Effects of leptin to cultured growth plate chondrocytes

OBJECTIVE: This study aimed to evaluate whether leptin has any effect on growth plate chondrocytes. METHODS: We studied the effects of exogenous leptin on cultured rabbit growth plate chondrocytes. This involved assessing [3H]thymidine incorporation, alkaline phosphatase (ALP) activity, proteoglycan production, leptin receptor (Ob-R) activity, and detection of Ob-R using Western blot analysis. RESULTS: The existence of Ob-R in growth plate chondrocytes was revealed by Western blot and Ob-R activity. Prior to semiconfluence, leptin increased [3H]thymidine incorporation while at the semiconfluent and early confluent stages, leptin promoted ALP activity and tended to promote proteoglycan production. CONCLUSION: Growth plate chondrocytes possess Ob-Rs, and leptin enhance chondrocyte proliferation and subsequent cell differentiation.     

Growth of normal oral keratinocytes and squamous cell carcinoma cells in a novel protein-free defined medium

Summary A novel protein-free synthetic medium was developed for the culture of normal human oral keratinocytes. This medium, designated PFM-7, supports the serial cultivation of primary or secondary normal oral keratinocytes in protein-free, chemically defined conditions. Normal oral keratinocytes in PFM-7 exhibited nearly equal growth in mass culture without noticeable changes in morphology, response to added growth factors, or gene expression of growth factors and their receptors, compared to cells in Keratinocyte-SFM containing epidermal growth factor and bovine pituitary extract. Furthermore, PFM-7 supported the serial subcultivation of human squamous cell carcinoma cells and enabled both normal and malignant oral squamous cells derived from the same patient to grow under the same protein-free defined conditions. These results indicate that PFM-7 can be used for precise investigations of growth mechanisms, cell products, and gene expression associated with carcinogenesis of human epidermal cells.     

Acute and Temporal Expression of Tumor Necrosis Factor (TNF)-α-stimulated Gene 6 Product,TSG6, in Mesenchymal Stem Cells Creates Microenvironments Required for Their Successful Transplantation into Muscle Tissue

Previously, we demonstrated that when mesenchymal stem cells (MSCs) from mouse ES cells were transplanted into skeletal muscle, more than 60% of them differentiated into muscles in the crush-injured tibialis anterior muscle in vivo, although MSCs neither differentiated nor settled in the intact muscle. Microenvironments, including the extracellular matrix between the injured and intact muscle, were quite different. In the injured muscle, hyaluronan (HA), heavy chains of inter-α-inhibitor (IαI), CD44, and TNF-α-stimulated gene 6 product (TSG-6) increased 24–48 h after injury, although basement membrane components of differentiated muscle such as perlecan, laminin, and type IV collagen increased gradually 4 days after the crush. We then investigated the microenvironments crucial for cell transplantation, using the lysate of C2C12 myotubules for mimicking injured circumstances in vivo. MSCs settled in the intact muscle when they were transplanted together with the C2C12 lysate or TSG6. MSCs produced and released TSG6 when they were cultured with C2C12 lysates in vitro. MSCs pretreated with the lysate also settled in the intact muscle. Furthermore, MSCs whose TSG6 was knocked down by shRNA, even if transplanted or pretreated with the lysate, could not settle in the muscle. Immunofluorescent staining showed that HA and IαI always co-localized or were distributed closely, suggesting formation of covalent complexes, i.e. the SHAP-HA complex in the presence of TSG6. Thus, TSG6, HA, and IαI were crucial factors for the settlement and probably the subsequent differentiation of MSCs.     

Taxonomy and phylogeny of <Emphasis Type="Italic">Cryptocoryneum</Emphasis> (Pleosporales,Dothideomycetes)

Species of Cryptocoryneum were taxonomically reassessed on the basis of morphological observations and the results of molecular phylogenetic analysis. Eighteen isolates of Cryptocoryneum species, namely two strains from Africa, three from Europe, and 13 from Japan, were phylogenetically analysed using sequences of nuclear rDNA internal transcribed spacers (ITS) and the partial sequence of the translation elongation factor 1α gene (TEF1). The phylogenetic analysis indicated that Cryptocoryneum species formed a monophyletic clade and were closely related to Lophiotrema (Lophiotremataceae) and Aquasubmersa (incertae sedis) in the Pleosporales (Dothideomycetes). We examined holotype specimens of C. fasciculatum, C. hysterioides, and Torula uniformis and concluded that these species are conspecific, with C. hysterioides having priority. Although C. hysterioides has long been regarded as a synonym of C. condensatum, we consider C. hysterioides to be a distinct species within the genus. We found several cryptic species that were morphologically similar to C. condensatum sensu lato, but that could be separated on the basis of conidial size and the number of conidial arms and conidial septa, characters that seem to be informative for species delimitation within Cryptocoryneum. A total of seven new species, namely C. akitaense, C. brevicondensatum, C. congregatum, C. japonicum, C. longicondensatum, C. paracondensatum, and C. pseudorilstonei, are described and illustrated. A key to species accepted in Cryptocoryneum is provided.     

Orally active factor Xa inhibitor: synthesis and biological activity of masked amidines as prodrugs of novel 1,4-diazepane derivatives

Factor Xa (fXa) is a serine protease, which plays a pivotal role in the coagulation cascade. To improve the oral anticoagulant activity of fXa inhibitors containing a 1,4-diazepane moiety as the P4 part, a prodrug strategy was examined. Among the compounds evaluated in this study, amidoxime prodrugs bearing an ester moiety, such as compounds 21 and 30, showed effective oral anticoagulant activity in mice.     

