Influence of mild hypothermia on delayed mitochondrial dysfunction after transient intrauterine ischemia in the immature rat brain.

The aim of this study was to determine the effect of different maternal thermal conditions during transient intrauterine ischemia on the mitochondrial respiratory activities in the immature rat brain. On 17 days of gestation, transient intrauterine ischemia was induced by 30 min of right uterine artery occlusion under hypothermic (33.5-34.5 degrees C, n=6), normothermic (36.5-37.5 degrees C, n=6), and hyperthermic conditions (39.5-40.5 degrees C, n=6). All of the pups were delivered by cesarean section at 21 days of gestation and cerebral neocortical tissue was sampled 1 h after delivery. The mitochondrial respiration was measured polarographically in homogenates. In the ischemic uterine horn, ADP-stimulated respiration of the normothermia and the hyperthermia groups decreased significantly to 73 and 74% of the non-ischemic controls, respectively. Since non-stimulated respiration remained unchanged, the respiratory control ratio (RCR) of the normothermia and the hyperthermia groups decreased significantly to 59 and 54% of the non-ischemic levels, respectively. In contrast, the mitochondrial respiratory activities of the hypothermia group showed no differences between the non-ischemic and the ischemic uterine horns. The results demonstrate that mild maternal hypothermia ameliorates the cerebral mitochondrial dysfunction in neonatal rats after intrauterine ischemia due to transient uterine artery occlusion and suggest that maternal thermal conditions, particularly during uteroplacental insufficiency, have important implications for the neuropathological outcome of the newborn.

Effect of the immunosuppressant drug FK506 on neonatal cerebral mitochondrial function and energy metabolism after transient intrauterine ischemia in rats.

Mitochondrial respiratory activities and energy metabolism were measured in neonatal rat brains to evaluate the influence of transient intrauterine ischemia on the near-term fetus and to assess the effect of the immunosuppressant drug FK506 treatment. Transient intrauterine ischemia was induced by 30 min of right uterine artery occlusion at 17 days of gestation in Wistar rats. The vehicle or 1.0 mg/kg of FK506 was administered after 1 h of recirculation. All of the pups were delivered by cesarean section at 21 days of gestation and samples of cerebral cortical tissue were obtained from pups at 1 h after birth. The mitochondrial respiration was measured polarographically in homogenates. For the analysis of ATP, ADP, and AMP, neonatal brains were frozen in situ and fluorometric enzymatic techniques were used. In the neonatal cortical tissue exposed to ischemia, mitochondrial respiratory activities and ATP concentrations decreased significantly to approximately 59 and 67% of those in normoxic controls, respectively. The deterioration of both mitochondrial respiratory activities and high-energy phosphates was prevented by FK506, given 1 h after the start of recirculation. The present results indicate that transient intrauterine ischemia is accompanied by mitochondrial dysfunction and cellular bioenergetic failure in the neonatal rat brain and suggest that treatment with FK506 prevents the deterioration, even when administered after the ischemic periods.

Maternal treatment with NMDA receptor antagonist (MK-801) attenuates delayed mitochondrial dysfunction after transient intrauterine ischemia in the neonatal rat brain.

PURPOSE: Mitochondrial respiratory activities were measured in neonatal rat brains to evaluate the influence of transient intrauterine ischemia on the near-term fetus and to assess the effect of dizocilpine maleate (MK-801), a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. METHODS: Transient intrauterine ischemia was induced by 30 min of right uterine artery occlusion at 17 days of gestation in Wistar rats. Vehicle (saline) or 0.5 mg/kg of MK-801 was administered after 1 h of recirculation. All of the pups were delivered by cesarean section at 21 days of gestation and samples of cerebral cortical tissue were obtained from pups at 1 h after birth. Adenosine diphosphate (ADP)-stimulated, nonstimulated, and uncoupled respirations were measured polarographically in homogenates. The respiratory control ratio was defined as ADP-stimulated divided by non-stimulated respiration. RESULTS: In the vehicle-treated group the neonatal cortical tissue exposed to ischemia showed a significant decrease in ADP-stimulated respiration and respiratory control ratio compared with these findings in normoxic control animals. The delayed mitochondrial respiratory dysfunction was prevented by MK-801, given 1 h after the start of recirculation (P < 0.05). CONCLUSION: The present results indicate that transient intrauterine ischemia in the near-term rat fetus is associated with delayed mitochondrial dysfunction in the neonatal brain; the results suggest that maternal treatment with MK-801 attenuates the deterioration, even when administered 1 h after the start of recirculation.

