Immunomagnetic separation of scum-forming bacteria using polyclonal antibody that recognizes mycolic acids

Mycolic acid-containing bacteria (mycolata) are thought to be involved in scum formation in aeration basins of activated sludge plants due to their ability to produce biosurfactants and their cell surface hydrophobicity. To isolate these bacteria, immunomagnetic separation (IMS) using an anti-mycolic acid polyclonal antibody was investigated. IMS that targeted Gordonia amarae SC1 exhibited a 100% recovery at 5x10(3) CFU ml(-1). At cell concentration of 7.8x10(6) CFU ml(-1), the recovery was lowered, but 80% of cells were still captured. Effect of bead concentrations on the recovery of SC1 at 10(6) CFU ml(-1) was examined. The results showed that addition of more than 6-7x10(6) beads for 1x10(6) CFU reached a maximum recovery (83%). Furthermore, the IMS procedure optimized with SC1 cells was tested with another mycolata. The results suggested that variation of the recovery for each mycolata is dependent on the specificity of the polyclonal antibody and that mycolata which are recognized by the antibody can be recovered by this procedure.     

Proliferative effects of angiotensin II and endothelin-1 on guinea pig gingival fibroblast cells in culture

We investigated whether phenytoin (PHT) and nifedipine (NIF) induce angiotensin II (Ang II) and endothelin-1 (ET-1) generation by cultured gingival fibroblasts derived from guinea pigs and whether Ang II and ET-1 induce proliferation of these cells. Immunohistochemical experiments showed that PHT (250 nM) and NIF (250 nM) increased the immunostaining intensities of immunoreactive Ang II and ET-1 (IRET-1) in these cells. Captopril (3 microM), an angiotensin-converting enzyme inhibitor, reduced these enhanced intensities to control levels. Ang II (100 nM) enhanced the immunostaining intensity of IRET-1. PHT (250 nM) and NIF (250 nM)-induced cell proliferation. Both PHT- and NIF-induced proliferation was inhibited by captopril (3 microM). Ang II (100 nM) and ET-1 (100 nM) also induced cell proliferation. Ang II-induced proliferation was inhibited by CV11974 (1 microM), an AT(1) receptor antagonist and saralasin (1 microM), an AT(1)/AT(2) receptor antagonist, but not by PD123,319 (1 microM), an AT(2) receptor antagonist. ET-1-induced proliferation was inhibited by BQ123 (10 microM), an ET(A) receptor antagonist, but not by BQ788 (1 microM), an ET(B) receptor antagonist. These findings suggest that PHT- and NIF-induced gingival fibroblast proliferation is mediated indirectly through the induction of Ang II and ET-1 and probably mediated through AT(1) and ET(A) receptors present in or on gingival fibroblasts.     

Overexpression of Arabidopsis response regulators,ARR4/ATRR1/IBC7 and ARR8/ATRR3, alters cytokinin responses differentially in the shoot and in callus formation

Arabidopsis ARR4/ATRR1/IBC7 and ARR8/ATRR3 are homologous genes of prokaryotic response regulators that are involved in the His-Asp phosphorelay signal transduction. We analyzed the function of these genes as response regulators using transgenic plants. Overexpression of ARR4 in cultured stems of the transgenics markedly promoted shoot formation in the presence of cytokinin, while overexpression of ARR8 repressed shoot formation and greening of calli. The expression level of cytokinin-inducible genes, cycD3 and cab increased in the ARR4 overexpressor but decreased in the ARR8 overexpressor. By contrast, two drought stress-inducible genes, rd29A and erd1, were expressed in both overexpressors as that in control plants. These results suggest that ARR4 and ARR8 are involved in cytokinin signal transduction, and that ARR4 functions as a positive-regulator, whereas ARR8 functions as a negative-regulator. Furthermore, microarray analysis showed that several genes were up-regulated in the ARR4 overexpressor. Consistent with these results, ARR4 and ARR8 might play important roles in the sensoring system of cytokinin signal transduction pathway in various developmental and environmental conditions and the regulation of gene expression.     

