Inhibition of RNA synthesis in yeast protoplasts by a peptide factor from Tetrahymena cells

Protoplasts of Schizosaccharomyces pombe, grown on a rich nutrient medium, were treated with a peptide factor isolated from cultures of the protozoan Tetrahymena pyriformis. The peptide factor is known to inhibit RNA synthesis in Tetrahymena. It has now been shown that the peptide factor also inhibits RNA synthesis in yeast protoplasts without affecting protein synthesis.     

Volume regulation in human fibroblasts: role of Ca2+ and 5-lipoxygenase products in the activation of the Cl− efflux

Trypsinized human skin fibroblasts in suspension perform regulatory volume decrease (RVD) after cell swelling in hypotonic medium. During RVD, ³⁶Cl^– efflux is dramatically increased and the cell membrane is depolarized, indicating the activation of Cl^– channels. This activation of Cl^– channels depends on extracellular as well as on intracellular Ca²⁺. The swelling-induced Cl^– efflux and the RVD response are inhibited by the 5-lipoxygenase inhibitor ETH 615-139. Finally, following hypotonic treatment, cellular pH decreases. The pH decrease does not involve the Cl^–/HCO ₃ ^– exchange because it is independent of the external Cl^– concentration.T. Mastrocola was recipient of a scientific fellowship from the Italian Consiglio Nazionale delle Ricerche (C.N.R.). This work was supported by Progetto Finalizzato Ingegneria Genetica, C.N.R., Roma, and by the Danish Natural Research Council.     

Expression levels of the metalloproteinase ADAM8 critically regulate proliferation,migration and malignant signalling events in hepatoma cells

Hepatocellular carcinoma (HCC) is one of the most common metastatic tumours. Tumour growth and metastasis depend on the induction of cell proliferation and migration by various mediators. Here, we report that the A Disintegrin and Metalloproteinase (ADAM) 8 is highly expressed in murine HCC tissues as well as in murine and human hepatoma cell lines Hepa1-6 and HepG2, respectively. To establish a dose-dependent role of different ADAM8 expression levels for HCC progression, ADAM8 expression was either reduced via shRNA- or siRNA-mediated knockdown or increased by using a retroviral overexpression vector. These two complementary approaches revealed that ADAM8 expression levels correlated positively with proliferation, clonogenicity, migration and matrix invasion and negatively with apoptosis of hepatoma cells. Furthermore, the analysis of pro-migratory and proliferative signalling pathways revealed that ADAM8 expression level was positively associated with expression of β1 integrin as well as with the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), Src kinase and Rho A GTPase. Finally, up-regulation of promigatory signalling and cell migration was also seen with a proteolytically inactive ADAM8 mutant. These findings reveal that ADAM8 is critically up-regulated in hepatoma cells contributes to cell proliferation and survival and furthermore induces pro-migratory signalling pathways independently of its proteolytic activity. By this, ADAM8 can promote cell functions most relevant for HCC growth and metastasis.     

The 'pair of sugar tongs' site on the non-catalytic domain C of barley alpha-amylase participates in substrate binding and activity

Some starch-degrading enzymes accommodate carbohydrates at sites situated at a certain distance from the active site. In the crystal structure of barley alpha-amylase 1, oligosaccharide is thus bound to the 'sugar tongs' site. This site on the non-catalytic domain C in the C-terminal part of the molecule contains a key residue, Tyr380, which has numerous contacts with the oligosaccharide. The mutant enzymes Y380A and Y380M failed to bind to beta-cyclodextrin-Sepharose, a starch-mimic resin used for alpha-amylase affinity purification. The K(d) for beta-cyclodextrin binding to Y380A and Y380M was 1.4 mm compared to 0.20-0.25 mm for the wild-type, S378P and S378T enzymes. The substitution in the S378P enzyme mimics Pro376 in the barley alpha-amylase 2 isozyme, which in spite of its conserved Tyr378 did not bind oligosaccharide at the 'sugar tongs' in the structure. Crystal structures of both wild-type and S378P enzymes, but not the Y380A enzyme, showed binding of the pseudotetrasaccharide acarbose at the 'sugar tongs' site. The 'sugar tongs' site also contributed importantly to the adsorption to starch granules, as Kd = 0.47 mg.mL(-1) for the wild-type enzyme increased to 5.9 mg.mL(-1) for Y380A, which moreover catalyzed the release of soluble oligosaccharides from starch granules with only 10% of the wild-type activity. beta-cyclodextrin both inhibited binding to and suppressed activity on starch granules for wild-type and S378P enzymes, but did not affect these properties of Y380A, reflecting the functional role of Tyr380. In addition, the Y380A enzyme hydrolyzed amylose with reduced multiple attack, emphasizing that the 'sugar tongs' participates in multivalent binding of polysaccharide substrates.     

