Does the regional oxygen uptake measured by near infrared spectroscopy reflect the phase II pulmonary oxygen uptake at the onset of exercise?

It has been hypothesized that the signals of near infrared spectroscopy (NIRS) would reflect muscle O(2) uptake (mVO(2)). Although it is not definite that NIRS signals accurately reflect mVO(2), there is every possibility that NIRS signals at least reflect regional O(2) uptake (rVO(2)). The phase II kinetics of pulmonary oxygen uptake (pVO(2)) is regarded as reflecting mVO(2) at the onset of exercise. To examine whether the rVO(2) on-kinetics measured by NIRS reflects the mVO(2) on-kinetics at the onset of exercise, we compared the rVO(2) as measured by NIRS with the phase II kinetics of pVO(2) at the onset of exercise. Twelve healthy male subjects cycled a Monark ergometer at three different intensities: below the ventilatory threshold (VT) level (below-VT), on the VT level (on-VT), and above the VT level (above-VT), for 6 minutes on three separate occasions. The rVO(2) was calculated from the concentration of oxyhemoglobin and deoxyhemoglobin, as measured by NIRS every 3 seconds. The pVO(2) was determined by the breath-by-breath method. A significant relationship between the amount of increases of pVO(2) and rVO(2) from rest to the end of exercise among all levels of exercise intensity was found (r=0.935, P<0.001). The time constants of rVO(2) (rVO(2)-Tc: below-VT: 6.514+/-2.159 s, on-VT: 7.760+/-2.035 s, above-VT: 9.532+/-2.342 s) were significantly faster than the time constants of pVO(2) (pVO(2)-Tc: below-VT: 23.8+/-4.4 s, on-VT: 25.9+/-5.1 s, above-VT: 26.3+/-5.7 s) (P<0.001). There was no significant relationship between rVO(2)-Tc and pVO(2)-Tc for each intensity (P>0.05). We conclude that the rVO(2) on-kinetics measured by NIRS does not necessarily reflect the mVO(2) kinetics at the onset of exercise.     

An enzymatic method of analysis for GDP-L-fucose in biological samples,involving high-performance liquid chromatography

To investigate the biological significance of GDP-L-fucose, we established a unique method for the determination of GDP-L-fucose levels in microsomal fractions, using an HPLC assay of alpha 1-6-fucosyltransferase (alpha1-6-FucT), an enzyme that catalyzes the synthesis of core fucosylation in N-glycans. A microsomal protein and a large excess of fluorescence-labeled synthetic oligosaccharide (a substrate) were incubated with a large excess of alpha1-6-FucT. The fluorescent intensity of the fucosylated reaction product, which was analyzed by isocratic reverse phase HPLC, was proportional to the level of GDP-L-fucose in the microsomal fractions over the range 0.20-10 pmol. This assay is applicable to the determination of the GDP-L-fucose content in various cancer cell lines as well as rat liver and would be useful in developing a better understanding of the fucosylation potential of such cells and tissues.     

Differential regulation of pituitary hormone secretion and gene expression by thyrotropin-releasing hormone. A role for mitogen-activated protein kinase signaling cascade in rat pituitary GH3 cells

We examined the possible involvement of mitogen-activated protein (MAP) kinase activation in the secretory process and gene expression of prolactin and growth hormone. Thyrotropin-releasing hormone (TRH) rapidly stimulated the secretion of both prolactin and growth hormone from GH3 cells. Secretion induced by TRH was not inhibited by 50 microM PD098059, but was completely inhibited by 1 microM wortmannin and 10 microM KN93, suggesting that MAP kinase does not mediate the secretory process. Stimulation of GH3 cells with TRH significantly increased the mRNA level of prolactin, whereas expression of growth hormone mRNA was largely attenuated. The increase in prolactin mRNA stimulated by TRH was inhibited by addition of PD098059, and the decrease in growth hormone mRNA was also inhibited by PD098059. Transfection of the cells with a pFC-MEKK vector (a constitutively active MAP kinase kinase kinase), significantly increased the synthesis of prolactin and decreased the synthesis of growth hormone. These data taken together indicate that MAP kinase mediates TRH-induced regulation of prolactin and growth hormone gene expression. Reporter gene assays showed that prolactin promoter activity was increased by TRH and was completely inhibited by addition of PD098059, but that the promoter activity of growth hormone was unchanged by TRH. These results suggest that TRH stimulates both prolactin and growth hormone secretion, but that the gene expressions of prolactin and growth hormone are differentially regulated by TRH and are mediated by different mechanisms.     

Macrophage binding and the uptake of oxidized low density lipoprotein are regulated by intracellular protein phosphorylation

The involvement of intracellular protein phosphorylation in macrophages in the binding and uptake of oxidized low density lipoprotein (oxLDL) was investigated. The treatment of fibronectin-unstimulated and stimulated mouse thioglycolate-induced macrophages with inhibitors of myosin light chain kinase, protein kinase C and protein tyrosine kinase resulted in decreased macrophage binding of oxLDL, macrophage foam cell formation, and whole intracellular protein phosphorylation. The treatment of fibronectin-unstimulated and stimulated macrophages with inhibitors of protein serine/threonine and tyrosine phosphatases caused enhanced macrophage binding of oxLDL, macrophage foam cell formation, and whole intracellular protein phosphorylation. Fibronectin, which stimulates macrophage activity, enhanced macrophage intracellular protein phosphorylation. Myosin light chain phosphorylation may be involved in the fibronectin stimulation of macrophages. Treatment of fibronectin-unstimulated and stimulated macrophages with thiophosphate, which forms thiophosphate esters of intracellular proteins that are not so susceptible to protein phosphatases, enhanced macrophage binding of oxLDL. The above results indicate that intracellular protein phosphorylation maintains and enhances macrophage binding and the uptake of oxLDL.     