Absence of Cajal-Retzius cells and subplate neurons associated with defects of tangential cell migration from ganglionic eminence in Emx1/2 double mutant cerebral cortex

Emx1 and Emx2, mouse orthologs of the Drosophila head gap gene, ems, are expressed during corticogenesis. Emx2 null mutants exhibit mild defects in cortical lamination. Segregation of differentiating neurons from proliferative cells is normal for the most part, however, reelin-positive Cajal-Retzius cells are lost by the late embryonic period. Additionally, late-born cortical plate neurons display abnormal position. These types of lamination defects are subtle in the Emx1 mutant cortex. In the present study we show that Emx1 and Emx2 double mutant neocortex is much more severely affected. Thickness of the cerebral wall was diminished with the decrease in cell number. Bromodeoxyuridine uptake in the germinal zone was nearly normal; moreover, no apparent increase in cell death or tetraploid cell number was observed. However, tangential migration of cells from the ganglionic eminence into the neocortex was greatly inhibited. The wild-type ganglionic eminence cells transplanted into Emx1/2-double mutant telencephalon did not move to the cortex. MAP2-positive neuronal bodies and RC2-positive radial glial cells emerged normally, but the laminar structure subsequently formed was completely abnormal. Furthermore, both corticofugal and corticopetal fibers were predominantly absent in the cortex. Most importantly, neither Cajal-Retzius cells nor subplate neurons were found throughout E11.5-E18.5. Thus, this investigation suggests that laminar organization in the cortex or the production of Cajal-Retzius cells and subplate neurons is interrelated to the tangential movement of cells from the ganglionic eminence under the control of Emx1 and Emx2.     

Electron microscopic studies of three gliding Mycoplasmas,Mycoplasma mobile,M. pneumoniae,and M. gallisepticum,by using the freeze-substitution technique

Freeze-substitution technique was applied to thin-sectioning electron microscopy of Mycoplasma mobile, M. pneumoniae, and M. gallisepticum, all of which can glide in the direction of the tapered cell end. M. mobile presented a flask-like cell morphology. An additional layer was found around the tapered end. The cell images of M. pneumoniae showed a protruding membrane extension, the attachment organelle, composed of a low density space inside the cells and featuring a filamentous dense core anchored to the terminal end. The detailed structures were more obvious than those observed by the conventional chemical fixation. The cells of M. gallisepticum presented irregular dense granules, in contrast to regular particles, which can be observed in the images of chemically fixed thin sections, in the rear portion of the cells. Received: 4 September 2001 / Accepted: 25 September 2001     

20-HETE contributes to the acute fall in cerebral blood flow after subarachnoid hemorrhage in the rat

This study examined the effects of blocking the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) on the acute fall in cerebral blood flow after subarachnoid hemorrhage (SAH) in the rat. In vehicle-treated rats, regional cerebral blood flow (rCBF) measured with laser-Doppler flowmetry fell by 30% 10 min after the injection of 0.3 ml of arterial blood into the cisterna magna, and it remained at this level for 2 h. Pretreatment with inhibitors of the formation of 20-HETE, 17-octadecynoic acid (17-ODYA; 1.5 nmol intrathecally) and N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (HET0016; 10 mg/kg iv), reduced the initial fall in rCBF by 40%, and rCBF fully recovered 1 h after induction of SAH. The concentration of 20-HETE in the cerebrospinal fluid rose from 12 +/- 2 to 199 +/- 17 ng/ml after SAH in vehicle-treated rats. 20-HETE levels averaged only 15 +/- 11 and 39 +/- 13 ng/ml in rats pretreated with 17-ODYA or HET0016, respectively. HET0016 selectively inhibited the formation of 20-HETE in rat renal microsomes with an IC(50) of <15 nM and human recombinant CYP4A11, CYP4F2, and CYP4F3 enzymes with an IC(50) of 42, 125, and 100 nM, respectively. These results indicate that 20-HETE contributes to the acute fall in rCBF after SAH in rats.     

