Maria Rita Micheli

BDNF and trkB mRNAs oscillate in rat brain during the light-dark cycle.

In this study, we investigated whether in basal conditions the different functional states occurring during a 24-h cycle are reflected by the expression of brain-derived neurotrophic factor (BDNF) and its receptor, trkB, in rat cerebral cortex and hippocampus. Using semiquantitative RT-PCR assay, the levels of both BDNF and trkB mRNAs were found to undergo significant variation in a 24-h period. The strongest variation was detected in the hippocampus, where the ratio between maximum and minimum levels was about 3.5 and 17.5 for BDNF and trkB, respectively. These findings provide the first evidence that, in the absence of any experimental manipulation, the expression of a neurotrophin and its receptor undergoes diurnal oscillation, possibly related to the physiological variations of the activity level.

Fingerprinting methods based on arbitrarily primed PCR.

Overview.- I DNA Extraction from Mammals.- II Insect DNA Extraction Protocol.- III Rapid DNA Extraction from Plants.- IV Preparation of Fungal Genomic DNA for PCR and RAPD Analysis.- V Extraction of Histoplasma capsulatum DNA for PCR.- VI DNA Extraction from Bacterial Cultures.- VII Arbitrarily Primed PCR and RAPDs.- VIII Random Amplified Polymorphic DNA Assay.- IX DNA Amplification Fingerprinting.- X Fluorescent Detection and Analysis of RAPD Amplicons Using the ABI PRISM DNA Sequencers.- XI Optimization of RAPD Fingerprinting.- XII Fingerprint Tailoring.- XIII Resolving DNA Amplification Products Using Polyacrylamide Gel Electrophoresis and Silver Staining.- XIV Denaturing Gradient Gel Electrophoresis for Enhanced Detection of DNA Polymorphisms.- XV Modified Temperature Sweep Gel Electrophoresis for the Separation of Arbitrarily Amplified DNA Fragments.- XVI High Throughput Scoring of RAPD Fragments Through the Use of Dot-Blot Hybridization.- XVII Recovering Amplified DNA from Silver Stained Gels.- XVIII Cloning of RAPD Markers.- XIX Sequencing of RAPD Markers.- XX Analysis of Tumor-Specific Genetic Alterations by Arbitrarily Primed PCR.- XXI Construction of Linkage Maps with RAPD Markers.- XXII Pseudo-Testcross Mapping Strategy Using RAPD Markers.- XXIII Estimating Nucleotide Divergence with RAPD Data.- XXIV RAPD and PAUP Analysis for Microbial Screening Programs.- XXV Production of Specific Probes for Microorganisms.- Overview.- XXVI Differential Display of Expressed mRNAs.- XXVII RNA Arbitrarily Primed PCR.- XXVIII Nonradioactive Differential Display of Messenger RNA.- XXIX Fluorescent Differential Display: A Fast and Safe Way for Reliable Differential Display Analysis.- XXX Slot Blot Hybridization Screening.- XXXI How To Find and Clone the Appropriate cDNA Fragments Generated in Differential mRNA Display by Using Northern Blot for cDNA Capture.- XXXII Verification of Differential Display Results by RNase Protection.- XXXIII Direct Automated Sequencing of DDRT-PCR Fragments.- XXXIV Ligation Linked PCR and Direct Sequencing of Differential Display Products.- XXXV Differential Display PCR Fragments as Probes for cDNA Cloning.- XXXVI Targeted RNA Fingerprinting.- XXXVII Generation of Tumor-Specific DEST Catalogue.- XXXVIII Analysis of Gene Expression in the Preimplantation Mouse Embryo Using mRNA Differential Display.- XXXIX Rapid Identification of Differentially Expressed Genes in Trypanosomes.

PHOSPHOLIPIDS AND NUCLEAR RNA

It has been demonstrated that in hepatocyte nuclei the chromatin phospholipid fraction is localized near the RNA in decondensed chromatin. The aim of the present study was to see if there is any linkage between phospholipids and other nuclear components. Isolated hepatocyte nuclei and nuclear membranes were treated with deoxyribonuclease and ribonuclease. No loss of phospholipids was observed after DNA digestion, whereas 48% was lost following enzymatic RNA removal. This loss of phospholipids, localized either near the membrane or inside the nucleus, was not homogeneous for all phospholipids: phosphatidylserine and sphingomyelin being the most affected. It can be concluded that 48% of nuclear phospholipids, in particular sphingomyelin, is lost with RNA removal. This result is discussed in view of a possible role of phospholipids in DNA synthesis and RNA transcription.

Expression and Modulation of the Intermediate- Conductance Ca2+-Activated K+ Channel in Glioblastoma GL-15 Cells

We report here the expression and properties of the intermediate-conductance Ca2+-activated K+ (IKCa) channel in the GL-15 human glioblastoma cell line. Macroscopic IKCa currents on GL-15 cells displayed a mean amplitude of 7.2±0.8 pA/pF at 0 mV, at day 1 after plating. The current was inhibited by clotrimazole (CTL, IC50=257 nM), TRAM-34 (IC50=55 nM), and charybdotoxin (CTX, IC50=10.3 nM). RT-PCR analysis demonstrated the expression of mRNA encoding the IKCa channel in GL-15 cells. Unitary currents recorded using the inside-out configuration had a conductance of 25 pS, a KD for Ca2+ of 188 nM at -100 mV, and no voltage dependence. We tested whether the IKCa channel expression in GL-15 cells could be the result of an increased ERK activity. Inhibition of the ERK pathway with the MEK antagonist PD98059 (25 µM, for 5 days) virtually suppressed the IKCa current in GL-15 cells. PD98059 treatment also increased the length of cellular processes and up-regulated the astrocytic differentiative marker GFAP. A significant reduction of the IKCa current amplitude was also observed with time in culture, with mean currents of 7.17±0.75 pA/pF at 1-2 days, and 3.11±1.35 pA/pF at 5-6 days after plating. This time-dependent downregulation of the IKCa current was not accompanied by changes in the ERK activity, as assessed by immunoblot analysis. Semiquantitative RT-PCR analysis demonstrated a ~35% reduction of the IKCa channel mRNA resulting from ERK inhibition and a ~50% reduction with time in culture.

