Adiponectin protects against myocardial ischemia-reperfusion injury through AMPK- and COX-2-dependent mechanisms

Obesity-related disorders are associated with the development of ischemic heart disease. Adiponectin is a circulating adipose-derived cytokine that is downregulated in obese individuals and after myocardial infarction. Here, we examine the role of adiponectin in myocardial remodeling in response to acute injury. Ischemia-reperfusion in adiponectin-deficient (APN-KO) mice resulted in increased myocardial infarct size, myocardial apoptosis and tumor necrosis factor (TNF)-alpha expression compared with wild-type mice. Administration of adiponectin diminished infarct size, apoptosis and TNF-alpha production in both APN-KO and wild-type mice. In cultured cardiac cells, adiponectin inhibited apoptosis and TNF-alpha production. Dominant negative AMP-activated protein kinase (AMPK) reversed the inhibitory effects of adiponectin on apoptosis but had no effect on the suppressive effect of adiponectin on TNF-alpha production. Adiponectin induced cyclooxygenase (COX)-2-dependent synthesis of prostaglandin E(2) in cardiac cells, and COX-2 inhibition reversed the inhibitory effects of adiponectin on TNF-alpha production and infarct size. These data suggest that adiponectin protects the heart from ischemia-reperfusion injury through both AMPK- and COX-2-dependent mechanisms.     

Mammalian 5-formyluracil-DNA glycosylase. 2. Role of SMUG1 uracil-DNA glycosylase in repair of 5-formyluracil and other oxidized and deaminated base lesions

In the accompanying paper [Matsubara, M., et al. (2003) Biochemistry 42, 4993-5002], we have partially purified and characterized rat 5-formyluracil (fU)-DNA glycosylase (FDG). Several lines of evidence have indicated that FDG is a rat homologue of single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1). We report here that rat and human SMUG1 (rSMUG1 and hSMUG1) expressed from the corresponding cDNAs indeed excise fU in single-stranded (ss) and double-stranded (ds) DNA. The enzymes also excised uracil (U) and uracil derivatives bearing an oxidized group at C5 [5-hydroxyuracil (hoU) and 5-hydroxymethyluracil (hmU)] in ssDNA and dsDNA but not analogous cytosine derivatives (5-hydroxycytosine and 5-formylcytosine) and other oxidized damage. The damage specificity and the salt concentration dependence of rSMUG1 (and hSMUG1) agreed well with those of FDG, confirming that FDG is rSMUG1. Consistent with the damage specificity above, hSMUG1 removed damaged bases from Fenton-oxidized calf thymus DNA, generating abasic sites. The amount of resulting abasic sites was about 10% of that generated by endonuclease III or 8-oxoguanine glycosylase in the same substrate. The HeLa cell extract and hSMUG1 exhibited a similar damage preference (hoU.G > hmU.A, fU.A), and the activities for fU, hmU, and hoU in the cell extract were effectively neutralized with hSMUG1 antibodies. These data indicate a dual role of hSMUG1 as a backup enzyme for UNG and a primary repair enzyme for a subset of oxidized pyrimidines such as fU, hmU, and hoU.     

Biochemical and molecular characterization of two phosphatidic acid-selective phospholipase A1s,mPA-PLA1alpha and mPA-PLA1beta

We have identified a novel phospholipase A1, named mPA-PLA1beta, which is specifically expressed in human testis and characterized it biochemically together with previously identified mPA-PLA1alpha. The sequence of mPAPLA1beta encodes a 460-amino acid protein containing a lipase domain with significant homology to the previously identified phosphatidic acid (PA)-selective PLA1, mPA-PLA1alpha. mPA-PLA1beta contains a short lid and deleted beta9 loop, which are characteristics of PLA1 molecules in the lipase family, and is a member of a subfamily in the lipase family that includes mPA-PLA1alpha and phosphatidylserine-specific PLA1. Both mPA-PLA1beta and mPA-PLA1alpha recombinant proteins exhibited PA-specific PLA1 activity and were vanadate-sensitive. When mPAPLA1beta-expressing cells were treated with bacterial phospholipase D, the cells produced lysophosphatidic acid (LPA). In both mPA-PLA1alpha and beta-expressing cells, most of the PA generated by the phospholipase D (PLD) treatment was converted to LPA, whereas in control cells it was converted to diacylglycerol. When expressed in HeLa cells most mPA-PLA1alpha protein was recovered from the cell supernatant. By contrast, mPA-PLA1beta was recovered almost exclusively from cells. Consistent with this observation, we found that mPA-PLA1beta has higher affinity to heparin than mPA-PLA1alpha. We also found that the membrane-associated mPA-PLA1s were insoluble in solubilization by 1% Triton X-100 and were detected in Triton X-100-insoluble buoyant fractions of sucrose gradients. The present study raises the possibility that production of LPA by mPA-PLA1alpha and -beta occurs on detergent-resistant membrane domains of the cells where they compete with lipid phosphate phosphatase for PA.     

Immunosuppressant FK506 induces neurite outgrowth in PC12 mutant cells with impaired NGF-promoted neuritogenesis via a novel MAP kinase signaling pathway

We obtained a drug-hypersensitive PC12 mutant cell (PC12m3), in which neurite outgrowth was strongly stimulated by various drugs such as FK506, calcimycin and cAMP, under the condition of NGF treatment. The frequency of neurite outgrowth stimulated by FK506 was approximately 40 times greater than by NGF alone. The effects of FK506 on neurite outgrowth in PC12m3 cells were inhibited by rapamycin, an FK506 antagonist, and by calcimycin, a calcium ionophore. PC12m3 cells had a strong NGF-induced MAP kinase activity, the same as PC12 parental cells. However, FK506-induced MAP kinase activity was detected only in PC12m3 cells. The activation of MAP kinase by FK506 in PC12m3 cells was markedly inhibited by rapamicin and calcimycin. FK506-induced MAP kinase activity was also inhibited by MAP kinase inhibitor U0126. These results demonstrate that drug-hypersensitive PC12m3 cells have a novel FK506-induced MAP kinase pathway for neuritogenesis.     

