Inhibition of proliferation and induction of differentiation of osteoblastic cells by a novel 1,25-dihydroxyvitamin D3 analog with an extensively modified side chain (CB1093)

1,25-Dihydroxyvitamin D₃ (1,25D) is involved in the regulation of proliferation and differentiation of a variety of cell types including cancer cells. In recent years, numerous new vitamin D₃ analogs have been developed in order to obtain favorable therapeutic properties. The effects of a new 20-epi analog, CB1093 (20-epi-22-ethoxy-23-yne-24a,26a,27a-trihomo-1α,25(OH)₂D₃), on the proliferation and differentiation of human MG-63 osteosarcoma cell line were compared here with those of the parent compound 1,25D. Proliferation of the MG-63 cells was inhibited similarly by 22%, 50% and 59% after treatment with 0.1 μM 1,25D or CB1093 for 48 h, 96 h, and 144 h, respectively. In transfection experiments, the compounds were equipotent in stimulating reporter gene activity under the control of human osteocalcin gene promoter. In cell culture experiments, however, CB1093 was more potent than 1,25D at low concentrations and more effective for a longer period of time in activating the osteocalcin gene expression at mRNA and protein levels. Also, a 6-h pretreatment and subsequent culture for up to 120 h without 1,25D or CB1093 yielded higher osteocalcin mRNA and protein levels with analog-treated cells than with 1,25D-treated cells. The electrophoretic mobility shift assay (EMSA) revealed stronger VDR-VDRE binding with analog-treated MG-63 cells than with 1,25D-treated cells. The differences in the DNA binding of 1,25D-bound vs. analog-bound VDR, however, largely disappeared when the binding reactions were performed with recombinant hVDR and hRXRβ proteins. These results demonstrate that the new analog CB1093 was equally or even more effective than 1,25D in regulating all human osteosarcoma cell functions ranging from growth inhibition to marker gene expression and that the differences in effectivity most probably resulted from interactions of the hVDR:hRXRβ-complex with additional nuclear proteins. J. Cell. Biochem. 70:414–424, 1998. © 1998 Wiley-Liss, Inc.     

Relationships between range of motion,Lo, and passive force in five strap-like muscles of the feline hind limb

The relationships between range of motion, optimal length for force production (l_o), and passive force provide useful insights into the structure and function of muscles but are unknown for most individual muscles. We measured these values and examined their relationships in five strap-like muscles of the cat hind limb: caudofemoralis, semitendinosus, sartorius anterior, tenuissimus, and biceps femoris anterior. The range of motion relative to l_o was found to vary significantly between different muscles and even between different specimens of the same muscle. The passive force-length (FL) curve was found to be correlated with both l_o and l_max (maximal in situ muscle length) but was correlated more strongly with l_max. The mean passive force produced by these muscles at l_max was less than 7% of estimated maximal isometric force, suggesting that passive force may not be important in these muscles during normal activation patterns. The variance in passive FL curves between specimens of the same muscle was found to be significantly lower when length was scaled by l_max as opposed to l_o. These results suggest that l_max may provide a more useful scaling factor for generic models of muscle. However, the passive length-tension properties of mammalian muscle appear to reflect a complex mix of structures at both the myofilament and connective tissue levels that may differ depending on muscle-fiber architecture and perhaps on the history of trophic influences on a particular specimen. © 1996 Wiley-Liss, Inc.     

Sea ice bacterial growth rate, growth efficiency and preference for inorganic nitrogen sources in the Baltic Sea

Seasonal Baltic Sea ice is structurally similar to polar sea ice and provides habitats for diverse ice organism assemblages that are integral to the biogeochemistry and ecology of the sea during winter. Temperature and inorganic nitrogen sources have been suggested to control bacterial growth, with increasing dependence on ammonium at low temperatures. To study the bacterial growth and preference for the nitrogen source, we conducted experiments at 0 and 4°C, using ammonium and nitrate as nitrogen sources at two coastal fast-ice stations in the Gulf of Finland and in the Gulf of Bothnia during three successive winters. The two study sites differ markedly in relation to the allochthonous dissolved organic matter supply from the catchment area. High levels of bacterial growth were recorded at both study sites, with community generation times of 15–37 h. The measured bacterial growth efficiencies of 20–58% suggest that the Baltic sea ice brines provide a rich medium for bacterial growth and efficient functioning of bacteria-based food webs. Our experiments with sea ice samples showed a preference for ammonium at both temperatures and high potential growth in both types of nitrogen supplies. No major differences in phosphorus depletion rates were found at the two temperatures, but rates were always highest when ammonium was added to the experiments. These experiments point out that ice maturity, presumably through changes in bacterial community structure, impacts nitrogen processes and that these processes are pronounced prior to melting of the ice.     

