Differential expression of thrombospondin,collagen, and thyroglobulin by thyroid-stimulating hormone and tumor-promoting phorbol ester in cultured porcine thyroid cells

In the present study, we have investigated the potential regulation of thyroglobulin (Tg) and extracellular matrix components synthesis by thyroid-stimulating hormone (TSH) and tetradecanoyl phorbol-13-acetate (TPA) on thyroid cells. Porcine thyroid cells isolated by trypsin-EGTA digestion of thyroid glands were maintained in serum containing medium on poly (L-lysine)-coated dishes. Cells differentiated into follicular or vesicular-like structures were distinguished by their ability to organify Na[¹²⁵l] and to respond to TSH stimulation. After an incubation of the cells with radiolabeled proline or methionine, two major proteins were identified, p450–480 and p290 (so named because of their molecular masses). Tg (p290) synthesis was demonstrated by the synthesis of [¹³¹l]-labeled polypeptides with electrophoretic properties identical to those of authentic Tg molecules. P450–480 resolved to Mr 190,000 under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) conditions. It was identified as thrombospondin by its reactivity with a monoclonal anti-human thrombospondin and by peptide sequencing of some of its tryptic fragments that displayed identity to thrombospondin l. Collagen synthesis was demonstrated by the formation of radioactive hydroxyproline and by the synthesis of pepsin-resistant polypeptides ranging from Mrs 120,000 to 200,000. When the cells were cultured in the presence of 100 nM TPA, the culture medium contents of thrombospondin and collagen were increased by 2.7 and 1.6-fold, respectively, whereas Tg content was decreased by a factor 3.9. In contrast, the acute treatment of control cells with TPA induced a decrease in both Tg and collagen content by factors 3.0 and 1.5, respectively, and an increase in thrombospondin content by a factor 2.5. In the presence of 100 nM TPA, TSH (1 mU/ml) did not counteract the stimulating effect of TPA on extracellular matrix components synthesis. In contrast, when cells were cultured in the presence of TSH alone at concentrations higher than 0.1 mU/ml, collagen and thrombospondin in the medium were decreased by a factor 2.0 and 1.9, respectively, and TSH preferentially activated Tg synthesis. However, no acute response to TSH was observed in cells incubated for 2 days without effectors (control cells). On TSH differentiated cells, TPA decreased both collagen and Tg accumulation by factor 1.2 and 1.8, respectively, whereas it increased the one of thrombospondin by a factor 2. These results, together with the stimulating effect of TPA on TSH mediated cell proliferation, argue for a role of thrombospondin in cell adhesion and migration events within the thyroid epithelium. © 1994 Wiley-Liss, Inc.     

Pharmacokinetics of Repeated Sodium Salicylate Administration to Laying Hens: Evidence for Time Dependent Increase in Drug Elimination from Plasma and Eggs

Salicylates were the first non-steroid anti-inflammatory drugs (NSAIDs) to be used in any species and are still widely used in humans and livestock. However, the data on their pharmacokinetics in animals is limited, especially after repeated administration. Evidence exist that in chickens (Gallus gallus) salicylate (SA) may induce its own elimination. The aim of this study was to investigate salicylate pharmacokinetics and egg residues during repeated administration of sodium salicylate (SS) to laying hens. Pharmacokinetics of SA was assessed during 14 d oral administration of SS at daily doses of 50 mg/kg and 200 mg/kg body weight to laying hens. On the 1^st, 7^th and 14^th d a 24 h-long pharmacokinetic study was carried out, whereas eggs were collected daily. Salicylate concentrations in plasma and eggs were determined using high-performance liquid chromatography with ultraviolet detection and pharmacokinetic variables were calculated using a non-compartmental model. Mean residence time (MRT), minimal plasma concentration (C_min, C_16h) and elimination half-life (T_1/2el) of SA showed gradual decrease in layers administered with a lower dose. Total body clearance (Cl_B) increased. Layers administered with the higher dose showed a decrease only in the T_1/2el. In the low dose group, SA was found only in the egg white and was low throughout the experiment. Egg whites from the higher dose group showed initially high SA levels which significantly decreased during the experiment. Yolk SA levels were lower and showed longer periods of accumulation and elimination. Repeated administration of SS induces SA elimination, although this effect may differ depending on the dose and production type of a chicken. Decreased plasma drug concentration may have clinical implications during prolonged SS treatment.     

