Localization of hematopoietic cells in the bullfrog (<Emphasis Type="Italic">Lithobates catesbeianus</Emphasis>)

Amphibians represent the first phylogenetic group to possess hematopoietic bone marrow. However, adult amphibian hematopoiesis has only been described in a few species and with conflicting data. Bone marrow, kidney, spleen, liver, gut, stomach, lung, tegument, and heart were therefore collected from adult Lithobates catesbeianus and investigated by light microscopy and immunohistochemical methods under confocal laser microscopy. Our study demonstrated active hematopoiesis in the bone marrow of vertebrae, femur, and fingers and in the kidney, but no hematopoietic activity inside other organs including the spleen and liver. Blood cells were identified as a heterogeneous cell population constituted by heterophils, basophils, eosinophils, monocytes, erythrocytic cells, lymphocytes, and their precursors. Cellular islets of the thrombocytic lineage occurred near sinusoids of the bone marrow. Antibodies against CD34, CD117, stem cell antigen, erythropoietin receptor, and the receptor for granulocyte colony-stimulating factor identified some cell populations, and some circulating immature cells were seen in the bloodstream. Thus, on the basis of these phylogenetic features, we propose that L. catesbeianus can be used as an important model for hematopoietic studies, since this anuran exhibits hematopoiesis characteristics both of lower vertebrates (renal hematopoiesis) and of higher vertebrates (bone marrow hematopoiesis).     

Ex vivo expansion of primitive hematopoietic cells for cellular therapies: An overview

Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, and autologous (patient) marrow that is depleted from prior chemo- or radiotherapy or has cancerous involvement. Anex vivo system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplant, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow and/or mobilized peripheral blood stem and progenitor cells required for transplantation, and reduce the time to white cell and platelet engraftment. The cloning of hematopoietic growth factors and the identification of appropriate conditions has enabled the development of successfulex vivo hematopoietic cell cultures. Purification systems based on the CD34 marker (which is expressed by the most primitive hematopoietic cells) have proven an essential tool for research and clinical applications. Present methods for hematopoietic cultures (HC) on stromal (i.e. accessory cells that support hematopoiesis) layers in flasks lack a well-controlled growth environment. Several bioreactor configurations have been investigated, and a first generation of reactors and cultures has reached the clinical trial stage. Our research suggests that perfusion conditions improve substantially the performance of hematopoietic reactors. We have designed and tested a perfusion bioreactor system which is suitable for the culture of non-adherent cells (without stromal cells) and readily scaleable for clinical therapies. Eliminating the stromal layer eliminates the need for a stromal cell donor, reduces culture time, and simplifies the culture system. In addition, we have compared the expansion characteristics of both mononuclear and CD34⁺ cells, since the latter are frequently assumed to give a superior performance for likely transplantation therapies.Abbreviations BFU0-E burst forming unit-erythroid - BM bone marrow - CB cord blood - CFU-C colony forming unit-culture - CFU-E colony forming unit-erythroid - CFU-F colony forming unit-fibroblast - CFU-GEMM colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte - CFU-GM colony forming unit-granulocyte, macrophage - CFU-Mix colony forming unit-mixed (also known as CFU-GEMM) - CML chronic myeloid leukemia - CSF colony stimulating factor - DMSO dimethyl sulfoxide - ECM extracellular matrix - EPO erythropoietin - FL fetal liver - HC hematopoietic culture - LTBMC long-term bone marrow culture - LTC-IC long-term culture initiating cell - LTHC long-term hematopoietic culture - MNC mononuclear cells - PB peripheral blood     

Kinetic studies of pyruvate kinase duringin vitro differentiation of GM-CFC haemopoietic precursor and bone marrow cells in mice