Cell kinetic study of normal human bone marrow hematopoiesis and acute leukemia using 7AAD/PY.

We have used the 7AAD/PY method to analyze the cell cycle status of normal human bone marrow hematopoiesis, and found that the cell kinetics differed. There were cells with relatively low levels of RNA in the S-phase (Type I) and a high level in the S-phase (Type 11). T-cells, B-cells, nucleated red cells and CD34+/CD19+ early B-cells in bone marrow were Type I, whereas myelomonocytic subset and CD34+/CD33-dim+ common myeloid cells were Type II. AC133+/CD38-dim cells, which were thought to be lineage-marker negative hematopoietic stem cells, had intermediate amounts of RNA in the S-phase between Type I and II (Type 0). Seventy-four cases of acute leukemia were also analyzed. Most of the T- and B-ALL cases were found to be Type I, most of the ANLL cases were Type II, and there were 10 cases that were Type 0. These findings yielded fundamental information about normal hematopoiesis and acute leukemia.

Interleukin-1 beta upregulates cardiac expression of vascular endothelial growth factor and its receptor KDR/flk-1 via activation of protein tyrosine kinases.

Vascular endothelial growth factor (VEGF) is not only an endothelial cell-specific angiogenic factor but also a potent mediator of vascular permeability. Interleukin-1 beta (IL-1 beta) is a pro-inflammatory cytokine that has numerous effects on the pathogenesis of the tissue injury. To explore the possible regulation of the VEGF system by IL-1 beta in the heart, we examined the regulation of expression of VEGF and KDR/flk-1 (one of the VEGF receptors) by IL-1 beta using cardiac myocytes and cardiac microvascular endothelial cells (CMEC). Both cardiac myocytes and CMEC substantially expressed VEGF mRNA and its expression was increased 3.6- and 2.4-fold by IL-1 beta, respectively. IL-1 beta-induced accumulations of VEGF mRNA in cardiac myocytes were abolished by the tyrosine kinase inhibitor genistein, whereas inhibition of protein kinase C (PKC) by staurosporin, calphostin C and phorbol ester-induced PKC depletion, and intracellular Ca2+ chelators did not affect the induction of VEGF mRNA by IL-1 beta. Relatively smaller amounts of KDR/flk-1 mRNA were detected in CMEC, but not in cardiac myocytes, and the analysis using quantitative reverse transcription-polymerase chain reaction revealed that IL-1 beta significantly stimulated the accumulation of KDR/flk-1 mRNA 3.0-fold. VEGF protein (23 kDa) levels in Western blot analysis were increased 4.2- and 3.4-fold by IL-1 beta in cardiac myocytes and CMEC, respectively. KDR/flk-1 protein (230 kDa) levels in CMEC were also increased 3.2-fold by IL-1 beta. In addition, pre-treatment of CMEC by IL-1 beta markedly enhanced VEGF-induced tyrosine phosphorylation of focal adhesion kinase compared with that in the unstimulated cells. These findings indicate that cardiac VEGF-KDR/flk-1 system is upregulated by IL-1 beta via activation of tyrosine kinases, suggesting that the IL-1 beta-modulated autocrine and/or paracrine system of VEGF has an important role in the process of angiogenesis in ischemic hearts.

Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor.