Three DNA polymerases of rat ascites hepatoma cells: properties of the enzymes and effect of RNA synthesis on the reactions.

Three different DNA polymerases have been isolated from rat ascites hepatoma cells [1--3]. The molecular weight of a DNA polymerase (polymerase C) purified from the soluble fraction of the cells was estimated to be 142 000 by sedimentation on a sucrose gradient, while the molecular weights of two DNA polymerases (polymerase P-1 and P-2) purified from nuclear membrane-chromatin fraction were estimated to be 117 000 and 44 000, respectively, by the same method. Under certain conditions, the poly (dT) strand of poly[(dA)-(dT)] was copied well by the polymerases, especially by the nuclear polymerases. Poly (dC) was a good template for the high molecular weight DNA polymerases C and P-1, but poly(dT) and poly(dA) were not effective templates. By addition of complementary oligoribonucleotides, the single-stranded deoxypolymers were copied by the high molecular weight polymerases C and P-1. When single-stranded fd phage DNA was used as template, the polymerization reactions by the high molecular weight polymerases were stimulated by the concomitant synthesis of RNA. This indicates that the oligoribonucleotide acts as a primer in these reactions.     

Visualisation of γH2AX Foci Caused by Heavy Ion Particle Traversal; Distinction between Core Track versus Non-Track Damage

Heavy particle irradiation produces complex DNA double strand breaks (DSBs) which can arise from primary ionisation events within the particle trajectory. Additionally, secondary electrons, termed delta-electrons, which have a range of distributions can create low linear energy transfer (LET) damage within but also distant from the track. DNA damage by delta-electrons distant from the track has not previously been carefully characterised. Using imaging with deconvolution, we show that at 8 hours after exposure to Fe (∼200 keV/µm) ions, γH2AX foci forming at DSBs within the particle track are large and encompass multiple smaller and closely localised foci, which we designate as clustered γH2AX foci. These foci are repaired with slow kinetics by DNA non-homologous end-joining (NHEJ) in G1 phase with the magnitude of complexity diminishing with time. These clustered foci (containing 10 or more individual foci) represent a signature of DSBs caused by high LET heavy particle radiation. We also identified simple γH2AX foci distant from the track, which resemble those arising after X-ray exposure, which we attribute to low LET delta-electron induced DSBs. They are rapidly repaired by NHEJ. Clustered γH2AX foci induced by heavy particle radiation cause prolonged checkpoint arrest compared to simple γH2AX foci following X-irradiation. However, mitotic entry was observed when ∼10 clustered foci remain. Thus, cells can progress into mitosis with multiple clusters of DSBs following the traversal of a heavy particle.     

Molecular phylogenetic assessment of the genus Hyphodiscus with description of Hyphodiscus hyaloscyphoides sp. nov.

Hyphodiscus hyaloscyphoides sp. nov. and its Catenulifera anamorph are described and illustrated. The teleomorph is morphologically most similar to H. hymeniophila but distinguished by dull white-colored apothecia. The anamorph is characterized by its minute conidiogenous structure with small subglobose conidia. The monophyly of Hyphodiscus was strongly supported in our molecular phylogenetic analyses using the D1–D2 region of large subunit of nuclear rDNA, but the phylogenetic relationships with other members of Hyaloscyphaceae were not strongly supported. In addition, Hyphodiscus hymeniophilus, one of the close relatives of H. hyaloscyphoides, turned out to be a heterogeneous assemblage based on the ITS region, which requires further research for taxonomic revision.     

Crystallization and preliminary diffraction studies of recombinant human granulocyte-stimulating factor (KW2228).

Human granulocyte colony-stimulating factor (hG-CSF) specifically stimulates proliferation of neutrophils. Two crystal forms of a mutant of hG-CSF expressed in Escherichia coli have been obtained using the hanging drop vapour diffusion method. One form is triclinic, space group P1, with cell dimensions a = 37.3 A, b = 46.4 A, c = 47.7 A, alpha = 105.5 degrees, beta = 98.0 degrees and gamma = 109.4 degrees. The other is monoclinic, space group C2, with cell dimensions a = 82.0 A, b = 49.2 A, c = 49.4 A and beta = 113.9 degrees. Both crystal forms diffract beyond 2.0 A and are suitable for X-ray analysis.     

Increased renin release by exogenous prostaglandin E1 in liver cirrhosis with and without ascites

To evaluate the sensitivity of the renin-angiotensin-aldosterone system in patients with liver cirrhosis, prostaglandin E1 was intravenously administered at the rate of 50 micrograms/hour for two hours to the 11 control subjects and 11 patients with liver cirrhosis (6 compensated and 5 decompensated). Basal plasma renin activity (PRA) in decompensated patients was significantly higher than those in control and compensated cirrhotics (P less than 0.01). Basal plasma aldosterone was also higher in decompensated than in control and compensated patients, but without significance. PGE1 had no virtual effect on PRA in control, but stimulated PRA in liver cirrhotics, in which statistical significance was only observed in decompensated (basal vs. one hour after PGE1: 2.4 +/- 0.9 ng/ml/min (mean +/- SE) vs. 6.9 +/- 2.1: P less than 0.025). The rate of renin release was significantly higher in compensated than in decompensated (327 +/- 50% vs. 143 +/- 26: P less than 0.05). Though PGE1 also increased plasma aldosterone in liver cirrhotics, statistical change was not seen. Fractional excretion of urinary sodium after PGE1 increased significantly in control (P less than 0.025), but not in liver cirrhotics. These results indicate that the renin-angiotensin-aldosterone system is easily activated by PGE1 in patients with liver cirrhosis and further suggest that the sensitivity of this system in compensated is more augmented than in decompensated patients.