Oxygen affinity of hemoglobin regulates O2 consumption,metabolism, and physical activity

The oxygen affinity of hemoglobin is critical for gas exchange in the lung and O(2) delivery in peripheral tissues. In the present study, we generated model mice that carry low affinity hemoglobin with the Titusville mutation in the alpha-globin gene or Presbyterian mutation in the beta-globin gene. The mutant mice showed increased O(2) consumption and CO(2) production in tissue metabolism, suggesting enhanced O(2) delivery by mutant Hbs. The histology of muscle showed a phenotypical conversion from a fast glycolytic to fast oxidative type. Surprisingly, mutant mice spontaneously ran twice as far as controls despite mild anemia. The oxygen affinity of hemoglobin may control the basal level of erythropoiesis, tissue O(2) consumption, physical activity, and behavior in mice.     

Regional concentration and chromatographic characterization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the brain of the bullfrog,Rana catesbeiana

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a regulatory neuropeptide which functions as a hypothalamic factor for pituitary hormone release, and as a neurotransmitter, neuromodulator and neurotrophic factor in both frogs and mammals. This study examined the quantitative distribution and chromatographic characterization of immunoreactive PACAP in the central nervous system (CNS) of the bullfrog, Rana catesbeiana, using an enzyme immunoassay (EIA), named avidin-biotin complex detectable EIA for PACAP, and high-performance liquid chromatographic (HPLC) analysis. The brain of adult bullfrogs contained relatively high levels of immunoreactive PACAP (344.63 pmol/g wet weight of tissue). The average concentrations of immunoreactive PACAP in the regions of the telencephalon, diencephalon, tectum, cerebellum, rhombencephalon, and spinal cord were 213.84, 767.14, 524.94, 192.71, 237.67, and 362.04 pmol/g wet weight of tissue, respectively. The concentrations of immunoreactive PACAP increased with the brain development during metamorphosis, and the concentration of immunoreactive PACAP in the brain of tadpoles at the end of metamorphosis was approximately 200 pmol/g wet weight of tissue. The predominant form of immunoreactive PACAP in the CNS of adult and tadpole was eluted closely with synthetic PACAP38, but another smaller immunoreactivity also appeared in a the fraction, which corresponded to the retention time of synthetic PACAP27, as analyzed by reverse-phase HPLC.     

Simultaneous on/off regulation of transgenes located on a mammalian chromosome with Cre-expressing adenovirus and a mutant loxP

The site-specific recombinase Cre has often been used for on/off regulation of expression of transgenes introduced into the mammalian chromosome. However, this method is only applicable to the regulation of a single gene and cannot be used to simultaneously regulate two genes, because site-specific recombination occurs from the target loxP sequence of one regulating unit to the loxP sequence of any other unit and would eventually disrupt the structure of both regulating units. We previously reported a mutant loxP sequence with a two base substitution called loxP V (previously called loxP 2272), with which wild-type loxP cannot recombine but with which the identical mutant loxP recombines efficiently. In the present study we isolated cell lines bearing two regulating units on a chromosome containing a pair of wild-type loxP sequences or mutant loxP V sequences. After infection with Cre-expressing recombinant adenovirus AxCANCre, expression of a gene in each regulating unit was simultaneously turned on and off. Southern analyses showed that both regulating units were processed simultaneously and independently, even after infection with a limited amount of AxCANCre. The results showed that simultaneous regulation of gene expression on a mammalian chromosome mediated by Cre can be achieved by using mutant loxP V and wild-type loxP. This method may be a useful approach for conditional transgenic/knockout animals and investigation of gene function involving two genes simultaneously. Another possible application is for preparation of a new packaging cell line of viral vectors through simultaneous production of toxic viral genes.     

