Regulation of cellulase synthesis in mycelial fungi: Participation of ATP and cyclic AMP

Summary ATP and cAMP in 4 strains of mycelial fungi were determined by luciferin-luciferase system and HPLC respectively. Cellulase synthesis was subject to the dual control of ATP and cAMP. No matter what carbon sourse was used, cellulase synthesis was repressed if intracellular ATP concentration was over 10^-7mg/ml. Exogenous cAMP could increase cellulase synthesis under depression conditions.     

Kinetics and mechanism of CO substitution of [η-CpFe(CO)3]PF6 with PPh3 in the presence of substituted pyridine N-oxide

The kinetics of substitution reactions of [η-CpFe(CO)₃]PF₆ with PPh₃ in the presence of R-PyOs have been studied. For all the R-PyOs (R = 4-OMe, 4-Me, 3,4-(CH)₄, 4-Ph, 3-Me, 2,3-(CH)₄, 2,6-Me₂, 2-Me), the reactions yeild the same product [η⁵-CpFe(CO)₂PPh₃]PF₆, according to a second-order rate law that is first order in concentrations of [η⁵-CpFe(CO)₃]PF₆ and of R-PyO but zero order in PPh₃ concentration. These results, along with the dependence of the reaction rate on the nature of R-PyO, are consistent with an associative mechanism. Activation parameters further support the bimmolecular nature of the reactions: ΔH^≠ = 13.4 ± 0.4 kcal mol⁻¹, ΔS^≠ = −19.1 ± 1.3 cal k⁻¹ mol⁻¹ for 4-PhPyO; ΔH^≠ = 12.3 ± 0.3 kcal mol⁻¹, ΔS^≠ = 24.7 ±1.0 cal K⁻¹ mol⁻¹ for 2-MePyO. For the various substituted pyridine N-oxides studied in this paper, the rates of reaction increase with the increasing electron-donating abilities of the substituents on the pyridine ring or N-oxide basicities, but decrease with increasing ¹⁷O chemical shifts of the N-oxides. Electronic and steric factors contributing to the reactivity of pyridine N-oxides have been quantitatively assessed.     

Caloric Restriction: Conservation of in Vivo Cellular Replicative Capacity Accompanies Life-Span Extension in Mice

In male mice of a long-lived hybrid strain (B6D2F1), long-term 40% caloric restriction (CR) extended both mean and maximum life spans by 36 and 20%, respectively, over that of ad libitum fed (AL) controls. Measurements of entry into S-phase were made in vivo of six different cell types in five different organs using 2-week exposures to BrdU. The labeling index (L.I.) in all organs studied was lower in young CR mice than in young AL fed mice. In most cases, the L.I. in AL mice fell to the levels of that in the CR mice by 13 months of age, and the two groups then remained so through old age. However, when the L.I. was measured in old CR mice which had been placed on the AL diet for a period of 4 weeks (this was termed refeeding (RF)), it was found to be above that of similar age AL or CR mice and almost at the level of young AL mice. This was still true, but to a lesser degree, in a repeat study using an 8-week period of RF. In a separate but parallel in vitro study (companion paper, this volume), the superiority of CR over AL for retention of cellular replication capacity was confirmed by clone size distribution measurements made in several cell types in mice of several age groups. These results indicate that: (1) the rate of cell replication in AL diet mice diminishes greatly by early middle age in all organ sites studied and then plateaus or declines much more slowly; (2) CR broadly preserves in vivo cellular replicative capacity but often requires the energy levels provided by a switch to AL feeding to demonstrate this late in life; (3) accordingly, the replicative deficit in AL fed mice appears to be cumulative and is significant only in old age. The mechanism(s) involved is yet to be discovered but may be related to, or even the same as, that which extends life spans in CR animals. Correspondingly, and with corroborative data from our in vitro companion study, (W. R. Pendergrass et al., 1995. Exp. Cell. Res. 217, 309-316), we suggest that cell populations sustain an accrual of biochemical damage or physiological alterations which increasingly limit their replicative capacity as the animal ages, and that CR reduces the accrual of this damage.     