P marks the spot: site-specific integrin phosphorylation regulates molecular interactions

Integrins are heterodimeric adhesion receptors at the cell membrane that function as two-way signaling devices. The short intracellular tails of integrins are devoid of catalytic activity, but are nevertheless important for adhesion and signaling, presumably, through interactions with cytoplasmic molecules. Recently, the structure of the intracellular tails has been investigated using NMR, giving important new insight into how integrins might be regulated, but many questions remain unanswered. Signaling by many cell-surface receptors involves protein phosphorylation; over the past few years, phosphorylation of the integrin tails at specific sites has started to emerge as a dynamic mechanism that regulates molecular interactions between integrins and cytoplasmic molecules. This phosphorylation might give rise to signaling specificity and fine-tuning of the integrin-mediated responses.     

VP22 does not significantly enhance enzyme prodrug cancer gene therapy as a part of a VP22-HSVTk-GFP triple fusion construct

BACKGROUND: VP22 is a herpes simplex virus type 1 (HSV-1) tegument protein that has been suggested to spread from cell to cell, alone or as a part of fusion proteins. Creating controversy, some reports indicate that VP22 cannot facilitate significant intercellular spreading. To study the capacity of VP22 to cause spreading and enhance thymidine kinase/ganciclovir cancer gene therapy, we constructed a novel triple fusion protein containing VP22, HSV thymidine kinase and green fluorescent protein (VP22-Tk-GFP). This fusion protein has three functional domains in the same polypeptide, thus making it possible to reliably compare the causality between transduction rate and cell killing efficiency in vitro and in vivo. METHODS: VP22-Tk-GFP was cloned into lenti- and adenoviral vectors and used for expression studies, analyses for VP22-mediated protein spreading, and to study the effect of VP22 to thymidine kinase/ganciclovir-mediated cytotoxicity. The function of VP22-Tk-GFP was also investigated in vivo. RESULTS: The triple fusion protein was expressed correctly in vitro, but intercellular trafficking was not observed in any of the studied cell lines. However, under certain conditions, VP22-Tk-GFP sensitized cells more efficiently to ganciclovir than Tk-GFP. In vivo there was a trend for increased inhibition of tumor growth with VP22-Tk-GFP when ganciclovir was present, but the difference with Tk-GFP was not statistically significant. CONCLUSIONS: Based on our results, VP22 fusion proteins do not seem to traffic intercellularly at detectable levels in most tumor cell types. Even though VP22 enhanced cytotoxicity in one cell line in vitro, the effect in vivo was modest. Therefore, our results do not support the utility of VP22 as an enhancer of enzyme prodrug cancer gene therapy.     

Inhibition of the osteoclast V-ATPase by small interfering RNAs

The multisubunit enzyme V-ATPase harbours isoforms of individual subunits. a3 is one of four 116 kDa subunit a isoforms, and it is crucial for bone resorption. We used small interfering RNA (siRNA) molecules to knock down a3 in rat osteoclast cultures. Labeled siRNA-molecules entered osteoclasts via endocytosis and knocked down the a3 mRNA. Bone resorption was decreased in siRNA-treated samples due to decreased acidification and osteoclast inactivation. Expression of a1 did not respond to decreased a3 levels, suggesting that a1 does not compensate for a3 in osteoclast cultures. Subunit a3 is thus an interesting target for novel nucleic acid therapy.     

What have we learnt about microarray analyses of atherogenesis?