Efficiency considerations for the purely tapered interference fit (TIF) abutments used in dental implants

A tapered interference fit provides a mechanically reliable retention mechanism for the implant-abutment interface in a dental implant. Understanding the mechanical properties of the tapered interface with or without a screw at the bottom has been the subject of a considerable amount of studies involving experiments and finite element (FE) analysis. In this paper, approximate closed-form formulas are developed to analyze the mechanics of a tapered interference fit. In particular, the insertion force, the efficiency, defined as the ratio of the pull-out force to insertion force, and the critical insertion depth, which causes the onset of plastic deformation, are analyzed. It is shown that the insertion force is a function of the taper angle, the contact length, the inner and outer radii of the implant, the static and the kinetic coefficients of friction, and the elastic modulii of the implant/abutment materials. The efficiency of the tapered interference fit, which is defined as the ratio of the pull-out force to insertion force, is found to be greater than one, for taper angles that are less than 6 deg when the friction coefficient is 0.3. A safe range of insertion forces has been shown to exist. The lower end of this range depends on the maximum pull-out force that may occur due to occlusion in the multiple tooth restorations and the efficiency of the system; and the upper end of this range depends on the plastic deformation of the abutment and the implant due to interference fit. It has been shown that using a small taper angle and a long contact length widens the safe range of insertion forces.     

Satellite cell proliferation in low frequency-stimulated fast muscle of hypothyroid rat

Satellite cell proliferation was assessed inlow-frequency-stimulated hypothyroid rat fast-twitch muscle by5-bromo-2'-deoxyuridine (BrdU) labeling and subsequent staining oflabeled muscle nuclei, and by staining for proliferating cell nuclearantigen (PCNA). BrdU labeling and PCNA staining were highly correlatedand increased approximately fourfold at 5 days of stimulation, decayedthereafter, but remained elevated over control in 10- and 20-daystimulated muscles. Myogenin mRNA was ~4-fold elevated at 5 days and1.5-fold at 10 days. Staining for myogenin protein yielded resultssimilar to that for PCNA and BrdU. Furthermore, a detailed examination of the pattern of myogenin staining revealed that the number of myogenin-positive nuclei was elevated in the fast pure IIB fiber population at 5 and 10 days of chronic low-frequencystimulation. By 20 days, myogenin staining was observed intransforming fast fibers that coexpressed embryonic and adult myosinheavy chain isoforms. In the slower fiber populations (i.e., IIA andI), myogenin-positive transforming fibers that coexpressed embryonicmyosin heavy chain, appeared already at 5 days. Thus the satellite cellprogeny on slower fibers seemed to proliferate less and to fuse earlierto their associated fibers than the satellite cell progeny on fast fibers. We suggest that the increase in muscle nuclei of the fast fibers might be a prerequisite for fast-to-slow fiber type transitions.

     

Growth and Alkaloid Patterns of Roots of Tabernaemontana pachysiphon and Rauvolfia mombasiana as Influenced by Environmental Factors

The influence of environmental factors on the indole alkaloid content and biomass of the roots of Tabernaemontana pachysiphon and Rauvolfia mombasiana, two species of considerable local medicinal use in tropical East Africa, was investigated. Both species, belonging to the Apocynaceae, are frequent constituents of the residual tropical forests, prefering sites of different ecological conditions. Experimental plants, raised from seeds, were grown for 16 months in a temperature- and humidity-controlled greenhouse. Environmental factors at variance were water and nutrient supply, and light intensity. At sufficient water and nutrient supply, the more drought and nutrient shortage-tolerating heliophilous Rauvolfia mombasiana showed increased alkaloid accumulation, concurrently with reduced root biomass. Under the same conditions, the drought-sensitive and higher levels of nutrient-requiring ombrophilous Tabernaemontana pachysiphon produced more root biomass but accumulated less alkaloids in the roots. The results indicate that the accumulation of indole alkaloids in the roots, as well as biomass allocation to the roots, is influenced in an opposite manner by the nutrient and water supply to the heliophilous and the ombrophilous species.     

Liquid chromatography tandem mass spectrometry method for the quantitation of NTBC (2-(nitro-4-trifluoromethylbenzoyl)1,3-cyclohexanedione) in plasma of tyrosinemia type 1 patients

In this study, we describe a bioanalytical method for quantification of NTBC in plasma of patients with hereditary tyrosinemia type 1 (HT-1) using high-performance liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). After protein precipitation with acetonitrile including Mesotrione as internal standard, separation of NTBC was achieved by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in selected reaction monitoring (SRM) mode. NTBC recovery in the developed method was found to be more than 90%. The lower limit of quantification was calculated to be 0.35 μM. The intra-day and inter-day precision of three different quality control samples (measured as RSD%) was less than 10% and 15%, respectively. The standard calibration curves showed good linearity within the range of 2.5–40 μM and the determined correlation coefficients were r² ≥ 0.995. This presented method is rapid, sensitive, specific and suitable for clinical practice and research.