Pyruvate kinase studies in the granulocyte-macrophage lineage duringin vitro differentiation have been performed using culture techniques on GM-CFC cells and a study has also been done in bone marrow cells.The enzyme exhibits biphasic behaviour with respect to both of its substrates in cells derived fromin vitro cultures at 5 and 7 days of incubation period. However in bone marrow cells these kinetics are only observed for ADP.The different kinetic behaviour of pyruvate kinase toward Fru-1,6-P₂, Ala, Phe and ATP in the three cellular populations allows us to conclude that the expression of pyruvate kinase is associated with the differentiation of these cells.Abbreviations GM-CFC granulocyte-macrophage colony forming cells - PK pyruvate kinase - CFU-E Colony Forming Units Erythroid - E_w Error weight - PEP phosphoenolpyruvate - Fru-1,6-P₂ fructose 1,6-bisphosphate - Ala L-alanine - Phe L-phenylanine - 5 GM granulocytemacrophage colonies obtained after 5 days incubation - 7 GM granulocyte-macrophage colonies obtained after 7 days incubation - h Hill coefficient - S_0,5 substrate concentration that yields half-maximal velocity     

Role of cytokines and extracellular matrix in the regulation of haemopoietic stem cells

The understanding of molecular mechanisms regulating the formation, growth and differentiation of haemopoietic stem cells has advanced considerably recently. Particular progress has been made in defining the cytokines, chemokines and extracellular matrix components which retain and maintain primitive haemopoietic cell populations in bone marrow. Furthermore, signal transduction pathways that are critical for haemopoiesis, both in vivo and in vitro, and that are activated by cytokines have also been identified and further characterised. The importance of these processes has, this year, been exemplified by the phenotypes of mice deficient in key signal transduction proteins and the discovery that mutations in the component proteins of some signalling pathways are linked to human diseases. Significant advances in understanding the molecular mechanisms for mobilisation of stem cells from bone marrow have also been made this year; this has potential importance for bone marrow transplantation.     

Immunomodulatory and cytoprotective role of RP-1 in γ-irradiated mice

RP-1 has been reported to provide protection against lethal -irradiation in mice. The present study was undertaken to understand its mechanism of action, especially with respect to modulation of radiation-induced changes in immune cell function, plasma antioxidant potential, cell cycle perturbations, apoptosis in mouse bone marrow cells, and micronuclei frequency in mice reticulocytes. 2 Gy reduced mitogenic response of splenic lymphocytes significantly at 48 h. Pre-irradiation RP-1 treatment significantly countered the radiation-induced loss of splenocyte proliferation. RP-1 treatment, with or without radiation, suppressed macrophage activation as compared to control. Irradiation decreased plasma antioxidant status significantly (p < 0.05) at 1 and 2 h (4.8 ± 0.224 and 4.9 ± 0.057 mM Fe²⁺) as compared to control (6.29 ± 0.733 mM Fe²⁺) that was countered by RP-1 pre-treatment significantly (p < 0.05). RP-1 and irradiation individually caused G₂ delay in bone marrow cells. RP-1 pre-treatment augmented radiation-induced G₂ delay and elicited significant (p < 0.05) recovery in S phase fraction at 48 h in comparison to irradiated group. Radiation-induced apoptosis (3%) was significantly higher than the control. RP-1 pre-treatment further enhanced apoptosis frequency (7.2%) in bone marrow cells. RP-1 pre-treatment significantly (p < 0.05) reduced (1.23%) the radiation-induced MN frequency (2.9%) observed at 48 h post-irradiation interval. Since the radioprotective manifestation of RP-1 is mediated through multiple mechanisms, needs further investigation.     

