A brain-specific decrease of the tyrosine hydroxylase protein in sepiapterin reductase-null mice--as a mouse model for Parkinson's disease

Sepiapterin reductase (SPR) is an enzyme that acts in the third and final step of tetrahydrobiopterin (BH4) biosynthesis. The human Spr gene locates within the region of 2.5 MB mapped to PARK3, an autosomal dominant form of familial Parkinson’s diseases. In order to explore the role of SPR in the metabolism of BH4, we produced and analyzed Spr-deficient mice. Most of Spr-null mice survived beyond two weeks. Whereas the BH4 contents in the homozygous mutant mice were greatly decreased than those in wild-type and heterozygous mice, the substantial amounts of BH4 were remained even 17 days after delivery. Spr-null mice exhibited severe monoamine deficiencies and a tremor-like phenotype after weaning. The amount of TH protein in the brain of Spr-null mice was less than 10% of wild-type, while TH protein in the adrenal, phenylalanine hydroxylase protein in the liver, and nNOS in the brain were not altered. These data suggest an essential role of SPR in the biosynthesis of BH4, and that the SPR gene could be a candidate gene for PARK3.     

Microarray-based global mapping of integration sites for the retrotransposon, intracisternal A-particle, in the mouse genome

Mammalian genomes contain numerous evolutionary harbored mobile elements, a part of which are still active and may cause genomic instability. Their movement and positional diversity occasionally result in phenotypic changes and variation by causing altered expression or disruption of neighboring host genes. Here, we describe a novel microarray-based method by which dispersed genomic locations of a type of retrotransposon in a mammalian genome can be identified. Using this method, we mapped the DNA elements for a mouse retrotransposon, intracisternal A-particle (IAP), within genomes of C3H/He and C57BL/6J inbred mouse strains; consequently we detected hundreds of probable IAP cDNA–integrated genomic regions, in which a considerable number of strain-specific putative insertions were included. In addition, by comparing genomic DNAs from radiation-induced myeloid leukemia cells and its reference normal tissue, we detected three genomic regions around which an IAP element was integrated. These results demonstrate the first successful genome-wide mapping of a retrotransposon type in a mammalian genome.     

Stimulation of NTS A1 adenosine receptors differentially resets baroreflex control of regional sympathetic outputs

Previously we showed that pressor and differential regional sympathoexcitatory responses (adrenal > renal >/= lumbar) evoked by stimulation of A(1) adenosine receptors located in the nucleus of the solitary tract (NTS) were attenuated/abolished by baroreceptor denervation or blockade of glutamatergic transmission in the NTS, suggesting A(1) receptor-elicited inhibition of glutamatergic transmission in baroreflex pathways. Therefore we tested the hypothesis that stimulation of NTS A(1) adenosine receptors differentially inhibits/resets baroreflex responses of preganglionic adrenal (pre-ASNA), renal (RSNA), and lumbar (LSNA) sympathetic nerve activity. In urethane-chloralose-anesthetized male Sprague-Dawley rats (n = 65) we compared baroreflex-response curves (iv nitroprusside and phenylephrine) evoked before and after bilateral microinjections into the NTS of A(1) adenosine receptor agonist (N(6)-cyclopentyladenosine, CPA; 0.033-330 pmol/50 nl). CPA evoked typical dose-dependent pressor and differential sympathoexcitatory responses and similarly shifted baroreflex curves for pre-ASNA, RSNA, and LSNA toward higher mean arterial pressure (MAP) in a dose-dependent manner; the maximal shifts were 52.6 +/- 2.8, 48.0 +/- 3.6, and 56.8 +/- 6.7 mmHg for pre-ASNA, RSNA, and LSNA, respectively. These shifts were not a result of simple baroreceptor resetting because they were two to three times greater than respective increases in baseline MAP evoked by CPA. Baroreflex curves for pre-ASNA were additionally shifted upward: the maximal increases of upper and lower plateaus were 41.8 +/- 16.4% and 45.3 +/- 8.7%, respectively. Maximal gain (%/mmHg) measured before vs. after CPA increased for pre-ASNA (3.0 +/- 0.6 vs. 4.9 +/- 1.3), decreased for RSNA (4.1 +/- 0.6 vs. 2.3 +/- 0.3), and remained unaltered for LSNA (2.1 +/- 0.2 vs. 2.0 +/- 0.1). Vehicle control did not alter the baroreflex curves. We conclude that the activation of NTS A(1) adenosine receptors differentially inhibits/resets baroreflex control of regional sympathetic outputs.     