The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of growth factor stimulation, and recent evidence suggests that G protein-coupled receptors transactivate growth factor receptors to transmit mitogenic effects. In the present study, we report the involvement of epidermal growth factor receptor (EGF-R) in Ang II-induced extracellular signal-regulated kinase (ERK) activation, c-fos gene expression, and DNA synthesis in cardiac fibroblasts. Ang II induced a rapid tyrosine phosphorylation of EGF-R in association with phosphorylation of Shc protein and ERK activation. Specific inhibition of EGF-R function by either a dominant-negative EGF-R mutant or selective tyrphostin AG1478 completely abolished Ang II-induced ERK activation. Induction of c-fos gene expression and DNA synthesis were also abolished by the inhibition of EGF-R function. Calmodulin or tyrosine kinase inhibitors, but not protein kinase C (PKC) inhibitors or downregulation of PKC, completely abolished transactivation of EGF-R by Ang II or the Ca2+ ionophore A23187. Epidermal growth factor (EGF) activity in concentrated supernatant from Ang II-treated cells was not detected, and saturation of culture media with anti-EGF antibody did not affect the Ang II-induced transactivation of EGF-R. Conditioned media in which cells were incubated with Ang II could not induce phosphorylation of EGF-R on recipient cells. Platelet-derived growth factor-beta receptor was not phosphorylated on Ang II stimulation, and Ang II-induced c-jun gene expression was not affected by tyrphostin AG1478. Our results demonstrated that in cardiac fibroblasts Ang II-induced ERK activation and its mitogenic signals are dominantly mediated by EGF-R transactivated in a Ca2+/calmodulin-dependent manner and suggested that the effects of Ang II on cardiac fibroblasts should be interpreted in association with the signaling pathways regulating cellular proliferation and/or differentiation by growth factors.

Leaf-opening Substance in the Nyctinastic Plant, Albizzia julibrissin Durazz.

cis-p-Coumaroylagmatine (1) was isolated from Albizzia julibrissin Durazz, a nyctinastic plant, as a leaf-opening substance. The compound was quite effective for opening the plant leaves at 5×10(-6) M at night, but was not effective for other nyctinastic plants. The bioactive fraction with leaf-closing activity was also separated from the plant extract. Although the leaf-opening activity of the plant extract changed between the day and night, the content of 1 was almost constant through a 24-h day. These results suggest that the change in content of an unknown leaf-closing factor induced balance between the two leaf-movement factors through a 24-h day.

Angiotensin type 2 receptors are reexpressed by cardiac fibroblasts from failing myopathic hamster hearts and inhibit cell growth and fibrillar collagen metabolism.

BACKGROUND: Angiotensin (Ang) II type 1 receptor (AT1-R) induces cardiomyocyte hypertrophy and fibroblast proliferation, whereas the physiological role of AT2-R in cardiac remodeling remains poorly defined. METHODS AND RESULTS: Using Bio14.6 cardiomyopathic (CM) hamsters, we found that AT2-R sites were increased by 153% during heart failure compared with F1B controls. AT1-R numbers were increased by 72% in the hypertrophy stage and then decreased to the control level during heart failure. Such differential regulation of AT2-R and AT1-R during heart failure was consistent with changes in the respective mRNA levels. Autoradiography and immunocytochemistry revealed that both AT2-R and AT1-R are localized at higher densities in fibroblasts present in fibrous regions. Surrounding myocardium predominantly expressed AT1-R, but the level of expression was less than that in fibrous regions. Cardiac fibroblasts isolated from CM hearts during heart failure but not from control hamsters expressed AT2-R (30 fmol/mg protein). Using the cardiac fibroblasts expressing AT2-R, we found that Ang II stimulated net collagenous protein production by 48% and pretreatment with an AT2-R antagonist, PD123319, evoked a further elevation (83%). Ang II-induced synthesis of fibronectin and collagen type I were enhanced by 40% and 53%, respectively, by pretreatment with PD123319. Ang II-induced DNA synthesis (assessed by [3H]thymidine uptake) was significantly increased by PD123319, and the AT2-R agonist CGP42112A reduced the serum-stimulated increase in cell numbers by 23%. Treatment with an AT1-R antagonist, TCV116, for 20 weeks inhibited progression of interstitial fibrosis by 28%, whereas with 44-week PD123319 treatment but not 20-week treatment, the extent of the fibrous region was increased significantly, by 29%. CONCLUSIONS: These findings demonstrate that AT2-R is re-expressed by cardiac fibroblasts present in fibrous regions in failing CM hearts and that the increased AT2-R exerts an anti-AT1-R action on the progression of interstitial fibrosis during cardiac remodeling by inhibiting both fibrillar collagen metabolism and growth of cardiac fibroblasts.