Rapid Evolution of the Male-Specific Antibacterial Protein Andropin Gene in Drosophila

Andropin, which encodes an antibacterial protein, is closely linked to the Cecropin gene cluster of D. melanogaster. Andropin and Cecropins are considered to have originated from one common ancestor. However, the expression pattern of Andropin is distinct from that of Cecropins, being restricted to the adult male ejaculatory duct. To elucidate the evolutionary process of Andropin, we have sequenced Andropin genes from D. melanogaster and its closely related species. In D. melanogaster, the nucleotide diversity of Andropin is remarkably low compared to that of Cecropin. In contrast, nonsynonymous substitutions of Andropin are conspicuously frequent between species. From genomic Southern analysis, Andropin-like genes are present in at least the melanogaster species subgroup. The series of present results suggests that Andropin was born in the course of constructing the Drosophila Cecropin gene family and then started to evolve rapidly, in contrast to Cecropins. Received: 10 August 2001 / Accepted: 29 October 2001     

Activation of nuclear Ca(2+)/calmodulin-dependent protein kinase II and brain-derived neurotrophic factor gene expression by stimulation of dopamine D2 receptor in transfected NG108-15 cells

We identified the isoforms of Ca(2+) /calmodulin-dependent protein kinase II (CaM kinase II) subunits in rat striatum. All four subunits of CaM kinase II alpha, beta, gamma and delta were detected including the isoforms of alphaB, gammaA, gammaA', gammaA.B, delta3 and delta7 with nuclear localization signal. We established NG108-15 cells with the stably expressed dopamine D2L receptor (D2LR, long form), which is an alternative splicing variant. The cells were termed NGD2L. Immunostaining demonstrated that D2LR was localized in plasma membranes. Calcium imaging with fluo-3 AM revealed that quinpirole, a D2R agonist, increased the intracellular Ca(2+), which was blocked by treatment with sulpiride and pertussis toxin in NGD2L cells, but not in mock cells. Furthermore, stimulation of D2LR with quinpirole in NGD2L cells activated the nuclear isoform of CaM kinase II. Stimulation of D2LR increased the expression of exon III- and IV-BDNF mRNA. Overexpression of CaM kinase II delta3 increased exon IV- but not exon III-BDNF mRNA. These results suggest that D2R is involved in the activation of the nuclear isoform of CaM kinase II and thereby in stimulation of gene expression through Ca(2+) signaling.     

Comparative genomics approach toward critical determinants for the imprinting of an evolutionarily conserved gene Impact

The Impact is an evolutionarily conserved gene subjected to genomic imprinting in mouse but not in human. A characteristic tandem repeat similar to those found in many other imprinted genes and an elevated expression level, both observed only for the mouse gene, are implicated in the evolution of imprinting, to which the repeat might have contributed via enhancement of the expression. To pursue the possibility further, we examined the correlation among the repeat, expression level, and imprinting of Impact in various mammals ranging from rodents, lagomorphs, carnivores, artiodactyls to primates. Intriguingly, rabbit Impact is abundantly expressed and imprinted like those of rodents, but is missing the repeat from its first intron like those of other mammals that express both alleles weakly. It thus seems that lineage-specific enhancement of gene expression rather than the tandem repeat per se played a critical role in the evolution of imprinting of Impact.     

Mapping the region of the alpha-type phospholipase A2 inhibitor responsible for its inhibitory activity

alpha-Type phospholipase A(2) inhibitory protein (PLIalpha) from the serum of the venomous snake Gloydius brevicaudus, GbPLIalpha,isone of the protective endogenous proteins that neutralizes its own venom phospholipase A(2) (PLA(2)), and it is a homotrimer of subunits having a C-type lectin-like domain. The nonvenomous snake Elaphe quadrivirgata has a homologous serum protein, EqPLIalpha-LP, that does not show any inhibitory activity against various snake venom PLA(2)s (Okumura, K., Inoue, S., Ikeda, K., and Hayashi, K. (2003) IUBMB Life 55, 539-545). By constructing GbPLIalpha-Eq- PLIalpha-LP chimeric proteins, we have mapped the residues important in conferring GbPLIalpha inhibitory activity on region 13-36 in the primary structure of GbPLIalpha. Noninhibitory EqPLIalpha-LP showed comparable inhibitory activity only when this region was replaced with that of GbPLIalpha. Further, mutational analysis of the candidate residues revealed that the individual GbPLIalpha to EqPLIalpha-LP residue substitutions N26K, K28E, D29N, and Y144S each produced a mutant GbPLIalpha protein with reduced inhibitory activity, with the single N26K substitution having the most significant effect. Residues 13-36 were suspected to be located in the helical neck region of the GbPLIalpha trimer. Therefore, the region of GbPLIalpha responsible for PLA(2) inhibition was distinct from the carbohydrate-binding site of the homologous C-type lectin.