Kinetics of nitrite oxidation in two Nitrobacter species grown in nitrite-limited chemostats

The influence of growth rate, the presence of acetate and variation in the dissolved oxygen concentration on the kinetics of nitrite oxidation was studied in suspensions of intact cells of Nitrobacter winogradskyi and Nitrobacter hamburgensis. The cells were grown in nitrite-limited chemostats at different dilution rates under chemolithotrophic and mixotrophic conditions. Growth of N. hamburgensis in continuous culture was dependent on the presence of acetate. Acetate hardly affected the maximal nitrite oxidation rate per cell (V _max), but displayed a distinctly negative effect on the saturation constants for nitrite oxidation (K _m ) of both Nitrobacter species. This effect was reversible; when acetate was removed from the suspensions the K _m -values for nitrite oxidation returned to their original values. A reduction of the dissolved oxygen concentration from 100% to 18% air saturation slightly decreased the V _max of chemolithotrophically grown N. winogradskyi cells, whereas a 2.3 fold increase was observed with mixotrophically grown cells of N. hamburgensis. It is suggested that the large variation in K _m encountered in field samples could be due to this observed phenotypic variability. The V _max per cell is not a constant, but apparently is dependent on growth rate and environmental conditions. This implies that potential nitrite oxidation activity and numbers of cells are not necessarily related. Considering their kinetic characteristics, it is unlikely that N. hamburgensis is able to compete succesfully with N. winogradskyi for limiting amounts of nitrite under mixotrophic conditions. However, at reduced partial oxygen tensions, N. hamburgensis may become the better competitor.     

Enhancement of proliferation and differentiation in bone marrow hematopoietic cells by Spirulina (Arthrospira) platensis in mice

This study evaluates whether Spirulina, including its components such as phycocyanin, enhances or sustains immune functions by promoting immune competent-cell proliferation or differentiation. The effects of Spirulina of a hot-water extract (SpHW), phycocyanin (Phyc), and cell-wall component extract (SpCW) on proliferation of bone marrow cells and induction of colony-forming activity in mice were investigated. The Spirulina extracts, SpHW, Phyc, and SpCW, enhanced proliferation of bone-marrow cells and induced colony-forming activity in the spleen-cell culture supernatant. Granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were detected in the culture supernatant of the spleen cells stimulated with the Spirulina extracts. Bone marrow-cell colony formation in soft-agar assay was also significantly induced by the blood samples and the culture supernatants of the spleen and Peyer's patch cells of the mice which ingested Spirulina extracts orally for 5 weeks in in vivo study. Ratios of neutrophils and lymphocytes in the peripheral blood and bone marrow, consequently, increased in the mice. Spirulina may have potential therapeutic benefits for improvement of weakened immune functions caused by, for example, the use of anticancer drugs.     

Differentiating megakaryocytes in myelodysplastic syndromes succumb to mitochondrial derangement without caspase activation

Myelodysplastic syndromes (MDS) constitute a preneoplastic condition in which potentially malignant cancer stem cells continuously die during differentiation. This MDS-associated cell death often involves caspase-3 activation, yet can also occur without caspase activation, for instance in differentiating megakaryocytes (MK). We investigated, the mechanisms through which MK from MDS patients undergo premature cell death. While polyploid, mature MK from healthy subjects or MDS patients manifested caspase-3 activation during terminal differentiation, freshly isolated, immature MK from MDS died without caspase-3 activation. Similarly, purified bone marrow CD34⁺ cells from MDS patients that were driven into MK differentiation in vitro died without caspase-3 activation at an immature stage, before polyploidization. The premature death of MDS MK was accompanied by the mitochondrial release of cytochrome c, Smac/DIABLO and endonuclease G, a caspase-independent death effector, as well loss of the mitochondrial membrane potential and plasma membrane phosphatidylserine exposure before definitive loss of viability. Thus, a stereotyped pattern of mitochondrial alterations accompanies differentiation-associated MK death in MDS. T. Braun and G. Carvalho contributed equally to this paper.     