Rate-dependent [K+](o) accumulation in canine right atria in vivo: electrophysiological consequences

Sudden increases in heart rate cause accumulation of K+ in the extracellular space. However, the exact relationship between rate and extracellular K+ concentration ([K+](o)) in vivo is unknown. We measured [K+](o) in right atria of anesthetized dogs by using K(+)-sensitive electrodes. Peak increase in [K+](o) ranged from 0.18 +/- 0.04 mM [means +/- SE; cycle length (CL) = 350 ms] to 0.80 +/- 0.09 mM (CL = 250 ms) above baseline (3.50 +/- 0.08 mM at CL = 380 ms; n = 5). During rapid pacing-induced atrial fibrillation, peak increase in [K+](o) averaged 0.80 +/- 0.07 mM (n = 5). Whole cell current-clamp measurements in single right atrial myocytes (n = 5) showed that raising [K+](o) from 3 to 5 mM in 1-mM steps progressively depolarized resting membrane potential and reduced both phase 0 action potential amplitude and maximal upstroke velocity. Multisite epicardial mapping (n = 4) demonstrated that sudden rate increases changed longitudinal conduction velocity (CV(L)) by -3.6 +/- 1.8% to -5.9 +/- 1.2% over a CL range of 330 to 250 ms. Our observations suggest that rate-related [K+](o) accumulation in vivo is of sufficient magnitude to modulate those cellular electrophysiological properties that determine atrial CV(L).     

A high-resolution 13C-nmr study of collagenlike polypeptides and collagen fibrils in solid state studied by the cross-polarization–magic angle-spinning method. Manifestation of conformation-dependent 13C chemical shifts and application to conformational characterization

We have recorded high-resolution ¹³C-nmr spectra of collagen fibrils in the solid state by the cross-polarization–magic-angle-spinning(CP–MAS)method and analyzed the spectra with reference to those of collagenlike polypeptides. We used two kinds of model polypeptides to obtain reference ¹³C chemical shifts of major amino acid residues of collagen (Gly, Pro, Ala, and Hyp): the 3₁-helical polypeptides [(Gly)_nII, (Pro)_nII, (Hyp)_n, and (Ala? Gly? Gly)_nII], and the triple-helical polypeptides [(Pro? Gly? Pro)_n and (Pro? Ala? Gly)_n]. Examination of the ¹³C chemical shifts of these polypeptides, together with our previous data, showed that the ¹³C chemical shifts of individual amino acid residues are the same, within experimental error (±0.5 ppm), among different polypeptides with different primary sequences, if the conformations are the same. We found that the ¹³C chemical shifts of Ala residues of the 3₁-helical (Ala? Gly? Gly)_n and triple-helical (Pro? Ala? Gly)_n are significantly displaced, compared with those of the α-helix, β-sheet, and silk I form, and can be utilized as excellent probes to examine conformational features of collagen-like polypeptides. Further, the ¹³C chemical shifts of Gly and Pro residues in the triple-helical polypeptides are substantially displaced from those found in (Gly)_nII and (Pro)_nII of the 3₁-helix, reflecting further conformational change from the 3₁-helix to the supercoiled triple helix. In particular, the ¹³C chemical shifts of Gly C ? O carbons of the triple-helical polypeptides are substantially displaced upfield (4.1–5.1 ppm), with respect to those of the 3₁-helical polypeptides. These displacements are interpreted by that Gly C ? O of the former is not involved in NH …? O ? C hydrogen bonds, while this carbon of the latter is linked by these kinds of hydrogen bonds. On the basis of these ¹³C chemical shifts, as reference data for the collagenlike structure, we were able to assign the ¹³C-nmr peaks of Gly, Ala, Pro, and Hyp residues of collagen fibrils, which are in good agreement with the values expected from the model polypeptides mentioned above. We also discuss a plausible conformational change of collagen fibrils during denaturation.     

Immunotherapy for experimental rat autoimmune thyroiditis using a novel immunosuppressant, FTY720

While autoimmune disease needs to be continuously treated via long term, administration of immunosuppressants conventional immunotherapy using drugs such as cyclosporin and tacrolimus may cause adverse effects. Recently, a novel agent, FTY720, which was structurally modified from a natural product, has been shown to have a moderate and different immunosuppressive effect from conventional drugs. In this study, we examined the effect of FTY720 on experimental autoimmune thyroiditis (EAT), which was induced in rats by neonatal thymectomy, followed by subsequent sublethal irradiation. Thyroid stimulating hormone (TSH) was measured before and at the end of drug therapy. Histological and hematological examinations were performed using the samples from sacrificed animals. High TSH levels in the animals returned to the normal range following treatment with FTY720. The severity of the thyroiditis was lower in the FTY720 group than in the control group. FTY720 markedly decreased the number of circulatory lymphocytes, and no infections episodes were observed under this treatment. Thus, FTY720 treatment might be preferred for continuous immunotherapy for autoimmune disease without adverse effects.