, some desmin, and basal lamina were prominent features. The special smooth muscle cells corresponded to c-Kit-immunopositive cells and so-called interstitial cells or interstitial cells of Cajal in other reports. Their population was larger in the flexure region and the proximal colon than in the distal colon. The circular muscle layer at the flexure region was thicker than in other regions. The contraction in the flexure region showed the highest frequency and regularity. The dense population of special smooth muscle cells at the flexure region and thicker muscle layer may make the mechanical contraction more regular. The antiperistalsis from the flexure region could be explained in relation to the highest frequency of the pulsating contraction.     

c-kit-Dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract

In vivo injection of a neutralizing, monoclonal antibody (ACK2) to the receptor tyrosine kinase (c-kit) disrupts the normal motility patterns of the mouse small intestine. Immunohistochemical studies showed that cells expressing c-kit-like immunoreactivity (c-kit-LI) decreased in numbers in response to ACK2, but the identity of these cells is unknown. We investigated the identity and development of the cells that express c-kit-LI in the mouse small intestine and colon. Cells in the region of the myenteric plexus and deep muscular plexus of the small intestine and in the subserosa, in the myenteric plexus region, within the circular and longitudinal muscle layers, and along the submucosal surface of the circular muscle in the colon were labeled with ACK2. The distribution of cells that express c-kit-LI was the same as that of interstitial cells (ICs). In whole-mount preparations cells with c-kit-LI were interconnected, forming a netword similar to the network formed by cells that stained with methylene blue, which has been used as a marker for ICs in the mouse gastrointestinal tract. Immunocytochemistry verified that ICs were labeled with ACK2. Multiple injections of animals with ACK2 between days 0 and 8 post partum (pp) caused a dramatic reduction in the number of ICs compared to control animals. From an ultrastructural point of view, the proliferation and development appeared to be suppressed in some classes of ICs, while others displayed an altered course of development. Functional studies showed that the decrease in ICs was accompanied by a loss of electrical rhythmicity in the small intestine and reduced neural responses in the small bowel and colon. Morphological experiments showed that c-kit-positive cells are ICs, and physiological evidence reinforced the concept that ICs are involved in generation of rhythmicity and translation of neural inputs in gastrointestinal smooth muscles. Controlling the development of ICs provides a powerful new tool for the investigation of the physiological role of these cells.     

Replication of Radiation-Induced Murine Leukemia Virus in Normal and Transformed Mouse Cells

Autonomous radiation-induced leukemia virus (RadLV) replication could be detected in mouse 3T3 cells by the development of interference with murine sarcoma virus (MSV), the appearance of covert helper activity for defective MSV, and by the induction of cytopathic effect type foci in MSV-transformed, leukemia virus-negative (S+L-) cells. A chronic infection of either 3T3 or S+L- cells with RadLV could be established. Both RadLV infectivity and helper activity were demonstrated in the same peak at a buoyant density of 1.16 g/cm(3). Additionally a soluble inhibitor of MSV focus formation was found which could be separated from infectious RadLV. Examination of cell clones derived from chronically infected 3T3 cells showed that essentially every cell was infected and produced both infectious RadLV and low levels of inhibitor. Quantitative comparisons of autonomously replicating RadLV in normal 3T3 and S+L- cells suggested that RadLV may consist of several populations of virus of varying replicative potential. Apparently 99% of RadLV can be assayed only as helper units in normal cells or as replicative units in S+L- cells. To explain the atypical results, a model for RadLV deficiency is proposed.     

Acute and Temporal Expression of Tumor Necrosis Factor (TNF)-α-stimulated Gene 6 Product,TSG6, in Mesenchymal Stem Cells Creates Microenvironments Required for Their Successful Transplantation into Muscle Tissue