Cytotoxicity in feline leukemia virus subgroup-c infected fibroblasts is mediated by adherent bone marrow mononuclear cells

Summary The pathogenesis of retrovirus-induced erythroid aplasia in cats is unknown. In studies to define mechanisms of cytotoxicity associated with retroviral infections, bone marrow mononuclear cells (BMMC) from healthy specific pathogenfree cats were co-cultured with uninfected feline embryonic fibroblasts (FEA cells) and FEA cells infected with feline leukemia virus (FeLV) of subgroup A (FEA-A) or subgroup C (FEA-C). Moderate to marked cytotoxicity (CPE) developed in co-cultures of BMMC and FEA-C cells on Days 5 to 7 of incubation but not in co-cultures of BMMC and FEA-A or BMMC and uninfected cells (FEA-CT). Cytotoxicity was associated with adherent cells of light density (1.056) from bone marrow and peripheral blood, which were positive for alpha naphthyl butyrate esterase activity. Stimulation of adherent cells with phorbol ester or addition of recombinant human tumor necrosis factor-alpha (rhTNF-α) caused similar CPE in FEA-CT cells. The TNF-α concentrations in the culture supernatants of BMMC+FEA-C were higher than those of BMMC+FEA-A or BMMC+FEA-CT, and addition of anti-TNF antibodies to the cultures blocked the CPE. These data support the hypothesis that macrophages exposed to FeLV-C cause CPE in co-cultures of BMMC and FEA cells by a mechanism involving TNF-α. It is suggested that TNF-α may be involved in the suppression of hematopoiesis in cats which develop FeLV-C induced erythroid aplasia.     

Immunological regression of metastatic cancer in the liver as a result of “in vivo xenogenization”

We attempted to induce the regression of liver metastatic tumor cellsin vivo by the administration to rats of Friend leukemia virus (FV) (in vivo xenogenization). The virus which was used in this experiment, FV, is highly immunogenic and does not normally cause disease in an adult rat. At first, we induced a FV viremia in tumor bearing rats in order to deliver the virus to the site of the tumor cells. FV viremia was induced by injecting 60 mg/kg cyclophosphamide (CY) i.v. after the administration of FV, and by transferring syngeneic bone marrow cells so that FV would be able to infect them and then replicate.In order that the tumor cells which were infected with virus should regress, it was necessary to break down their tolerance to FV antigens. As adoptive immunotherapy we therefore, transferred syngeneic spleen cells from rats which had been immunized with FV to tumor bearing rats. The result of this experiment was that these tumor bearing rats infected with FV which had received either normal syngeneic spleen cells or no spleen cells as controls died from liver metastasis (8 out of 9 rats (89%) and 15 out of 17 (88%) respectively). On the other hand, only 4 out of the 15 (27%) tumor bearing rats which were infected with FV and which received FV-immune spleen cells died from liver metastasis.These sets of data indicate that thein vivo xenogenization of tumor cells are indeed able to induce the regression of metastic tumor cells.     

Effects of degranulation of mast cells on proliferation of mesenchymal cells in the mesentery of mice

Summary A mast-cell activator, compound 48/80, causes proliferation of mesenchymal cells in the mesentery of rats. Its effect on W/W ^vmice deficient in mast cells was tested to determine whether the proliferation is mediated in the degranulation of mast cells. Incorporation of [³H]thymidine into mesenchymal cells in the mesentery of these mice with or without compound 48/80 was very small compared to their normal litter mates. However, bone marrow transplantation markedly enhanced the effect of compound 48/80, and resulted in an incorporation of [³H]thymidine almost comparable to that observed in normal mice. Our results provide evidence that mesenchymal cell proliferation is caused by a product secreted by mast cells when stimulated by compound 48/80.Supported by a Grant-in-Aid for Scientific Research, No. 366, from the Japanese Ministry of Health and WelfareThe authors are indebted to Drs. Motomu Minamiyama and Yukio Hirata for valuable advices, and to Miss Mitsuko Inoue for technical assistance     