Timing of early developmental events in embryos of a tropical sea urchin Echinometra mathaei

Egg volume of a tropical sea urchin Echinometra mathaei is about one half that of other well-known species. We asked whether such a small size of eggs affected the timings of early developmental events or not. Cleavages became asynchronous from the 7th cleavage onward, and embryos hatched out before completion of the 9th cleavage. These timings were one cell cycle earlier than those in well-known sea urchins, raising the possibility that much earlier events, such as the increase in adhesiveness of blastomeres or the specification of dorso-ventral axis (DV-axis), would also occur earlier by one cell cycle. By examining the pseudopodia formation in dissociated blastomeres, it was elucidated that blastomeres in meso- and macromere lineages became adhesive after the 4th and 5th cleavages, respectively. From cell trace experiments, it was found that the first or second cleavage plane was preferentially employed as the median plane of embryo; the DV-axis was specified mainly at the 16-cell stage. Timings of these events were also one cell cycle earlier than those in Hemicentrotus pulcherrimus. The obtained results suggest that most of the early developmental events in sea urchin embryos do not depend on cleavage cycles, but on other factors, such as the nucleo-cytoplasmic ratio.     

Normovolemic hemodilution with Hb vesicle solution attenuates hypoxia in ischemic hamster flap tissue

The aim of this study was to test whether oxygenation in acutely ischemic, collateralized tissue may be improved by normovolemic hemodilution with a solution containing liposome-encapsulated human Hb (HbV). A skin flap model in anesthetized hamsters was used, which consisted of two parts receiving either anatomic or collateral perfusion. Microhemodynamics were investigated with intravital microscopy. Partial tissue oxygen tension was measured with a Clark-type microprobe. Hemodilution was obtained by exchanging 50% of the total blood volume with HbV suspended in 8% human serum albumin (HSA8) or 6% Dextran 70 (Dx70). The size of the vesicles was 276 nm, the P(50) was 22 mmHg, and the Hb concentration of the solutions was 7.5 g/dl. Colloid osmotic pressure and viscosity were 49.9 mmHg and 8.7 cP for HbV-Dx70 and 40.0 mmHg and 2.9 cP for HbV-HSA8, respectively. Hemodilution with HbV-Dx70 led to an increase in microvascular blood flow in the ischemic microvessels to maximally 158% (median, P < 0.01), whereas blood flow remained virtually unchanged after hemodilution with HbV-HSA8. In the ischemic tissue, oxygen tension was improved from 11.9 to 17.0 mmHg (P < 0.01) after hemodilution with HbV-Dx70 but remained virtually unchanged after hemodilution with HbV-HSA8. Our study suggests that the oxygenation in acutely ischemic, collateralized tissue may be improved by normovolemic hemodilution with HbV suspended in Dx70. The effect was achieved by an increase in microcirculatory blood flow related to the rheological properties of the suspending medium.     

Dolastatin 11 connects two long-pitch strands in F-actin to stabilize microfilaments

Dolastatin 11, a drug isolated from the Indian Ocean sea hare Dolabella auricularia, arrests cytokinesis in vivo and increases the amount of F-actin to stabilize F-actin in vitro, like phalloidin and jasplakinolide. However, according to the previous biochemical study, the binding of dolastatin 11 to F-actin does not compete with that of phalloidin, suggesting that the binding sites are different. To understand the mechanism of F-actin stabilization by dolastatin 11, we determined the position of bound dolastatin 11 in F-actin using the X-ray fiber diffraction from oriented filament sols. Our analysis shows that the position of dolastatin 11 is clearly different from that of phalloidin. However, these bound drugs are present in the gap between the two long-pitch F-actin strands in a similar way. The result suggests that the connection between the two long-pitch F-actin strands might be a key for the control of F-actin stabilization.     