The Nematode Eukaryotic Translation Initiation Factor 4E/G Complex Works with a trans-Spliced Leader Stem-Loop To Enable Efficient Translation of Trimethylguanosine-Capped RNAs

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5′ end through the eIF4F initiation complex binding to the 5′ m⁷G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5′ end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m⁷G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5′ untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.Cap-dependent translation initiation in eukaryotes is a complex process involving many factors and serves as the primary mechanism for eukaryotic translation (37, 44). The first step in the initiation process, recruitment of the m⁷G (7-methylguanosine)-capped mRNA to the ribosome, is widely considered the rate-limiting step. It begins with recognition of and binding to the m⁷G cap at the 5′ end of the mRNA by the eukaryotic translation initiation factor 4F (eIF4F) complex, which contains three proteins: eIF4E (a cap-binding protein), eIF4G (a scaffold protein with RNA binding sites), and eIF4A (an RNA helicase). eIF4G''s interaction with eIF3, itself a multisubunit complex that interacts with the 40S ribosome, facilitates the actual recruitment of capped RNA to the ribosome. With the help of several other initiation factors, the small ribosomal subunit scans the mRNA from 5′ to 3′ until a translation initiation codon (AUG) in appropriate context is identified and an 80S ribosomal complex is formed, after which the first peptide bond is formed, thus ending the initiation process (37, 44). The AUG context can play an important role in the efficiency of translation initiation (23, 44). The length, structure, and presence of AUGs or open reading frames in the mRNA 5′ untranslated region (UTR) can negatively affect cap-dependent translation and ribosomal scanning. In general, long and highly structured 5′ UTRs, as well as upstream AUGs leading to short open reading frames, can impede ribosome scanning and lead to reduced translation (23, 44). In addition, 5′ UTRs less than 10 nucleotides (nt) in length are thought to be too short to enable preinitiation complex assembly and scanning (24). Thus, several attributes of the mRNA 5′ UTR are known to negatively affect translation initiation, whereas only the AUG context and the absence of negative elements are known to have a positive effect on translation initiation (44).Two of the important mRNA features associated with cap-dependent translation, the cap and the 5′ UTR, are significantly altered by an RNA processing event known as spliced leader (SL) trans splicing (3, 8, 17, 26, 36, 47). This takes place in members of a diverse group of eukaryotic organisms, including some protozoa, sponges, cnidarians, chaetognaths, flatworms, nematodes, rotifers, crustaceans, and tunicates (17, 28, 39, 55, 56). In SL trans splicing, a separately transcribed small exon (16 to 51 nucleotides [nt]) with its own cap gets added to the 5′ end of pre-mRNAs. This produces mature mRNAs with a unique cap and a conserved sequence in the 5′ UTR. In metazoa, the m⁷G cap is replaced with a trimethylguanosine (TMG) cap (m^2,2,7GpppN) (27, 30, 46, 49). In nematodes, ∼70% of all mRNAs are trans spliced and therefore have a TMG cap and an SL (2). In general, eukaryotic eIF4E proteins do not effectively recognize the TMG cap (35). This raises the issues of how the translation machinery in trans-splicing metazoa effectively recognizes TMG-capped trans-spliced mRNAs, what role the SL sequence plays in translation initiation, and how the conserved translation initiation machinery has adapted to effectively translate trans-spliced mRNAs.Previous work has shown that efficient translation of TMG-capped messages in nematodes requires the SL sequence (22 nt) immediately downstream of the cap (5, 25, 29). In the current studies, we sought to understand the manner in which the SL enhanced the translation of TMG-capped mRNAs. Using a cell-free nematode in vitro translation system, we carried out mutational analyses that define the specific sequences in the SL that are required and sufficient for efficient translation of TMG-capped mRNAs. These analyses led to the discovery of a small, discrete stem-loop immediately adjacent to the TMG cap in trans-spliced messages required for efficient translation. Notably, the sequences involved in the base pairing of the stem are highly conserved in alternative SL sequences found in nematodes. We further show that the nematode eIF4E/G complex plays a major role in facilitating the SL enhancement of TMG-capped mRNA that likely occurs after the initial cap-binding step. The results demonstrate the importance of specific enhancing elements in the 5′ UTR and adaptation in the eIF4F complex necessary for optimal cap-dependent translation.     

Purification of extracellular laccase from Cerrena unicolor

Cerrena unicolor was found to produce large amounts of extracellular laccase when grown aerobically on the optimized Lindenberg and Holm medium in fermenter culture with an automatic pH control. The laccase from this source was purified to homogeneity by a rapid procedure, using ion-exchange chromatography, affinity chromatography, and chromatofocusing. The enzymes isoforms were recovered with a 65- to 92-fold increase in specific activity and a yield for Ia1?=?6.7%; Ia2?=?27.5%; Ib?=?9.7%; and IIa1?=?21%. The molecular mass of the purified enzymes proved to be 45, 47, 54, and 62?kD, respectively, as determined by size-exclusion high-performance liquid chromatography (HPLC). The isoelectric points were in the range of 4.7 to 4.2, and the carbohydrate content in the purified enzymes was between 1.6 and 3.5%.     