古林箐秋海棠叶斑结构对叶色的影响

该研究以古林箐秋海棠(Begonia gulinqingensis)为材料,通过分析叶片形态特征、上表皮光学特性、组织结构、叶绿素含量及叶绿素荧光参数(F_v/F_m),探讨了叶片色斑的形成原因。结果表明:(1)古林箐秋海棠叶斑发生频率和数量无明显规律,但发生部位相对稳定,叶斑主要发生在正对叶柄的两条主脉之间。(2)斑区有两种光反射模式,点状反射和多角形反射,栅栏组织细胞呈近等轴的圆形,排列疏松,与上表皮细胞间存在空隙;非斑区只有点状反射模式,栅栏组织细胞为漏斗型,排列紧密,与上表皮细胞间不存在空隙。(3)斑区和非斑区叶绿体均有密集的堆积基粒和丰富的类囊体膜,斑区叶绿素a、b及总叶绿素含量仅比非斑区分别低24.9%、25.2%、25.1%。(4)叶绿素荧光参数(F_v/F_m)值斑区为0.793,非斑区为0.790。虽然斑区叶绿素含量比非斑区略低,但叶绿体结构完整,且叶绿素荧光参数与非斑区无显著差异。斑区上表皮与栅栏组织细胞间的空隙可使光线到达绿色组织时发生二次反射,在叶片表皮细胞边缘形成白色多边形光反射使该区域相对周围正常叶片区域偏白,基于上述结果可推测古林箐秋海棠的淡绿色块斑形成与特殊的叶片结构有关。     

N-andO-glycosylation of glycoprotein C synthesized by Herpes simplex virus type 1-infected ricin resistant cells

The progeny of Herpes simplex virus type 1 (HSV-1) grown in ricin-resistant 14 cells (Ric^R14) lackingN-acetylglucosaminyltransferase I was released in the extracellular medium at a very low rate. By using a monoclonal antibody immobilized on Sepharose we purified from HSV-1-infected Ric^R14 cells a viral glycoprotein (gC), which carries bothN-andO-linked oligosaccharides. Glycopeptides obtained from [³H]mannoselabeled gC by Pronase digestion were entirely susceptible to endo--N-acetylglucosaminidase H, and the major oligosaccharide released was Man₄GlcNAc. The accumulation of this high-mannose species was related to the enzymic defect of the host cells and to the long retention of the viral glycoprotein within the cells. The extent ofO-glycosylation evaluated in [¹⁴C]glucosamine-labeled gC from Ric^R14 cells as compared to that of gC from wild type cells did not appear to be significantly modified.Abbreviations Con A concanavalin A - BHK cells baby hamster kidney cells - HSV Herpes simplex virus     

Inactivation of Amphidinium sp. in ballast waters using UV/Ag-TiO2+O3 advanced oxidation treatment

Ballast water poses a biological threat to the world’s waterways by transferring aquatic species from one body of water to another. This study investigates the use of combined ultraviolet (UV)/Ag-TiO₂ + ozone (O₃) processes for treating ballast water using Amphidinium sp. as an indicator microorganism. Sufficient Amphidinium sp. cells in ballast waters can be inactivated using O₃ alone, UV irradiation alone (with or without an Ag-TiO₂ coating), and combined treatments. For the low inactivation ratio (<40%) regime, the effects of ozonation and photocatalysis were observed to be cumulative. The combined UV/Ag-TiO₂ + O₃ treatment produced excess hydroxyl radicals and total residual oxidants (TROs), and readily damaged cell membranes to release intracellular substances. The comparison tests revealed that the combined treatments synergistically inactivate Escherichia coli in ballast waters. However, the combined process did not synergistically inactivate Amphidinium sp. cells. Inactivating different aqua species in ballast waters needs distinct treatment methods and dosages.     

