Comparative Analysis of Different Approaches to Investigate Cell Kinetics

Abstract. The potential of different methods to investigate proliferative activity of cell populations was analysed for non-Hodgkin's lymphomas. Cells in S phase and all cycling cells were determined on cell suspensions obtained from fresh lymph node material by [³H]-thymidine autoradiography ([³H]TdR LI), a monoclonal antibody to bromodeoxyuridine (BrdU LI), and the monoclonal antibody Ki67. A good correlation was observed between the values of [³H]TdR LI and BrdU LI ( r _s= 0.90; P < 0.01), [³H]TdR LI and S phase ( r _s= 0.62; P < 0.01) and [³H]TdR LI and Ki67 ( r _s= 0.64; P < 0.01) in individual lymphomas. Using the median values obtained from the different approaches as cut-off points to define slowly and rapidly proliferating tumours, the best agreement was observed between [³H]TdR LI and BrdU LI (91%) and poorer agreements, even though statistically significant, were observed between [³H]TdR LI and S phase (73%) or Ki67 (76%). In conclusion, the kinetic information derived from different approaches was more or less concordant and newly proposed approaches should be directly and carefully verified for their prognostic relevance before using them as alternatives to conventional methods.     

Characterization of novel long-chain 1,2-diols in Thermus species and demonstration that Thermus strains contain both glycerol-linked and diol-linked glycolipids.

In this study, we purified and characterized tetra- and triglycosyl glycolipids (GL-1 and GL-2, respectively) from two different colonial forms of Thermus scotoductus X-1, from T. filiformis Tok4 A2, and from T. oshimai SPS-11. Acid hydrolysis of the purified glycolipids liberated, in addition to the expected long-chain fatty acids, two components which were identified by gas chromatography-mass spectrometry as 16-methylheptadecane-1,2-diol and 15-methylheptadecane-1,2-diol. Fast atom bombardment mass spectrometry of the intact glycolipids indicated that a major proportion consisted of components with glycan head groups linked to long-chain 1,2-diols rather than to glycerol, although in all cases glycerol-linked compounds containing similar glycan head groups were also present. As in other Thermus strains, the polar head group of GL-1 from T. filiformis Tok4 A2 and from T. scotoductus X-1 colony type t2 was a glucosylgalactosyl-(N-acyl)glucosaminylglucosyl moiety. However, GL-2 from T. scotoductus X-1 colony type t1 and from T. oshimai SPS-11 was a truncated analog which lacked the nonreducing terminal glucose. Long-chain 1,2-diols have been previously reported in the polar lipids of Thermomicrobium roseum and (possibly) Chloroflexus aurantiacus, but to our knowledge, this is the first report of their detection in other bacteria and the first account of the structural determination of long-chain diol-linked glycolipids.     

Effect of a moderate intensity exercise training protocol on the metabolism of macrophages and lymphocytes of tumour-bearing rats

It is commonly accepted that moderate intensity exercise is beneficial to the immune system. We tested the influence of a moderate intensity training protocol (8 weeks) upon immune system function in Wistar tumour-bearing (TB) rats. The metabolism of glucose and glutamine in lymphocytes and macrophages was assessed, together with some functional parameters (hydrogen peroxide production and lymphocyte proliferative response). These substrates were chosen since they represent the most important energetic and synthetic metabolites for these cellular types. The training protocol caused a decrease of 17.4 per cent in the production of H(2)O(2) by macrophages, as well as a decrease in glucose consumption (25 per cent) and lactate production (47.1 per cent), and an increase in the production of labelled CO(2) from the oxidation of [U-(14)C]-glucose, in TB rats. The training protocol was also able to induce changes in the maximal activity of some key enzymes in the metabolism of glucose and glutamine, a reduction of hexokinase (68.8 per cent) activity and an increase in the activity of citrate synthase (10.1 per cent) in TB rats. The training protocol increased the proliferative response of lymphocytes cultivated in the absence of mitogens (75 per cent), of those cultivated in the presence of ConA (38.2 per cent) and in the presence of LPS (45.0 per cent). These cells also showed an increase in the maximal activity of some key enzymes of the glycolytic and glutaminolytic pathways. Our data demonstrated that the training protocol was able to induce an increase in aerobic utilisation of both substrates in lymphocytes and macrophages. The training protocol was also able to prevent several changes in glucose and glutamine metabolism that are normally present in sedentary TB rats. These changes in immune cell metabolism induced by the training protocol were able to increase TB rat survival.     