PURPOSE OF REVIEW: This review covers recent developments in microarray studies in the field of atherogenesis research. RECENT FINDINGS: During the past year microarrays have been applied to the analysis of pathogenic mechanisms of several atherosclerosis risk factors, including ageing, hypertension, obesity, and cytomegalovirus infection. In addition, gene expression patterns during the development of in-stent restenosis have been examined. Several studies have also explored the pleiotropic effects of statin therapy. As a technical improvement, the combination of laser microdissection with microarrays in human samples has been reported. SUMMARY: Microarray analyses have given important new information about atherogenesis. It is anticipated that microarray studies will significantly contribute to further discoveries in the field.     

Occurrence and molecular characterization of cultivable mesophilic and thermophilic obligate anaerobic bacteria isolated from paper mills

The aim of this work was to characterize the cultivable obligate anaerobic bacterial population in paper mill environments. A total of 177 anaerobically grown bacterial isolates were screened for aerotolerance, from which 67 obligate anaerobes were characterized by automated ribotyping and 41 were further identified by partial 16S rDNA sequencing. The mesophilic isolates indicated 11 different taxa (species) within the genus Clostridium and the thermophilic isolates four taxa within the genus Thermoanaerobacterium and one within Thermoanaerobacter (both formerly Clostridium). The most widespread mesophilic bacterium was closely related to C. magnum and occurred in three of four mills. One mill was contaminated with a novel mesophilic bacterium most closely related to C. thiosulfatireducens. The most common thermophile was T. thermosaccharolyticum, occurring in all four mills. The genetic relationships of the mill isolates to described species indicated that most of them are potential members of new species. On the basis of identical ribotypes clay could be identified to be the contamination source of thermophilic bacteria. Automated ribotyping can be a useful tool for the identification of clostridia as soon as comprehensive identification libraries are available.     

Novel heparan sulfate structures revealed by monoclonal antibodies

The sulfated glycosaminoglycan heparan sulfate (HS) is found ubiquitously on cell surfaces, in the extracellular matrix, and intracellularly as HS proteoglycans. Because of the structural heterogeneity of HS, tissue-derived HS preparations represent a mixture of HS chains originating from different cell types and tissue loci. Monoclonal anti-HS antibodies have been employed to detect the localization of specific HS epitopes in tissues, but limited information has been available on the saccharide structures recognized by the antibodies. We have studied the saccharide epitope structures of four anti-HS antibodies, HepSS1, JM13, JM403, and 10E4, which all recognize distinct HS species as demonstrated by different patterns of immunoreactivity upon staining of embryonic rat and adult human tissues. The epitopes recognized by JM13 and HepSS1 were found almost exclusively in basement membrane HS, whereas JM403 and 10E4 reacted also with cell-associated HS species. The binding of HepSS1, JM403, and 10E4 to HS was dependent on the GlcN N-substitution of the polysaccharide rather than O-sulfation. HepSS1 thus interacted with N-sulfated HS domains, JM403 binding was critically dependent on N-unsubstituted GlcN residues, and 10E4 bound to "mixed" HS domains containing both N-acetylated and N-sulfated disaccharide units. By contrast, JM13 binding seemed to require the presence of 2-O-sulfated glucuronic acid residues.     

The Drosophila hugin gene codes for myostimulatory and ecdysis-modifying neuropeptides

In a genomic screen we isolated the Drosophila gene hugin (hug, cytology 87C1-2) by cross-hybridisation to a human glial cell line-derived neurotrophic factor cDNA. Upon cDNA sequence analysis and in vitro expression assays, the hugin gene was found to encode a signal peptide containing proprotein that was further processed in Schneider-2 cells into peptides similar to known neuropeptides. Two of the peptides were similar to FXPRL-amides (pyrokinins) and to the ecdysis-triggering hormone, respectively. The former displayed myostimulatory activity in a bioassay on the cockroach hyperneural muscle preparation, as well as in the Drosophila heart muscle assay. Hugin is expressed during the later half of embryogenesis and during larval stages in a subgroup of neurosecretory cells of the suboesophageal ganglion. Ubiquitous ectopic hugin expression resulted in larval death predominantly at or shortly after ecdysis from second to third instar, suggesting that at least one of the posttranslational cleavage products affects molting of the larva by interfering with the regulation of ecdysis.