Comparison of the crown dimensions between the maxillary second deciduous molar and the first permanent molar.

The morphologic relationship of the crown structures of the maxillary second deciduous molar (m2) and the first permanent molar (M1) was investigated with odontometric methods. Materials used were 124 male dental casts taken from Chinese living in Kaohsiung (Taiwan). The mesiodistal and buccolingual diameters, cusp sizes, and intercusp distances were measured. The mean crown diameters were larger in M1 than in m2. The tooth crown proportions were similar in m2 and M1. The intercusp distance was compressed buccolingually in m2. With respect to the relative cusp size, the paracone was not significantly different between m2 and M1. The metacone was relatively larger in m2 than M1, while lingual cusps (protocone and hypocone) were relatively smaller in m2 than in M1. Principal component analysis was performed to investigate factors influencing the variation in crown dimensions of the two teeth. Four components were extracted: (1) buccolingual intercusp distances, (2) crown proportion (mesiodistal and buccolingual diameters), (3) mesiobuccal and distolingual compression, and (4) protocone and mesiodistal intercusp distance. Analysis of principal component scores indicated that the crown proportions of m2 and M1 were similar. The m2 had a smaller protocone and more buccolingually compressed intercusp distances than did M1. The morphologic characteristics of the crown of m2 indicate that it is more primitive and has developed less than M1.

Studies on the reduction indices of the deciduous teeth of Chinese living in Taiwan.

The reduction index of deciduous teeth was investigated statistically. Materials used were plaster casts of the deciduous dentition taken from Chinese children living in Taiwan. The reduction indices showed no sexual differences. In the maxillary teeth, reduction indices of the bucco-lingual diameters (BL) had the largest values, followed by those of the mesio-distal diameters (MD), and those of the crown area (AREA) which were the least. In contrast to the maxillary teeth, in the mandibular teeth, the reduction indices of MD and those of BL had nearly the same values although the reduction indices of MD were slightly larger than those of BL in females (p < 0.05). With respect to the difference between the maxilla and the mandible, in deciduous incisors the reduction index of MD in the mandible was larger than that in the maxilla (p < 0.01). While in BL the reverse relation was noted. In the deciduous molars the reduction indices of BL and AREA in the maxilla were larger than those in the mandible (p < 0.01). The reduction indices showed no significant difference among the Mongoloid populations sampled. This result may be explained by the primitiveness of deciduous teeth.

Germanium intoxication with sensory ataxia.

Sensory ataxia in inorganic germanium intoxication is rare. A 63-year-old housewife had taken inorganic germanium preparations at a dosage of 36 mg a day for about 6 years (total dose about 80 g). She subsequently developed difficulty in writing and gait disturbance with peripheral neuropathy and renal involvement. Germanium, which is not usually detected in the non-germanium user, was accumulated in her hair and nails, permitting a diagnosis of inorganic germanium intoxication. The peripheral neuropathy and renal injury were not reversible after discontinuing the preparation. Pneumonia and sepsis then supervened and the patient died. Autopsy findings showed degeneration and loss of the dorsal root ganglion cells and degeneration of the dorsal column of the spinal cord. Two previously reported cases presented with ataxia. These patients took germanium for long periods and/or large quantities like our case. It was supposed that sensory ataxia was induced by chronic and dose dependent toxicity of inorganic germanium.

[A case of successful emergency coronary artery bypass grafting surgery for failed coronary angioplasty using percutaneous cardiopulmonary support].

This is the first Japanese case of the successful emergency coronary artery bypass grafting (CABG) surgery for failed percutaneous transluminal coronary angioplasty (PTCA) using percutaneous cardiopulmonary support (PCPS). A 81-year-old woman with old myocardial infarction and angina pectoris is presented. Her coronary angiogram showed the 90% and 50% stenosis of the right coronary artery (RCA) and the total occlusion of the left anterior descending artery (LAD). PTCA for the 90% stenosis of RCA was performed. But, during the balloon dilation, her heart rate and blood pressure decreased. PTCA was stopped. As her chest pain was worse, re-PTCA was tried, using PCPS. Under PCPS (3 l/min), the balloon dilation was performed safely and smoothly. But, the unexpected dissection of RCA occurred, and became larger rapidly. After 85 minutes, the emergency CABG was performed. By using PCPS, the stable hemodynamics was given till the operation. CABG to RCA and LAD was performed safely. After the surgery, the patient progressed well. PCPS was a very useful cardiopulmonary assist device.