A jumping gene in Streptomyces coelicolor A3(2)

SummaryThe difficulty in mapping the gene for chloramphenicol resistance (cmlR) in Streptomyces coelicolor A3(2) stock strains is possibly due to its location on different sites of the chromosome in various mixed subelones. Fresh isolates from CmlR strains show single unequivocal locations of cmlR. The same holds for CmlR strains derived as revertants from CmlS variants. The two best established sites for cmlR are one between cys A and met A, the other at right of arg A, possibly in the right empty arc of the map (Fig. 2). The cmlR gene was assumed to be on a transposon (SCTn1), together with a gene for arginine-succinate synthase (argG), a gene for chromosome transfer (tra) and a gene for aereal mycelium formation (amy). In a CmlR revertant, the cmlR gene appears disjoined from argG (Fig. 5), thus showing the ability of SCTnl to be split and partially transposed. The possible wide occurrence of transposons in the genus Streptomyces is discussed.

Muscle actin isoforms are differentially expressed in human satellite cells isolated from donors of different ages.

Myogenesis is mainly sustained by a subpopulation of myogenic cells known as satellite cells (SC). In this paper we studied α‐smooth muscle (αSMA) and α‐sarcomeric muscle (αSRA) actin isoform expression in cultures of human satellite cells (HSC) isolated from skeletal muscle biopsies from a 5‐day‐old newborn, a 34‐year‐old young adult and a 71‐year‐old donor. Myogenicity of cultures was assessed using immunocytochemical detection of desmin and myosin heavy chain. Time‐course expression of αSRA and αSMA were studied with both immunocytochemistry and western blotting procedures. Although αSMA was never detected in whole skeletal muscle, both αSMA and αSRA were detected in proliferating and differentiating HSC derived from donors of all examined ages. The expression level experiments showed that αSRA was gradually up‐regulated during HSC differentiation, but no significant differences were observed between newborn, young, and elderly HSC cultures. Our data demonstrated that HSC, isolated from subjects of different ages, re‐expressed αSMA, but its levels and expression pattern varied considerably in the newborn with respect to the young adult and elderly donors. These results are discussed in relation to the myogenic differentiation capability of HSC during human muscle senescence.

Modulation of BDNF and TrkB expression in rat hippocampus in response to acute neurotoxicity by diethyldithiocarbamate.

In this study, we examined the expression profile of brain-derived neurotrophic factor (BDNF) and its receptor TrkB in adult rat hippocampus following acute administration of diethyldithiocarbamate (DDTC), a neurotoxic compound which was previously shown to induce microglia activation and cell death. Semiquantitative RT-PCR analysis detected significant variations of BDNF mRNA levels in whole hippocampus homogenates, with a peak at 24h after DDTC injection. Increased BDNF protein expression was demonstrated by immunohistochemistry in various hippocampal subfields. The most relevant increase was observed in the hilus of the dentate gyrus where BDNF levels at 120h were found to be almost four times those of basal levels. Full-length TrkB (TrkB.FL) encoding mRNA was also shown to undergo an earlier increase in the hippocampus of DDTC-treated rats. TrkB immunostaining with an antibody binding both full-length and truncated (TrkB.T) isoforms was found to increase at 120h in the hippocampal CA2 and CA3 regions. These results demonstrate that DDTC modulates the expression of BDNF and its receptor in the adult rat hippocampus and suggest a possible involvement of this neurotrophin in the protective response to DDTC-induced neuronal damage.

Properties of transposon SCTn1 of Streptomyces coelicolor A3(2).

SummaryChloramphenicol resistance (Cmlr) of Streptomyces coelicolor A3(2) behaves like a transposon locus, not being localisable in any region of the map and yet being transferable in crosses at a rate comparable to that of chromosomal markers. It can, also be transposed onto a plasmid (SCP1) and back to the chromosome. Some traits, such as arginino-succinate synthase production (ArgG), aerial mycelium formation (AmyA), resistance to tetracycline and to rifamycin C appear to be joined to Cml in three processes: co-mutation, i.e. simultaneous loss, post-mutation, i.e. spontaneous loss at high, frequency in subclones from Cmls strains, co-transfer, i.e. joint transfer with the cml locus in crosses or during infection by the aggregate SCP1::SCTn1 plasmid. All these processes have been consistently observed with special attention to the argG locus.

Molecuar biology of hepatocellular carcinoma and hepatitis B virus association

SummaryThe involvement of the hepatitis B virus in the pathogenesis of hepatocellular carcinoma was initially suggested on the basis of epidemiological studies. In recent years several kinds of experimental evidence have supported this hypothesis; however, the role played by hepatitis B virus in hepatocarcinogenesis still needs to be elucidated. Several groups of researchers are presently involved in establishing whether hepatitis B virus makes a specific genetic contribution to carcinogenesis or predisposes to neoplastic transformation by causing chronic inflammation and cell regeneration. A comprehensive examination of the data available in the literature suggests that the two hypotheses may not be mutually exclusive.

Bacteriophage T4 gene 28

The complete nucleotide sequence of bacteriophage T4D gene 28 has been determined. Gene 28 product is a structural component of the viral baseplate for which an enzymatic activity has also been proposed.