Regulatory Factors of Lymphocyte-Lymphocyte Interaction: II. Characterization of Human Mitogenic Factor Derived from the Culture Supernatant of a Mouse-Human Hybridoma

Cell hybridization of phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes (PBL) with murine lymphoma (EL-4) provided three hybridomas (MHH-16, MHH-20, and MHH-22) which spontaneously produced human mitogenic factor (MF). MHH-16 was serially subcloned by limiting dilution procedures, which resulted in maintaining two subclones producing human MF spontaneously for more than one year (PQL-3 and PQL-5 subcloned lines). Human MF (MHH-MF) derived from supernatants of PQL-5 line cultures had a molecular weight (m.w.) of about 26,000–30,000 daltons (the major peak) with a minor peak with an m.w. of 15,000 daltons on Sephadex G-100 chromatography, and at a high concentration of NaCl (1 m), the activity of the 26,000–30,000-m.w. fraction became weak and that of the 15,000-m.w. fraction became predominant. MHH-MF had an isoelectric point of pH 5.0–6.5. On DEAE-cellulose chromatography, MHH-MF was eluted at a fairly low salt concentration (sodium phosphate buffer 0.02 M, pH 8.0, NaCl 10 mm). After periodate treatment of this MHH-MF, the mitogenic activity almost disappeared. MHH-MF was relatively unstable to heating at 56 C for 20 min. In the presence of tunicamycin (0.3μg/ml), an inhibitor of N-linked glycosylation, the synthesized MHH-MF showed a decrease in m.w. as follows: the major peak shifted from 26,000–30,000 to 23,000 daltons and the minor peak from 15,000 to 10,000 daltons on Sephadex G-100 chromatography. In internal labeling experiments with [³H]leucine, the ³H-labeled MF was partially purified, with mitogenic activity as a guide. This ³H-labeled MHH-MF fraction could be absorbed by PHA blasts but not by normal PBL. On SDS-PAGE under reducing conditions, only the radioactive peak of the 15,000-dalton fraction was recovered. MHH-MF obtained from the hybridoma culture supernatants may be a dimer of the 15,000-dalton fraction and a glycoprotein.     

Morphological pathway of flagellar assembly in Salmonella typhimurium.

The process of flagellar assembly was investigated in Salmonella typhimurium. Seven types of flagellar precursors produced by various flagellar mutants were purified by CsCl density gradient protocol. They were characterized morphologically by electron microscopy, and biochemically by two-dimensional gel electrophoresis. The MS ring is formed in the absence of any other flagellar components, including the switch complex and the putative export apparatus. Four proteins previously identified as rod components, FlgB, FlgC, FlgF, FlgG, and another protein, FliE, assemble co-operatively into a stable structure. The hook is formed in two distinct steps; formation of its proximal part and elongation. Proximal part formation occurs, but elongation does not occur, in the absence of the LP ring. FlgD is necessary for hook formation, but not for LP-ring formation. A revised pathway of flagellar assembly is proposed based on these and other results.     

X-ray equatorial analysis of crab striated muscle in the relaxed and rigor states

The structure of muscle projected along the fiber axis was studied by equatorial X-ray diffraction. The clectron-density distributions in axial projection of muscle were derived by the Fourier syntheses to a resolution of 7 nm in the relaxed and rigor states. The structure of the thick filament backbone (diameter about 21.5 nm) has a nearly smooth cylindrical surface and a low electron-density core (diameter about 7 nm) in the center. In the relaxed state, the center of gravity of the myoXXXin heads is situated at a radius of 19.6 nm from the center of the thick filament, lying just between the surface of the thick filament backbone and the surface of the thin filament (diameter about 8.4 nm). From the electron-density distributions in two slates. the amount of mass transfer from the thick filament to the thin filament was estimated. It was in accordance with that predicted from the structure derived bv the X-ray layer-line analyses.     

Correlation between Supercoiling and Conformational Motions of the Bacterial Flagellar Filament

The bacterial flagellar filament is a very large macromolecular assembly of a single protein, flagellin. Various supercoiled states of the filament exist, which are formed by two structurally different conformations of flagellin in different ratios. We investigated the correlation between supercoiling of the protofilaments and molecular dynamics in the flagellar filament using quasielastic and elastic incoherent neutron scattering on the picosecond and nanosecond timescales. Thermal fluctuations in the straight L- and R-type filaments were measured and compared to the resting state of the wild-type filament. Amplitudes of motion on the picosecond timescale were found to be similar in the different conformational states. Mean-square displacements and protein resilience on the 0.1 ns timescale demonstrate that the L-type state is more flexible and less resilient than the R-type, whereas the wild-type state lies in between. Our results provide strong support that supercoiling of the protofilaments in the flagellar filament is determined by the strength of molecular forces in and between the flagellin subunits.     