The differential ability of HLA B*5701+ long-term nonprogressors and progressors to restrict human immunodeficiency virus replication is not caused by loss of recognition of autologous viral gag sequences

Although the HLA B(*)5701 class I allele is highly overrepresented among human immunodeficiency virus (HIV)-infected long-term nonprogressors (LTNPs), it is also present at the expected frequency (11%) in patients with progressive HIV infection. Whether B57(+) progressors lack restriction of viral replication because of escape from recognition of highly immunodominant B57-restricted gag epitopes by CD8(+) T cells remains unknown. In this report, we investigate the association between restriction of virus replication and recognition of autologous virus sequences in 27 B(*)57(+) patients (10 LTNPs and 17 progressors). Amplification and direct sequencing of single molecules of viral cDNA or proviral DNA revealed low frequencies of genetic variations in these regions of gag. Furthermore, CD8(+) T-cell recognition of autologous viral variants was preserved in most cases. In two patients, responses to autologous viral variants were not demonstrable at one epitope. By using a novel technique to isolate primary CD4(+) T cells expressing autologous viral gene products, it was found that 1 to 13% of CD8(+) T cells were able to respond to these cells by gamma interferon production. In conclusion, escape-conferring mutations occur infrequently within immunodominant B57-restricted gag epitopes and are not the primary mechanism of virus evasion from immune control in B(*)5701(+) HIV-infected patients. Qualitative features of the virus-specific CD8(+) T-cell response not measured by current assays remain the most likely determinants of the differential abilities of HLA B(*)5701(+) LTNPs and progressors to restrict virus replication.     

MAP kinase-mediated proliferation of DLD-1 carcinoma by the stimulation of protease-activated receptor 2

Protease-activated receptor-2 (PAR-2) has been demonstrated to be highly expressed in the gastrointestinal tract. In the present study, we investigated the effects of PAR-2 stimulation on the cell signaling and proliferation of DLD-1, a human colon carcinoma cell line, in comparison with the PAR-1 stimulation. PAR-2 stimulation by agonist peptide SLIGKV concentration-dependently induced the increase in [Ca2+]i and the proliferation of DLD-1 whereas the inverse peptide LSIGKV did not. Trypin (10(-9) M), an agonist protease for PAR-2, also enhanced the proliferation of DLD-1. The proliferative response of DLD-1 to PAR-2 stimulation was associated with the transient phosphorylation of MEK and MAP kinase, but not p38 MAP kinase and JNK. Inhibition of MEK by PD98059 (50 microM) completely inhibited the proliferation-stimulating effects as well as the phosphorylation of MAP kinase induced by PAR-2 agonist peptide (100 microM) and trypsin (10(-9) M). The prolonged treatment with PAR-2 agonist peptide for more than one hour was required for the enhanced proliferative response, suggesting the existence of unknown long-lasting cooperative signaling with MAP kinase cascade. PAR-1 stimulation by the agonist peptide SFLLRN (100 microM) or thrombin (10(-8) M) produced Ca2+ signaling, however, the stimulation neither produced the cell proliferative response nor the activation of MEK-MAP kinase cascade. These results indicated that Ca2+ signaling induced by PARs activation was not enough for inducing the cell proliferation in DLD-1 cells and that stimulation of PAR-2 can induce the activation of MEK-MAP kinase cascade, leading to the growth promoting response.     

Urotensin II-induced activation of extracellular signal-regulated kinase in cultured vascular smooth muscle cells: involvement of cell adhesion-mediated integrin signaling

Urotensin II (UII), a cyclic dodecapeptide, is a potent mammalian vasoconstrictive substance recently shown to induce proliferation of vascular smooth muscle cells (VSMCs). However, little is known about mechanisms involved in UII-induced mitogenic response such as cell proliferation. To investigate the intracellular signaling pathways involved in this process, we examined the effects of UII on activation of extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK) in VSMCs. UII stimulated in time- and dose-dependent manners the phosphorylation level of ERK. In contrast, UII failed to alter the phosphorylation level of FAK. Although angiotensin II-induced ERK phosphorylation was noted even in suspended cells, UII failed to induce an increase in ERK phosphorylation in such cells. On the other hand, UII induced an increase in the phosphorylation level of ERK, but not FAK, in cells adherent to fibronectin. Furthermore, UII-induced proliferation of VSMCs was inhibited by ERK kinase inhibitor PD98059. Our results suggested that activation of integrin-mediated signaling pathways play a critical role in UII-induced phosphorylation of ERK, leading to proliferation of VSMCs, which does not involved increased phosphorylation of FAK.