Melatonin and the pea aphid,Acyrthosiphon pisum

Pea aphids, Acyrthosiphon pisum, were fed on artificial diet containing various concentrations of melatonin. Under long-day conditions (16h light:8h dark) their progeny included males and virginoparous/oviparous (asexual/sexual) intermediate females, which normally occur only in short days or around critical night-length. Endogenous melatonin in pea aphids was measured by radioimmunoassay and verified by parallelism with a melatonin standard curve and by thin layer chromatography. However, melatonin titres showed large variations and although they tended to be higher during the scotophase than during the photophase they were not significantly different. The possibility of melatonin being involved in photoperiodism is discussed.     

Identification of Influenza C Virus Phosphoproteins

The HMV-II cells infected with influenza C virus were labeled with inorganic [³²P]phosphate to identify phosphorylated proteins. Analysis by radioimmunoprecipitation with antiviral serum or monoclonal antibodies revealed that three major structural proteins of the virus, hemagglutinin-esterase (HE), nucleoprotein (NP), and matrix protein (M1) are all phosphorylated in both infected cells and virions. It was also observed that, in the presence of trypsin (10 μg/ml), the unphosphorylated form of the HE glycoprotein was cleaved efficiently whereas the phosphorylated form was not, raising the possibility that phosphorylation of HE may influence its susceptibility to degradation by proteolytic enzymes.     

Stabilization of double-stranded oligonucleotides using backbone-linked disulfide bridges.

A convenient, practical route to the synthesis of disulfide-bridged oligonucleotides has been developed. Aliphatic linkers with terminal thiol groups have been attached to the phosphodiester backbones of partially or fully complementary oligonucleotide sequences and oxidized to yield covalently closed oligonucleotides with disulfide bridges. This procedure has been used to prepare a duplex with disulfide bridges at both ends and stem-loop sequences with single disulfide bridges. Oxidation of a self-complementary duplex possessing terminal thiol groups produced both hairpin and duplex structures with disulfide bridges, the relative proportions of each being dependent upon the reaction conditions. These bridged hairpin and duplex structures were shown to be interconvertible by reduction and re-oxidation. The melting profiles of disulfide-bridged oligonucleotides were compared with the same sequences without bridges and with sequences possessing triethylene glycol bridges, and in all cases the introduction of disulfide bridges resulted in a considerable increase in thermal stability. EcoRI endonuclease was capable of cleaving a disulfide-bridged duplex possessing a recognition site for this enzyme, thus supporting a lack of distortion of the recognition site. The disulfide bridges could be cleaved using a large excess of DTT to regenerate the corresponding sulfhydryl compounds. A study of the serum stabilities of disulfide-bridged oligonucleotides showed that the bridged duplexes were much more stable than their unmodified counterparts, whereas the rate of degradation of the stem-loop structures was more dependent upon the size of the loop than the presence or absence of the disulfide bridge. In summary, we have described a novel methodology, employing commercially available reagents, for the stabilization of oligonucleotide duplexes or stem-loop structures by disulfide bridge formation.     

Discrete differences between strains of different Rhizobium spp. for competitive nodule occupancy on beans

Rhizobium etli strain TAL182 and R. leguminosarum bv phaseoli strain 8002, both of which produce melanin pigment, were tested for their nodulation competitiveness on beans by paired inoculation with two strains which do not produce melanin: R. tropici strain CIAT899 and Rhizobium sp. strain TAL1145. An assay was developed to distinguish nodules formed by the melanin-producing and non-producing strains. Strain TAL182 had discrete competitive superiority over CIAT899 and TAL1145 for nodulation of beans. Nodulation competitiveness was not correlated with the ability to produce melanin pigment or the host range of the Rhizobium strains tested.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gillmore 402, Honolulu, HI 96822, USA