Ammonium uptake by Chlorella

The preincubation of Chlorella cells with glucose caused a tenfold increase of the maximal uptake rate of ammonium without change in the K _m (2 M). A similar stimulation of ammonium uptake was found when the cells were transferred to nitrogen-free growth medium. The time-course of uptake stimulation by glucose revealed a lag period of 10–20 min. The turnover of the ammonium transport system is characterized by a half-life time of 5–10 h, but in the presence of light 30% of uptake activity stayed even after 50 h. 6-Deoxyglucose was not able to increase the ammonium uptake rate. These data together were interpreted as evidence for induction of an ammonium transport system by a metabolite of glucose. Mechanistic studies of the ammonium transport system provided evidence for the electrogenic uptake of the ammonium ion. The charge compensation for NH ₄ ⁺ entry was achieved by immediate K⁺ efflux from the cells, and this was followed after 1 min by H⁺ extrusion. Ammonium accumulated in the cells; the rate of uptake was sensitive to p-trifluoromethoxy-carbonylcyanide-phenylhydrazon and insensitive to methionine-sulfoxime. Uptake studies with methylamine revealed that methylamine transport is obviously catalyzed by the ammonium transport system and, therefore, also increased in glucose-treated Chlorella cells.Abbreviation p.c. packed cells     

Lack of adenosine A3 receptors causes defects in mouse peripheral blood parameters

The role of the adenosine A₃ receptor in hematopoiesis was studied using adenosine A₃ receptor knockout (A₃AR KO) mice. Hematological parameters of peripheral blood and femoral bone marrow of irradiated and untreated A₃AR KO mice and their wild-type (WT) counterparts were investigated. Irradiation of the mice served as a defined hematopoiesis-damaging means enabling us to evaluate contingent differences in the pattern of experimentally induced hematopoietic suppression between the A₃AR KO mice and WT mice. Defects were observed in the counts and/or functional parameters of blood cells in the A₃AR KO mice. These defects include statistically significantly lower values of blood neutrophil and monocyte counts, as well as those of mean erythrocyte volume, mean erythrocyte hemoglobin, blood platelet counts, mean platelet volume, and plateletcrit, and can be considered to bear evidence of the lack of a positive role played by the adenosine A₃ receptor in the hematopoietic system. Statistically significantly increased values of the bone marrow parameters studied in A₃AR KO mice (femoral bone marrow cellularity, granulocyte/macrophage progenitor cells, and erythrocyte progenitor cells) can probably be explained by compensatory mechanisms attempting to offset the disorders in the function of blood elements in these mice. The pattern of the radiation-induced hematopoietic suppression was very similar in A₃AR KO mice and their WT counterparts.     

Prolyl oligopeptidase participates in cell cycle progression in a human neuroblastoma cell line

Prolyl oligopeptidase (POP) is a post-proline cleaving enzyme, which is widely distributed in various organs, with high levels in the brain. In this study, we investigated the effects of a selective POP inhibitor, 3-({4-[2-(E)-styrylphenoxy]butanoyl}-l-4-hydroxyprolyl)-thiazolidine (SUAM-14746), on the growth of NB-1 human neuroblastoma cells. SUAM-14746 treatment for 24–72 h suppresses the growth of NB-1 cells without cell death in a dose-dependent manner (10–60 μM). Similar suppressive effects were observed with another POP inhibitor benzyloxycarbonyl-thioprolyl-thioprolinal. The SUAM-14746-induced growth inhibition in NB-1 cells was associated with pronounced G₀/G₁ arrest and reduced levels of phosphorylated retinoblastoma protein (pRb), cyclin E, and cyclin dependent kinase (CDK) 2, and increased levels of the CDK inhibitor p27^kip1 and the tumor suppressor p53. SUAM-14746 also induced transient inhibition of S and G₂/M phase progression, which was correlated with retardation of the decrease in the levels of cyclins A and B. Moreover, RNAi-mediated knockdown of POP also led to inhibition of NB-1 cell growth and the effect was accompanied by G₀/G₁ arrest. These results indicate that POP is a part of the machinery that controls the cell cycle.     