An enzymatic strategy for site-specific immobilization of functional proteins using microbial transglutaminase

A novel strategy for site-specific immobilization of recombinant proteins was investigated using microbial transglutaminase (MTG). Alkaline phosphatase (AP) was selected as a model protein and tagged with a short peptide (MKHKGS) at the N-terminus to provide a reactive Lys residue for MTG. On the other hand, casein, a well-known substrate for MTG, was chemically attached onto a polyacrylic resin to provide reactive Gln residues for the enzymatic immobilization of the recombinant AP. As a result, we succeeded in MTG-mediated functional immobilization of the recombinant AP onto casein-coated polyacrylic resin. It was found that the immobilized AP prepared using MTG exhibited much higher specific activity than that prepared by chemical modification. Moreover, enzymatic immobilization gave an immobilized formulation with higher stability upon repeated use than that obtained by physical adsorption. Use of this ability of MTG in posttranslational protein modification will provide us with a benign, site-specific immobilization method for functional proteins.     

In vivo estimation of contraction velocity of human vastus lateralis muscle during "isokinetic" action.

To determine the shortening velocities of fascicles of the vastus lateralis muscle (VL) during isokinetic knee extension, six male subjects were requested to extend the knee with maximal effort at angular velocities of 30 and 150 degrees /s. By using an ultrasonic apparatus, longitudinal images of the VL were produced every 30 ms during knee extension, and the fascicle length and angle of pennation were obtained from these images. The shortening fascicle length with extension of the knee (from 98 to 13 degrees of knee angle; full extension = 0 degrees ) was greater (43 mm) at 30 degrees /s than at 150 degrees /s (35 mm). Even when the angular velocity remained constant during the isokinetic range of motion, the fascicle velocity was found to change from 39 to 77 mm/s at 150 degrees /s and from 6 to 19 mm/s at 30 degrees /s. The force exerted by a fascicle changed with the length of the fascicle at changing angular velocities. The peak values of fascicle force and velocity were observed at approximately 90 mm of fascicle length. In conclusion, even if the angular velocity of knee extension is kept constant, the shortening velocity of a fascicle is dependent on the force applied to the muscle-tendon complex, and the phenomenon is considered to be caused mainly by the elongation of the elastic element (tendinous tissue).     

Increased liver temperature efficiently augments human cellular immune response: T-cell activation and possible monocyte translocation

Hyperthermia (HT), in combination with other conventional therapeutic modalities, has become a promising approach in cancer therapy. In addition to heat-induced apoptosis, an augmented immunological effect is considered to be a benefit of hyperthermic treatment over chemo- or radiotherapy. Here, we investigated the effect of regional HT targeting the liver on immune cells, especially T cells and antigen-presenting cells, which are important in recognizing and eliminating tumor cells and pathogens such as viruses. In healthy volunteers exposed to such regional HT, both CD4⁺ and CD8⁺ T cells that express an activation marker CD69 increased transiently at 1 h post-treatment, with a subsequent decrease to base levels at 6 h after the treatment. At 24 h post-treatment, the percentage of CD69-positive cells significantly increased again but only among CD8⁺ T cells. IFN-γ production from PHA-stimulated peripheral blood mononuclear cells was gradually and significantly increased in the 2 days following the heating procedure, peaking at 36 h post-treatment. Furthermore, we found marked increases in plasma levels of IL-1β and IL-6 starting at 24 h post-treatment. With regard to the number of each leukocyte subpopulation, a transient and dramatic decrease in the number of a subset of monocytes, CD14⁺ CD16⁻ cells, was observed at 1 h after the hyperthermic treatment, suggesting that the regional HT aimed at the liver may have influenced the extravasation of blood monocytes. No significant changes in T-cell activities or monocyte counts were observed in the volunteers exposed to heating of the lungs or the legs. These results suggest that heating of the liver may efficiently induce cellular immune responses to liver cancers.     

Activation of Toll-like receptor 2 impairs hypoxic pulmonary vasoconstriction in mice

Toll-like receptors (TLRs) mediate inflammation in sepsis, but their role in sepsis-induced respiratory failure is unknown. Hypoxic pulmonary vasoconstriction (HPV) is a unique vasoconstrictor response that diverts blood flow away from poorly ventilated lung regions. HPV is impaired in sepsis and after challenge with the TLR4 agonist lipopolysaccharide (LPS). Unlike TLR4 agonists, which are present only in Gram-negative bacteria, TLR2 agonists are ubiquitously expressed in all of the major classes of microorganisms that cause sepsis, including both Gram-positive and Gram-negative bacteria and fungi. We tested the hypothesis that (S)-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys(4)-OH, trihydrochloride (Pam3Cys), a TLR2 agonist, impairs HPV and compared selected pulmonary and systemic effects of Pam3Cys vs. LPS. HPV was assessed 22 h after challenge with saline, Pam3Cys, or LPS by measuring the increase in the pulmonary vascular resistance of the left lung before and during left lung alveolar hypoxia produced by left mainstem bronchus occlusion (LMBO). Additional endpoints included arterial blood gases during LMBO, hemodynamic parameters, weight loss, temperature, physical appearance, and several markers of lung inflammation. Compared with saline, challenge with Pam3Cys caused profound impairment of HPV, reduced systemic arterial oxygenation during LMBO, weight loss, leukopenia, and lung inflammation. In addition to these effects, LPS-challenged mice had lower rectal temperatures, metabolic acidosis, and were more ill appearing than Pam3Cys-challenged mice. These data indicate that TLR2 activation impairs HPV and induces deleterious systemic effects in mice and suggest that TLR2 pathways may be important in sepsis-induced respiratory failure.     