Heat-Resistance and Heat-Shock Response in the Nosocomial Pathogen <Emphasis Type="Italic">Enterococcus faecium</Emphasis>

We have characterized the heat-shock response of the nosocomial pathogen Enterococcus faecium. The growth of E. faecium cells was analyzed at different temperatures; little growth was observed at 50°C, and no growth at 52°C or 55°C. In agreement, a marked decrease of general protein synthesis was observed at 52°C, and very light synthesis was detected at 55°C. The heat resistance of E. faecium cells was analyzed by measuring the survival at temperatures higher than 52°C and, after 2 h of incubation, viable cells were still observed at 70°C. By Western blot analysis, two heat-induced proteins were identified as GroEL (65 kDa) and DnaK (75 kDa). Only one isoform for either GroEL or DnaK was found. The gene expression of these heat-shock proteins was also analyzed by pulsed-labeled experiments. The heat-induced proteins showed an increased rate of synthesis during the first 5 min, reaching the highest level of induction after 10 min and returning to the steady-state level after 20 min of heat treatment. Received: 29 March 2002 / Accepted: 5 July 2002     

Diversity of gene cassettes and the abundance of the class 1 integron-integrase gene in sediment polluted by metals

The integron-gene cassette system has typically been associated with antibiotic-resistant pathogens. However, the diversity of gene cassettes and the abundance of class 1 integrons outside of the clinical context are not fully explored. Primers targeting the conserved segments of attC recombination sites were used to amplify gene cassettes from the sediment of the Mina stream, which exhibited a higher degree of stress to metal pollution in the dry season than the rainy season. Of the 143 total analyzed sequences, 101 had no matches to proteins in the database, where cassette open reading frames could be identified by homology with database entries. There was a predominance of sequences encoding essential cellular functions. Each season that was sampled yielded a specific pool of gene cassettes. Real-time PCR revealed that 8.5 and 41.6 % of bacterial cells potentially harbored a class 1 integron in the rainy and dry seasons, respectively. In summary, our findings demonstrate that most of the gene cassettes have no ascribable function and, apparently, historically metal-contaminated sediment favors the maintenance of bacteria containing the intI1 gene. Thus, the diversity of gene cassettes is far from being fully explored deserving further attention.     

Time-calibrated molecular phylogeny reveals a Miocene–Pliocene diversification in the Amazon miniature killifish genus <Emphasis Type="Italic">Fluviphylax</Emphasis> (Cyprinodontiformes: Cyprinodontoidei)

Species of Fluviphylax are widely distributed over the Amazon and Orinoco river drainages and are among the smallest fish in the neotropics, inhabiting areas near the margin of slow-flowing clear and black water streams and lakes. Here, we present the first multigene molecular phylogeny of Fluviphylax, including all five nominal species of Fluviphylax and three undescribed species. The analysis included fragments of one mitochondrial and five nuclear genes, totaling 5880 bp. The dataset was analyzed using maximum parsimony, maximum likelihood, and Bayesian inference approaches providing high-supported well-solved trees. A time-calibrated analysis was performed providing information on the origin and diversification of the miniature genus in the Amazon. We estimate that Fluviphylax lineage splits from its sister group, the Anablepidae and Poeciliidae (Poeciliinae sensu Parenti, 1981), during the Late Eocene, about 36.6 Mya; but lineage diversification started only in the Middle Miocene, about 16 Mya, during the formation of the Pebas system. Subsequent splits within Fluviphylax occurred in the Late Miocene–Pliocene, between 10 and 6 Mya and during the Pliocene, and were probably influenced by paleogeographical events such as the breaching of the Purus arch, the rise of the Vaupés arch, the uplift of the Fitzcarrald arch, and the capture of the Contigo and Uraricoera river drainages by the Branco River. The present time-calibrated analysis provides the first insight on the evolution of one of the smallest vertebrate taxa in the Amazon and Orinoco river drainages.     

Opioid antinociception and positive reinforcement are mediated by different types of opioid receptors

Fentanyl (FEN) and diprenorphine's (DIPR) potentials for analgesia and reinforcement were assayed using rats. Analgesia was measured by the classic tail-flick test. The test germane to opioid reinforcement involved measuring pressing rates for direct electrical stimulation of the lateral hypothalamus and ventral tegmental area. FEN, as does morphine and heroin, produced strong analgesia and enhanced pressing rates for brain stimulation. DIPR produced no analgesia and antagonized FEN's analgesia. DIPR, at doses antagonizing FEN's analgesia, enhanced pressing for brain stimulation. DIPR's enhancement of pressing was antagonized by naloxone (100 micrograms/kg). When FEN and DIPR were given concurrently, pressing for brain stimulation was not reduced and was greater than after FEN alone was given. These data support a conclusion that different types of receptors are associated with opioid analgesia and reinforcement.     

Experimental models of maternal–fetal interface and their potential use for nanotechnology applications

During pregnancy, the placenta regulates the transfer of oxygen, nutrients, and residual products between the maternal and fetal bloodstreams and is a key determinant of fetal exposure to xenobiotics from the mother. To study the disposition of substances through the placenta, various experimental models are used, especially the perfused placenta, placental villi explants, and cell lineage models. In this context, nanotechnology, an area of study that is on the rise, enables the creation of particles on nanometric scales capable of releasing drugs aimed at specific tissues. An important reason for furthering the studies on transplacental transfer is to explore the potential of nanoparticles (NPs), in new delivery strategies for drugs that are specifically aimed at the mother, the placenta, or the fetus and that involve less toxicity. Due to the fact that the placental barrier is essential for the interaction between the maternal and fetal organisms as well as the possibility of NPs being used in the treatment of various pathologies, the aim of this review is to present the main experimental models used in studying the maternal–fetal interaction and the action of NPs in the placental environment.