Cellular density regulation of plasminogen gene expression in mouse hepatocytes

The liver produces a variety of proteins including plasminogen. Plasminogen is pro-enzyme that is converted into plasmin by plasminogen activator. Plasmin has a broad substrate spectrum and participates in several biological processes, such as fibrinolysis, tissue remodeling, cell migration, angiogenesis and embryogenesis. In the present study, the regulation of plasminogen expression in mouse hepatocytes was investigated in the primary culture system. Expression level of plasminogen mRNA in the culture at the low cell density condition (0.2 x 10(5) cells / cm(2)) was compared with that at the high cell density condition (1.0 x 10 (5) cells / cm(2)). In the low cell density culture, the expression level of plasminogen mRNA decreased by a time-dependent manner. However, mRNAs for albumin and alpha(2)-antiplasmin were not influenced by the low cell density culture. On the other hand, in the high cell density culture, plasminongen mRNA expressed constantly as well as albumin and alpha(2)-antiplasmin mRNAs. Thus, the decrease in plasminogen mRNA expression could specifically occur when the density of hepatocytes was low. The down-regulation of plasminogen mRNA in the low cell density culture is not observed in the presence of cycloheximide, suggesting that the de novo protein synthesis is required for the regulatory mechanism. These findings indicate that the expression of plasminogen mRNA from hepatocyte is dependent on the cell density and the stimulation by cell-cell contact may be an important factor for the constitutive expression of plasminogen gene in hepatocytes.     

The role of bone marrow-derived cells in bone fracture repair in a green fluorescent protein chimeric mouse model

We investigated the role of bone marrow cells in bone fracture repair using green fluorescent protein (GFP) chimeric model mice. First, the chimeric model mice were created: bone marrow cells from GFP-transgenic C57BL/6 mice were injected into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10Gy from a cesium source. Next, bone fracture models were created from these mice: closed transverse fractures of the left femur were produced using a specially designed device. One, three, and five weeks later, fracture lesions were extirpated for histological and immunohistochemical analyses. In the specimens collected 3 and 5 weeks after operation, we confirmed calluses showing intramembranous ossification peripheral to the fracture site. The calluses consisted of GFP- and osteocalcin-positive cells at the same site, although the femur consisted of only osteocalcin-positive cells. We suggest that bone marrow cells migrated outside of the bone marrow and differentiated into osteoblasts to make up the calluses.     

Adipogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice-including relationship of sex differences

We have previously demonstrated that adipose-derived stromal cells (ASCs) as well as bone marrow-derived stromal cells (BSCs) differentiate into a variety of cell lineages both in vitro and in vivo. Both types are considered to include mesenchymal stem cells. Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have also previously reported the plasticity of BSCs and ASCs. In this study, we focused on adipogenic differentiation in vitro by ASCs harvested from GFP transgenic mice. Moreover, preadipocytes and mature adipocytes were harvested at the same time, and the cells were cultured to compare them with ASCs. Inguinal fat pads from GFP transgenic mice were used for the isolation of ASCs, preadipocytes, and mature adipocytes. After expansion to three passages of ASCs, the cells were incubated in an adipogenic medium for two weeks. Adipogenic differentiation of ASCs was assessed by Oil Red O staining and the expression of the adipocyte specific peroxisome proliferative activated receptor gamma2 (PPAR-gamma2) gene. These ASCs stained positively, and expression of PPAR-gamma2 was detected. Moreover, we also tried to characterize the influence of sex differences on the adipogenic differentiation of ASCs harvested from both male and female mice. This was assessed by the expression levels of the PPAR-gamma2 gene using real-time PCR. The results showed that the expression levels of ASCs harvested from female mice were a maximum of 2.89 times greater than those harvested from male mice. This suggests that the adipogenic differentiation of ASCs is closely related to sex differences.     

Why egg-caring males of Isaza (<Emphasis Type="Italic">Gymnogobius isaza</Emphasis>, Gobiidae) refuse additional females: preliminary field observations

Like many other gobies, males of the Isaza goby, Gymnogobius isaza endemic to Lake Biwa, Japan, conduct parental care of eggs at nests, and females are likely to choose mates while visiting nests. The reproductive strategy should induce polygyny, but Isaza males never accept additional females in one breeding cycle. Sampling data of broods indicated that the egg mass areas were much smaller than the nest sizes, suggesting that nest size is not the limiting factor in obtaining further eggs from additional females. The brood size greatly decreased as the duration of care progressed. Few individuals including caring males ate eggs, and heterospecific egg predators were rarely observed. Sixty percent of egg masses at the middle stages of egg development were infected with aquatic fungi, some being covered with a fungus mat that drastically reduced survivorship. Infected egg masses contained more eggs than non-infected ones at the same stage, indicating that large egg masses are prone to be frequently destroyed by fungi. It is likely that the activity of parental males is lowered during the long care periods at low water temperature in early spring. Such lowered activity of caring males might be responsible for infections in large broods that would have needed more care. We propose the hypothesis that male rejection of additional females may be related to optimal brood size, which will be less susceptible to fungus infection and produce more hatching young than otherwise. This hypothesis will explain not only male avoidance of additional females but also some unique reproductive behaviors of this fish such as some females spawning of a portion of of the mature eggs in one nest.