Neocortical Expansion Due to Increased Proliferation of Basal Progenitors Is Linked to Changes in Their Morphology

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Osteopontin Deficiency Accelerates Spontaneous Colitis in Mice with Disrupted Gut Microbiota and Macrophage Phagocytic Activity

Background

Osteopontin (OPN) is a multifunctional protein expressed in a variety of tissues and cells. Recent studies revealed increased OPN expression in the inflamed intestinal tissues of patients with inflammatory bowel disease (IBD). The role of OPN in the pathophysiology of IBD, however, remains unclear.

Aims

To investigate the role of OPN in the development of intestinal inflammation using a murine model of IBD, interleukin-10 knock out (IL-10 KO) mice.

Methods

We compared the development of colitis between IL-10 KO and OPN/IL-10 double KO (DKO) mice. OPN expression in the colonic tissues of IL-10 KO mice was examined by fluorescence in situ hybridization (FISH) analysis. Enteric microbiota were compared between IL-10 KO and OPN/IL-10 DKO mice by terminal restriction fragment length polymorphism analysis. The effect of OPN on macrophage phagocytic function was evaluated by phagocytosis assay.

Results

OPN/IL-10 DKO mice had an accelerated onset of colitis compared to IL-10 KO mice. FISH analysis revealed enhanced OPN synthesis in the colonic epithelial cells of IL-10 KO mice. OPN/IL-10 DKO mice had a distinctly different enteric bacterial profile with a significantly lower abundance of Clostridium subcluster XIVa and a greater abundance of Clostridium cluster XVIII compared to IL-10 KO mice. Intracellular OPN deletion in macrophages impaired phagocytosis of fluorescence particle-conjugated Escherichia coli in vitro. Exogenous OPN enhanced phagocytosis by OPN-deleted macrophages when administered at doses of 1 to 100 ng/ml, but not 1000 ng/ml.

Conclusions

OPN deficiency accelerated the spontaneous development of colitis in mice with disrupted gut microbiota and macrophage phagocytic activity.     

2′‐Deoxymugineic acid promotes growth of rice (Oryza sativa L.) by orchestrating iron and nitrate uptake processes under high pH conditions

Poaceae plants release 2′‐deoxymugineic acid (DMA) and related phytosiderophores to chelate iron (Fe), which often exists as insoluble Fe(III) in the rhizosphere, especially under high pH conditions. Although the molecular mechanisms behind the biosynthesis and secretion of DMA have been studied extensively, little information is known about whether DMA has biological roles other than chelating Fe in vivo. Here, we demonstrate that hydroponic cultures of rice (Oryza sativa) seedlings show almost complete restoration in shoot height and soil‐plant analysis development (SPAD) values after treatment with 3–30 μm DMA at high pH (pH 8.0), compared with untreated control seedlings at normal pH (pH 5.8). These changes were accompanied by selective accumulation of Fe over other metals. While this enhanced growth was evident under high pH conditions, DMA application also enhanced seedling growth under normal pH conditions in which Fe was fairly accessible. Microarray and qRT‐PCR analyses revealed that exogenous DMA application attenuated the increased expression levels of various genes related to Fe transport and accumulation. Surprisingly, despite the preferential utilization of ammonium over nitrate as a nitrogen source by rice, DMA application also increased nitrate reductase activity and the expression of genes encoding high‐affinity nitrate transporters and nitrate reductases, all of which were otherwise considerably lower under high pH conditions. These data suggest that exogenous DMA not only plays an important role in facilitating the uptake of environmental Fe, but also orchestrates Fe and nitrate assimilation for optimal growth under high pH conditions.     

Multi-step neoplastic transformation of normal human fibroblasts by Co-60 gamma rays and Ha-ras oncogenes

As reported previously (Namba et al., 1985; Namba, 1985), normal human fibroblasts were transformed into immortal cells with abnormal karyotypes by Co-60 gamma-ray irradiation. These immortally transformed cells (KMST-6) showed no clonability in soft agar and were not tumorigenic. However, by treatment with Ha-ras oncogenes derived from a human lung carcinoma or Harvey murine sarcoma virus, the KMST-6 cells acquired elevated clonability in soft agar and transplantability in nude mice. All the tumors produced grew progressively without showing regression and killed the mice. The tumors were also serially transplantable into other mice. The Ha-ras oncogene alone did not convert normal human fibroblasts into either immortal or tumorigenic cells. Our current data suggest that gamma rays worked as an initiator of carcinogenesis in normal human cells, giving rise to chromosome aberrations and immortality, and the Ha-ras oncogene played a role in the progression of the immortally transformed cell population to a neoplastic one showing enhanced colony formation in soft agar and tumorigenicity in nude mice.