[The effect of kallikrein and caffeine on the sperm survival of cryopreserved semen in patients with oligozoospermia].

Cryopreservation of semen from patients with oligozoospermia (sperm counts less than 20 million/ml) generally results in a severe decline in the percentage of motile spermatozoa after thawing in comparison with normozoospermia. We studied the effects of two metabolically active compounds, namely kallikrein and caffeine, on the survival of frozen-stored spermatozoa, in particular, from patients with oligozoospermia. A modified Ackerman's solution was used as the protective medium, and the specimens were frozen using a programmed freezer. We evaluated the post-thaw survival rate of spermatozoa by sperm counts on addition of agents. 1) When used the modified Ackerman's solution only, the post-thaw survival rate in the cases of oligozoospermia was significantly lower than that in the cases of normozoospermia. On the other hand, between these groups, there were no significant differences in the post-thaw activity rates of spermatozoa. 2) When added 1 KE/ml kallikrein and 7.5 mM caffeine (final volume) to the protective medium, the survival rate in the cases of oligozoospermia increased significantly, so that no significant differences were seen in the survival rates between the groups of oligozoospermia and normozoospermia. The effect of caffeine was better than that of kallikrein. 3) It was suggested that kallikrein and caffeine activated the non-motile live spermatozoa after thawing especially in the cases of oligozoospermia.

[Postoperative percutaneous coronary angioplasty for stenosed aortocoronary vein bypass].

This paper was presented to demonstrate the efficacy of postoperative PTCA for varying degree of stenosis aortocoronary bypass with saphenous vein grafts. The following conclusions were drown. 1. The success rate of postoperative PTCA for 15 grafts showed 88.8%. 2. The rate of success for PTCA was much more effective in patients within one year after bypass operations. The authors concluded that postoperative PTCA can be done with excellent rate of success. Therefore, postoperative cine-coronary angiography should be done to evaluate the state of the graft and to decide to perform possible postoperative PTCA in order to keep long time patency rate of saphenous vein grafts.

Mechanical loading stimulates cell hypertrophy and specific gene expression in cultured rat cardiac myocytes. Possible role of protein kinase C activation.

To examine the molecular mechanisms by which mechanical stimuli induce cardiac hypertrophy and specific gene expression, we cultured rat neonatal cardiocytes in deformable dishes and imposed an in vitro mechanical load by stretching the adherent cells. Myocyte stretching increased total cell RNA content and mRNA levels of c-fos and skeletal alpha-actin. Nuclear run-off transcription assay revealed that this increase in c-fos mRNA level by stretching at least partially reflects changes in the transcriptional status. The transfected chloramphenicol acetyltransferase gene linked to upstream sequences of the fos gene indicated that sequences containing a serum response element were required for efficient transcription by stretching and that sequences containing a cAMP/calcium response element might not be involved in the c-fos response to myocyte stretching. The accumulation of c-fos mRNA by stretching was suppressed by protein kinase C inhibitors at the transcriptional level and inhibited markedly by down-regulation of protein kinase C. Moreover, myocyte stretching increased inositol phosphate levels, and activation of protein kinase C by phorbol esters stimulated the expression of c-fos and skeletal alpha-actin genes. These findings suggest that mechanical stimuli (myocyte stretching) might directly induce cardiac hypertrophy and specific gene expression possibly via protein kinase C activation.

Molecular mechanism of cardiac hypertrophy.