Previously, we demonstrated that when mesenchymal stem cells (MSCs) from mouse ES cells were transplanted into skeletal muscle, more than 60% of them differentiated into muscles in the crush-injured tibialis anterior muscle in vivo, although MSCs neither differentiated nor settled in the intact muscle. Microenvironments, including the extracellular matrix between the injured and intact muscle, were quite different. In the injured muscle, hyaluronan (HA), heavy chains of inter-α-inhibitor (IαI), CD44, and TNF-α-stimulated gene 6 product (TSG-6) increased 24–48 h after injury, although basement membrane components of differentiated muscle such as perlecan, laminin, and type IV collagen increased gradually 4 days after the crush. We then investigated the microenvironments crucial for cell transplantation, using the lysate of C2C12 myotubules for mimicking injured circumstances in vivo. MSCs settled in the intact muscle when they were transplanted together with the C2C12 lysate or TSG6. MSCs produced and released TSG6 when they were cultured with C2C12 lysates in vitro. MSCs pretreated with the lysate also settled in the intact muscle. Furthermore, MSCs whose TSG6 was knocked down by shRNA, even if transplanted or pretreated with the lysate, could not settle in the muscle. Immunofluorescent staining showed that HA and IαI always co-localized or were distributed closely, suggesting formation of covalent complexes, i.e. the SHAP-HA complex in the presence of TSG6. Thus, TSG6, HA, and IαI were crucial factors for the settlement and probably the subsequent differentiation of MSCs.     

Asymmetric bioreduction of a bisaryl ketone to its corresponding (S)-bisaryl alcohol, by the yeast Rhodotorula pilimanae ATCC 32762

The screening of 310 microbial strains yielded eight as suitable biocatalysts for the asymmetric bioreduction of a highly hindered bisaryl ketone to its corresponding alcohols. The production of both enantiomers with elevated optical purity (ee>96%) was achieved by different microorganisms. When scaling up the asymmetric bioreduction process in laboratory bioreactors (23 l scale), the production of preparative amounts (1.5 g) of the (S) enantiomer with elevated optically purity (ee>96%) was achieved when employing the yeast Rhodotorula pilimanae ATCC 32762. Achieving this asymmetric bioreduction with enantiocomplementarity in employing such a hindered substrate is remarkable and highlights the potential of such biological approach.     

Strain Differences in the Immunogenicity of Aggregated Human IgG and the Adjuvant Action of Lipopolysaccharide on the Low-Responder Strain of Mice

Strain differences in the antibody response to human IgG (HGG) were observed when aggregated HGG was injected intravenously. Lipopolysaccharide (LPS) administered subsequently markedly enhanced the antibody response to HGG in low responder C57BL/6 mice as compared with that in high responder DDD, C3H/He or (C57BL/6 × DDD)F₁ mice. Aggregate-free preparation of HGG at a dose of 0.5 mg induced immunological tolerance in all strains of mice tested. LPS injected subsequently converted tolerogenic, aggregate-free HGG into immunogen in DDD mice but not in C57BL/6 mice. To determine the correlation between adjuvanticity and mitogenicity of LPS, spleen cells from normal mice were cultured in the presence of LPS and ³H-thymidine uptake was measured. Spleen cells of DDD mice incorporated three times as much ³H-thymidine as those of C57BL/6 mice. There seems no strong correlation between both activities of LPS. The data obtained are discussed in terms of strain differences in the macrophage function for processing the antigen.     

Synergetic effects of Ca2+ and Cu2+ on phase transition in phosphatidylserine membranes

In complexes of divalent metals with large exchange rate constant (K_H2O) of the coordinated H₂O, such as Ca²⁺ and Cu²⁺, the cubic structure in the ligand field is usually unstable and conformation changes are easily induced. We observed the molecular motion of phosphatidylserine (PS) in an amphipathic solvent (water / methanol / chloroform) by ¹H-NMR and ESR using Ca²⁺ and / or Cu²⁺, which has a similar K_H2O to that of Ca²⁺. We found that Ca²⁺ did not hinder the molecular movements of PS. However, Cu²⁺ reduced the movements of both headgroups and the double bonds in the fatty acids of PS. By addition of both Ca²⁺ and Cu²⁺, phase transition to a soft solid phase in the PS membrane was observed at room temperature. The results indicate that the headgroups are clustered in two-dimensional network with each ligand field displaced from the aqueous phase to the water / oil interface. The structure changes of the polar headgroups after the binding of divalent cations are considered to trigger the phase transition of this acidic phospholipid membrane.