A comparison of the genotoxicity of ethylnitrosourea and ethyl methanesulfonate in lacZ transgenic mice (Muta™Mouse)

We compared the induction of gene mutations and chromosomal aberrations by ethylating agents in lacZ transgenic mice (Muta™Mouse). Chromosomal aberrations were detected by the peripheral blood micronucleus assay. Gene mutations were detected in the lacZ transgene. A small amount of blood was sampled from a tail vessel during the expression time for fixation of gene mutations in vivo; this enabled us to detect and compare clastogenicity and gene mutations in the identical mouse. Single intraperitoneal injections of ENU (50–200 mg/kg) and EMS (100–400 mg/kg) strongly induced micronucleated reticulocytes (MN) detectable in peripheral blood 48 h after treatment. The maximum MN frequencies induced were 6.6% and 3.3% for ENU (100 mg/kg) and EMS (400 mg/kg), respectively (the control value was 0.3%). lacZ mutant frequency (MF) was analyzed in bone marrow and liver 7 days after treatment. Spontaneous MFs were 2.0–4.6x10⁻⁶. MF in bone marrow was increased by ENU to 3.4x10⁻⁵ at 200 mg/kg and induced by EMS to 1.8x10⁻⁵ at 400 mg/kg. In liver, however, both chemicals at their highest doses induced only slight increases in MF. The induction of both micronuclei and lacZ mutations in bone marrow by both ENU and EMS correlated better with O⁶-ethylguanine adducts than with N7-ethylguanine adducts. The mutants (19 for ENU and 12 for EMS) were subjected to DNA sequence analysis. Among EMS-induced mutations, 75% were GC to AT transitions, which were probably caused by O⁶-ethylguanine. Among ENU-induced mutations, in contrast, 40% occurred as AT base pair substitutions (6 AT to TA transversions and 2 AT to GC transitions) (no such mutations were induced by EMS). These results, together with the known reactivity of ENU to thymine suggest that thymine adducts play a significant role in the ENU mutagenesis.     

Intact rat superior mesenteric artery endothelium is an electrical syncytium and expresses strong inward rectifier K+ conductance

Background and purpose

Vascular endothelial and smooth muscle cell phenotypes may change dramatically after isolation and in cell cultures. This study was designed to investigate gap junctions coupling in an integrated intact preparation and to test if K_IR channels modulate resting membrane conductance in “in situ” endothelial cells (EC), and acetylcholine (ACh)-evoked relaxation of the rat superior mesenteric artery.

Experimental approach

Whole cell blind patch recordings of ionic currents from in situ EC, dye-coupling experiments, and functional studies were performed in rat superior mesenteric artery.

Key results

EC were dye-coupled through gap junctions. 18β-glycyrretinic acid (25 μM) decreased outward and inward currents, the 80% decay of time and time constant of the capacitative transients, capacitance, and increased input resistance. Barium chloride (30 μM) decreased resting and ACh-evoked inward currents, the sensitivity of ACh-evoked relaxation, and decreased both the sensitivity and the maximal relaxation to S-nitroso-N-acetyl penicillamine in arteries with, but not in arteries without endothelium.

Conclusions

The present results suggest that the EC layer of this large artery is electrically coupled, and that K_IR channels regulate resting inward conductance, hence suggesting that they are of importance for resting membrane potential in in situ EC. Moreover, EC K_IR channels are involved in ACh-evoked relaxation.     

Effect of molecular hydrogen and carbon dioxide on chemo-organotrophic growth of Acetobacterium woodii and Clostridium aceticum

During growth of Acetobacterium woodii on fructose, glucose or lactate in a medium containing less than 0.04% bicarbonate, molecular hydrogen was evolved up to 0.1 mol per mol of substrate. Under an H₂-atmosphere growth of A. woodii with organic substrates was completely inhibited whereas under an H₂/CO₂-atmosphere rapid growth occurred. Under these conditions H₂+CO₂ and the organic substrate were utilized simultaneously indicating that A. woodii was able to grow mixotrophically. Clostridium aceticum differed from A. woodii in that H₂ was only evolved in the stationary phase, that the inhibition by H₂ was observed at pH 8.5 but not at pH 7.5, anf that in the presence of fructose and H₂+CO₂ only fructose was utilized.The hydrogenase activity of fructose-grown cells of C. aceticum amounted to only 12% of that of H₂+CO₂-grown cells. With A. woodii a corresponding decrease of the activity of this enzyme was not observed.