Pressure overload induces cardiac hypertrophy and reexpression of contractile protein isogenes. To ascertain the molecular mechanism of these events, we examined the expression of cellular oncogenes and the early change in the translational activity of specific cardiac mRNA by two-dimensional gel electrophoresis of in vitro translational products. Pressure overload increased the expression levels of c-fos, c-myc, and c-Ha-ras genes. The relative predominance of 8 species out of over 400 translational products was increased by pressure overload while that of 2 translational products was decreased. We cloned four pressure-overload-responsive cDNA clones by differential dot blot hybridization. The expression pattern of each cDNA clone in the pressure-overloaded hearts was similar to that in fetal hearts. To examine whether mechanical stimuli directly induce specific gene expression in the heart, we cultured rat neonatal cardiocytes in elastic silicone dishes and stretched these adherent cells. Myocytes stretching stimulated amino acid uptake and expression of the c-fos gene, which was blocked by protein kinase C inhibitors. These results suggest that there are some early responsive genes in cardiac hypertrophy and that mechanical loading directly stimulates gene expression possibly via protein kinase C activation.

Molecular cloning of gene sequences from rat heart rapidly responsive to pressure overload.

Although pressure overload induces cardiac hypertrophy and reexpression of contractile protein isogenes, the molecular mechanism of both is not well understood. We demonstrated the early change in the translational activity of specific cardiac messenger RNA by two-dimensional gel electrophoresis of in vitro translational products. A total of over 400 translational products were detected by fluorography, and the relative predominance of eight species was increased by pressure overload whereas that of two translational products was decreased. We cloned four pressure-overload-responsive complementary DNA clones, pPO-1, -4, -5, and -6, by differential dot blot hybridization. Two clones were increased from the early period after the imposition of pressure overload; the other two were decreased. The expression pattern of each complementary DNA clone in the pressure-overloaded hearts was similar to that in fetal hearts. Pressure overload also induced the additional messenger RNA, which hybridized with pPO-4. This mRNA was also recognized in fetal, neonatal, and adult spontaneously hypertensive rat hearts. The induction of this transcript by pressure overload was not suppressed but, rather, stimulated by cycloheximide. These results suggest that there are some genes that rapidly respond to pressure overload and that may play some role in the development of cardiac hypertrophy.

Stretching cardiac myocytes stimulates protooncogene expression.

Recently cellular protooncogenes have been found to be induced as an early response to pressure overload in cardiac hypertrophy. To examine whether mechanical stimuli directly induce specific gene expression in the heart, we cultured rat neonatal cardiocytes in elastic silicone dishes and stretched these adherent cells. Myocyte stretching stimulated expression of the protooncogene, c-fos, in a stretch length-dependent manner, followed by an increase in amino acid incorporation into proteins. c-fos mRNA levels were enhanced within 15 min by cardiocyte stretching, peaked at 30 min, and declined to undetectable levels by 240 min. In the presence of cycloheximide, a greater increase in c-fos mRNA was seen by stretching. The transfected chloramphenicol acetyltransferase gene linked to upstream sequences of the fos gene including its promoter was also activated by stretching cardiac myocytes. These results suggest that mechanical loading directly regulates gene transcription without the participation of humoral factors in cardiocytes.

Molecular cloning and characterization of a Ca2+ + Mg2+-dependent adenosine triphosphatase from rat cardiac sarcoplasmic reticulum. Regulation of its expression by pressure overload and developmental stage.

To investigate the regulation of expression of cardiac Ca2+ + Mg2+-dependent ATPase (Ca2+-ATPase) in sarcoplasmic reticulum (SR), we isolated cDNA (pHA6) encoding a Ca2+-ATPase of rat cardiac SR. The clone consisted of 2,311 mRNA-derived nucleotides, which covered half the coding region and the entire 3'-untranslated regions. The nucleotides and deduced amino acid sequences of pHA6 showed striking homology, 89 and 98%, respectively, to those of rabbit Ca2+-ATPase of the slow-twitch form. Northern blot analyses revealed that the mRNA levels of Ca2+-ATPase were decreased by pressure overload and became 32% of sham in 1 mo. During the developmental stage the mRNA levels were very low in the fetal period and steeply increased around birth. These changes in mRNA levels were correlated with the corresponding protein levels. These results suggest that the expression of cardiac Ca2+-ATPase in SR is regulated by pressure overload and the developmental stage, at least in part, at the pretranslational level.