Multitracer study on biosorption of trace elements in yeastSaccharomyces cerevisiae

To estimate the applicability of yeastSaccharomyces cerevisiae as a radioisotope accumulator, biosorption of 18 radionuclides (isotopes of Be, Na, Sc, V, Cr, Mn, Fe, Co, Zn, As, Se, Rb, Sr, Y, Zr, Tc, Ru, and Rh) by the cells was tested using a multitracer technique. This technique enables us to examine the uptake of various elements under an identical condition. We showed that the yeast has ability to take up radioactive tracers of Be, Sc, V, Cr, Mn, Fe, Co, Zn, As, Se, Sr, Y, Zr, Ru, and Rh from an aqueous environment. Tracers of Be, Sc, Mn, Co, Zn, Se, Sr, Y, and Zr were distributed in both cell walls and other cell parts. Moreover, tracers of Sc, Mn, Co, Zn, Se, Sr, and Y were present as soluble and insoluble forms, and most of the Be and Zr were present as an insoluble form in the cells.     

Effects of nano particles on antigen-related airway inflammation in mice

Background

Particulate matter (PM) can exacerbate allergic airway diseases. Although health effects of PM with a diameter of less than 100 nm have been focused, few studies have elucidated the correlation between the sizes of particles and aggravation of allergic diseases. We investigated the effects of nano particles with a diameter of 14 nm or 56 nm on antigen-related airway inflammation.

Methods

ICR mice were divided into six experimental groups. Vehicle, two sizes of carbon nano particles, ovalbumin (OVA), and OVA + nano particles were administered intratracheally. Cellular profile of bronchoalveolar lavage (BAL) fluid, lung histology, expression of cytokines, chemokines, and 8-hydroxy-2''-deoxyguanosine (8-OHdG), and immunoglobulin production were studied.

Results

Nano particles with a diameter of 14 nm or 56 nm aggravated antigen-related airway inflammation characterized by infiltration of eosinophils, neutrophils, and mononuclear cells, and by an increase in the number of goblet cells in the bronchial epithelium. Nano particles with antigen increased protein levels of interleukin (IL)-5, IL-6, and IL-13, eotaxin, macrophage chemoattractant protein (MCP)-1, and regulated on activation and normal T cells expressed and secreted (RANTES) in the lung as compared with antigen alone. The formation of 8-OHdG, a proper marker of oxidative stress, was moderately induced by nano particles or antigen alone, and was markedly enhanced by antigen plus nano particles as compared with nano particles or antigen alone. The aggravation was more prominent with 14 nm of nano particles than with 56 nm of particles in overall trend. Particles with a diameter of 14 nm exhibited adjuvant activity for total IgE and antigen-specific IgG₁ and IgE.

Conclusion

Nano particles can aggravate antigen-related airway inflammation and immunoglobulin production, which is more prominent with smaller particles. The enhancement may be mediated, at least partly, by the increased local expression of IL-5 and eotaxin, and also by the modulated expression of IL-13, RANTES, MCP-1, and IL-6.     

Role of metallothionein in antigen-related airway inflammation

Metallothionein (MT) is a protein that can be induced by inflammatory mediators and participates in cytoprotection. However, its role in antigen-related inflammation remains to be established. We determined whether intrinsic MT protects against antigen-related airway inflammation induced by ovalbumin (OVA) in MT-I/II null (MT [-/-]) mice and in corresponding wild-type (WT) mice. MT (-/-) mice and WT mice were intratracheally challenged with OVA (1 mug per body) biweekly four times. Twenty-four hours after the last OVA challenge, significant increases were shown in the numbers of total cells, eosinophils, and neutrophils in bronchoalveolar lavage fluid from MT (-/-) mice than in those from WT mice. The protein level of interleukin-1beta (IL-1beta) was significantly greater in MT (-/-) mice than in WT mice after OVA challenge. Immunohistochemical analysis showed that the formations of 8-oxy-deoxyguanosine and nitrotyrosine in the lung were more intense in MT (-/-) mice than in WT mice after OVA challenge. These results indicate that endogenous MT is a protective molecule against antigen-related airway inflammation induced by OVA, at least partly, via the suppression of enhanced lung expression of IL-1beta and via the antioxidative properties. Our findings suggest that MT may be a therapeutic target for the treatment of antigen-related airway inflammatory diseases such as bronchial asthma.