A novel pattern of follicular epithelium morphogenesis in higher dipterans

In fly ovaries, the follicular epithelium surrounding germline cells diversifies into several morphologically distinct cell subpopulations. This complex process is crucial for the formation of a regionally complex eggshell and establishment of polarity of the future embryo. Morphogenetic changes accompanying patterning of the follicular epithelium have been best characterized in the model fly, Drosophila melanogaster. Here, we analyze follicular epithelium diversification in the ovaries of Tachypeza nubila, a brachyceran fly closely related to the group Cyclorrhapha, which also includes Drosophila. We provide morphological evidence that in Tachypeza, the diversification process differs from that described in the Drosophila model system in several important respects: (i) follicle cells differentiate into five subpopulations (versus eight in Drosophila); (ii) only one of these subpopulations (i.e. border cells) is migratory (versus four in Drosophila); (iii) the main body follicle cells form a uniform epithelium with no distinct border between follicle cells covering the nurse cell compartment and the oocyte; (iv) chorionic material is deposited not only on the surface of the oocyte but also on the nurse cells; (v) there is no centripetal migration of the follicle cells; (vi) the resulting eggshell is morphologically simple with no regional specializations except for the micropylar apparatus at the anterior pole of the oocyte. Our findings provide novel insights into the evolution of the follicle cell patterning and functioning in dipterans. A critical analysis of these processes in different dipteran groups strongly indicates that in Tachypeza, follicular epithelium diversification follows a distinct pattern, novel for higher dipterans.     

Trends in solventless sample preparation techniques for environmental analysis

The paper presents recent trends in solventless sample preparation techniques for environmental analysis. First, a general classification of solventless methods is given. Next, three of them, treated as preferable techniques, i.e. SPME, SDME and HS, are presented in detail, with respect to their usability and effectiveness for environmental samples. Examples of all discussed techniques are given in the tables.     

Protective action of melatonin against oxidative DNA damage: chemical inactivation versus base-excision repair

Melatonin is a hormone-like substance that has a variety of beneficial properties as regulator of the circadian rhythm and as anti-inflammatory and anti-cancer agent. The latter activity can be linked with the ability of melatonin to protect DNA against oxidative damage. It may exert such action either by scavenging reactive oxygen species or their primary sources, or by stimulating the repair of oxidative damage in DNA. Since such type of DNA damage is reflected in oxidative base modifications that are primarily repaired by base-excision repair (BER), we tried to investigate in the present work whether melatonin could influence this DNA-repair system. We also investigated the ability of melatonin to inactivate hydrogen peroxide, a potent source of reactive oxygen species. Melatonin at 50 microM and its direct metabolite N(1)-acetyl-N(2)-formyl-5-methoxykynuramine reduced DNA damage induced by hydrogen peroxide at approximately the same ratio. Melatonin stimulated the repair of DNA damage induced by hydrogen peroxide, as assessed by the alkaline comet assay. However, melatonin at 50 microM had no impact on the activity in vitro of three glycosylases playing a pivotal role in BER: Endo III, Fpg and ANPG 80. On the other hand, melatonin chemically inactivated hydrogen peroxide, reducing its potential to damage DNA. And finally, melatonin did not influence the repair of an a-basic (AP) site by cellular extracts, as was evaluated by a functional BER assay in vitro. In conclusion, melatonin can have a protective effect against oxidative DNA damage by chemical inactivation of a DNA-damaging agent as well as by stimulating DNA repair, but key factors in BER, viz. glycosylases and AP-endonucleases, do not seem to be affected by melatonin. Further study with other components of the BER machinery and studies aimed at other DNA-repair systems are needed to clarify the mechanism underlying the stimulation of DNA repair by melatonin.     

The 2'-O-ribose methyltransferase for cap 1 of spliced leader RNA and U1 small nuclear RNA in Trypanosoma brucei

mRNA cap 1 2'-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2'-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2'-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2'-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3'-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.     

Hoc protein regulates the biological effects of T4 phage in mammals

We previously investigated the biological, non-antibacterial effects of bacteriophage T4 in mammals (binding to cancer cells in vitro and attenuating tumour growth and metastases in vivo); we selected the phage mutant HAP1 that was significantly more effective than T4. In this study we describe a non-sense mutation in the hoc gene that differentiates bacteriophage HAP1 and its parental strain T4. We found no substantial effects of the mutation on the mutant morphology, and its effects on electrophoretic mobility and hydrodynamic size were moderate. Only the high ionic strength of the environment resulted in a size difference of about 10 nm between T4 and HAP1. We compared the antimetastatic activity of the T2 phage, which does not express protein Hoc, with those of T4 and HAP1 (B16 melanoma lung colonies). We found that HAP1 and T2 decreased metastases with equal effect, more strongly than did T4. We also investigated concentrations of T4 and HAP1 in the murine blood, tumour (B16), spleen, liver, or muscle. We found that HAP1 was rapidly cleared from the organism, most probably by the liver. Although HAP1 was previously defined to bind cancer cells more effectively (than T4), its rapid elimination precluded its higher concentration in tumours. Maria Zembala and Janusz Boratynski contributed equally to this work.     

Conserved amino acids in each subunit of the heteroligomeric tRNA m1A58 Mtase from Saccharomyces cerevisiae contribute to tRNA binding

In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNA(Met)i The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-L-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind tRNA(Met)i, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present.