DNA rearrangements in a hybrid plasmid carrying the redB imm region of coliphage lambda

The hybrid plasmid consisting of pSC101 and the redB--N--imm region of phage lambda cI857 persists in cells grown at 30 degrees C but not in cells grown at 37 degrees C. In the latter case the plasmid was found to undergo several modifications. Restriction maps of these new plasmids indicate the following modifications: (1) the insertion of an IS1 element into gene N carried by the lambda fragment; (2) a mutation in the pL oL site of the same fragment, and (3) four large deletions (30 to 50% of the hybrid plasmid) which remove almost the entire lambda fragment. For the latter deletions, one endpoint seems to be fixed in the same restriction fragment of pSC101 while the other endpoint assumes four different positions on the lambda fragment; this might suggest a site-specific recombination event.     

Cytochrome c as an electron shuttle between the outer and inner mitochondrial membranes

Addition of exogenous NADH to rotenone- and antimycin A-treated mitochondria, in 125 mM KCl, results in rates of oxygen uptake of 0.5-1 and 10-12 nanoatoms of oxygen X mg protein-1 X min-1 in the absence and presence of cytochrome c, respectively. During oxidation of exogenous NADH there is a fast and complete reduction of cytochrome b5 while endogenous or added exogenous cytochrome c become 10-15% and 100% reduced, respectively. The reoxidation of cytochrome b5, after exhaustion of NADH, precedes that of cytochrome c. NADH oxidation is blocked by mersalyl, an inhibitor of NADH-cytochrome b5 reductase. These observations support the view of an electron transfer from the outer to the inner membrane of intact mitochondria. Both the rate of exogenous NADH oxidation and the steady state level of cytochrome c reduction increase with the increase of ionic strength, while the rate of succinate oxidation undergoes a parallel depression. These observations suggest that the functions of cytochrome c as an electron carrier in the inner membrane and as an electron shuttle in the intermembrane space are alternative. It is concluded that aerobic oxidation of exogenous NADH involves the following pathway: NADH leads to NADH-cytochrome b5 reductase leads to cytochrome b5 leads to intermembrane cytochrome c leads to cytochrome oxidase leads to oxygen. It is suggested that the communication between the outer and inner membranes mediated by cytochrome c may affect the oxidation-reduction level of cytosolic NADH and the related oxidation-reduction reactions.     

Isolation and organization of calf ribosomal DNA.

Ribosomal DNA (rDNA) from calf was isolated by three density gradient centrifugations. The first centrifugation in Cs2S04/BAMD was used to obtain partially resolved dG+dC-rich fractions from total DNA. The second and third centrifugations, in Cs2S04/Ag+, led to the isolation of an rDNA fraction characterized by a symmetrical band in CsCl, p = 1.724 g/cm3. This new procedure appears to be generally suitable for the isolation of rDNA and other dG+dC-rich repeated genes. The organization of isolated calf rDNA has been studied by restriction enzyme digestion and by hybridization with cloned rDNA from Xenopus laevis. The repeat unit of calf rDNA has a molecular weight of 21x10(6) and is split by EcoR1 into two fragments, 16x10(6) and 5.0x10(6), and by BamHI into seven fragments. EcoRI and BamHI sites have been mapped. Most of the 18S and 28S RNA genes and the transcribed spacer are contained in the small EcoRI fragment, while the non-transcribed spacer is localized in the large EcoRI fragment. This spacer showed length heterogeneity within a single individual; such heterogeneity is limited to two regions of the spacer.     

Experimental hybridization within the genus Triturus (Urodela: Salamandridae)

The spermatogenesis of 9 F₁ hybrids of Triturus cristatus carnifex × T. vulgaris meridionalis was studied in squash preparations of testicular fragments, treated by the C-staining method. The chromosome number of these hybrids was examined in spermatogonial metaphases and found to be diploid. The two parental sets were always recognized, which means that a regular, although heterospecific, amphimixis occurred (2n=n+n). Meiotic prophase I is greatly altered owing to a failure of typical chromosome pairing and chiasma formation. At metaphase I and/or meta-anaphase I, the effects of the hybrid combination of the 2 specific parental sets are clearly visible. Most primary spermatocytes contain only univalents. A few show chromosome associations (bivalents, trivalents and, more rarely, quadrivalent chains) besides univalents. Such associations are of 2 types: (a) intragenomal associations = associations of 2 chromosomes by a terminal (a¹) or subterminal chiasma (a²); (b) intergenomal associations = associations of 2 chromosomes by a terminal (b¹) or subterminal chiasma (b²). Univalents segregate at random while the associations often lag on the equatorial plane or migrate entire to a spindle pole. Primary spermatocytes with chromosome multivalents can encounter greater difficulties in accomplishing the first cytokinesis. Secondary spermatocytes are numerically and qualitatively unbalanced; however, some of them undergo spermiogenesis and can give rise to a small number of sperms, generally abnormal and never united in bundles. — Problems related to the occurrence of anomalous chiasmata and of intra- and inter-genomal homologies are discussed.     

Sesquiterpenes from Lactarius blennius

Six sesquiterpene lactones were isolated from Lactarius blennius. The structures of two new sesquiterpenes, blennin A and blennin B were determinated by spectroscopic methods and the structure of the seco-compound, blennin C, is revised. The two known furan sesquiterpenes and lactarorufin A were also identified.     

Cu(II) ion interaction with teicoplanin-vancomycin’s analog

Teicoplanin, a member of the “last chance” antibiotic family has a similar structure and the same mechanism of action as parent drug vancomycin, which is proved to be an effective binder of Cu(II) ions. However, the potentiometric and spectroscopic studies (UV-visible, CD, NMR) have shown that the modification of the N-terminal structure of the peptide backbone in teicoplanin affects considerably the binding ability towards Cu(II) ions. While vancomycin forms almost instantly the stable 3 N complex species involving the N-terminal and two amide nitrogen donors, in case of teicoplanin only two nitrogen donors derived from the N-terminal amino group and adjacent peptide bond are coordinated to Cu(II) ion within the whole pH range studied. The major factor influencing the binding mode is most likely the structure of the N-terminus of the peptide unit in the antibiotic ligand.     

Nusinersen treatment and cerebrospinal fluid neurofilaments: An explorative study on Spinal Muscular Atrophy type 3 patients

The antisense oligonucleotide Nusinersen has been recently licensed to treat spinal muscular atrophy (SMA). Since SMA type 3 is characterized by variable phenotype and milder progression, biomarkers of early treatment response are urgently needed. We investigated the cerebrospinal fluid (CSF) concentration of neurofilaments in SMA type 3 patients treated with Nusinersen as a potential biomarker of treatment efficacy. The concentration of phosphorylated neurofilaments heavy chain (pNfH) and light chain (NfL) in the CSF of SMA type 3 patients was evaluated before and after six months since the first Nusinersen administration, performed with commercially available enzyme-linked immunosorbent assay (ELISA) kits. Clinical evaluation of SMA patients was performed with standardized motor function scales. Baseline neurofilament levels in patients were comparable to controls, but significantly decreased after six months of treatment, while motor functions were only marginally ameliorated. No significant correlation was observed between the change in motor functions and that of neurofilaments over time. The reduction of neurofilament levels suggests a possible early biochemical effect of treatment on axonal degeneration, which may precede changes in motor performance. Our study mandates further investigations to assess neurofilaments as a marker of treatment response.