18O-Labeling of guanosine monophosphate upon hydrolysis of cyclic guanosine 3':5'-monophosphate by phosphodiesterase

The hydrolysis of cGMP by phosphodiesterase was conducted in [18O]water to determine the site of bond cleavage and the stoichiometry of 18O incorporation into 5'-GMP. Three different forms of phosphodiesterase including a calmodulin-calcium-dependent enzyme in its basal and activated states were examined. The hydrolysis of cGMP catalyzed by each of the forms of phosphodiesterase proceeded with incorporation of 1 18O atom recoverable in the phosphate moiety of each molecule of 5'-GMP generated. No molecular species of phosphate deriving from the 5'-GMP generated containing two or three 18O were detectable. These results indicate that the phosphodiesterase-catalyzed hydrolysis of cGMP proceeds by nucleophilic substitution at phosphorus resulting in P-O bond cleavage. The stoichiometry of 18O incorporation indicates that the reaction proceeds without phosphate-water oxygen exchange when the hydrolytic reaction is catalyzed by diverse forms of phosphodiesterase in the basal or activated state. These considerations of the phosphodiesterase reaction help to establish the validity of monitoring the rate of enzyme-catalyzed hydrolysis of cGMP as a function of the rate of 18O-labeling of the phosphate of 5'-GMP when the reaction proceeds in a medium of predetermined 18O enrichment.     

A comparison of the actions of trypsin and pepsin on porcine immunoglobulin M and their effects on biological activity

The action of trypsin at 55 degree C and pH 8.3 on pig IgM anti-Salmonella has been compared with the action of pepsin at 37 degree C and pH 4.6. Both processes cause the gradual removal of Fab arms and Cmu2 domains to produce eventually an (Fc)5 fragment. However, during tryptic digestion Fab arms are preferentially removed from the same subunit, whereas peptic digestion causes random removal from any subunit. At intermediate stages of digestion both processes produce partially fragmented molecules which consist of an (Fc)5 portion still attached to limited numbers of Fab arms. Both processes cause a gradual decrease in the ability of molecules to agglutinate Salmonella, but complement fixation by the complexes declines much more rapidly. A stage is reached where molecules having four Fab arms can still agglutinate but there is no complement fixation. However, the remaining arms on the tryptic molecules are distributed in pairs on the same subunit, whereas those on the peptic molecules are distributed randomly. Hence the number of remaining Fab arms, rather than their distribution, appears to be the critical factor which influences biological activity. A possible explanation for this is discussed.     

Changes in the levels of wheat- and barley-germ agglutinin during embryogenesis in vivo,in vitro and during germination

Radioimmunoassay has been used to measure levels of wheat-germ agglutinin and barley-germ agglutinin during embryogenesis and germination. The two lectins exhibited similar patterns of accumulation during grain maturation in vivo and both decreased to low levels after imbibition of harvest-ripe grains for 3 d. Precocious germination of immature wheat and barley embryos excised and cultured in vitro could be prevented either by inclusion of abscisic acid or mannitol in the culture medium. Changes in the level of wheat-germ agglutinin induced by in vitro culture depended on the maturation stage of the embryo. No direct correlation was found between application of exogenous abscisic acid and accumulation of the lectin.     

Genetic studies on capsid-length determination in bacteriophage T4. I. Isolation and partial characterization of second-site revertants of a gene 23 mutation affecting capsid length.

The T4 mutation ptg19-80 affects the mechanism of capsid-length determination. It is located in gene 23, which encodes the major structural protein of the capsid. The mutation results in the production of abnormal-length capsids in high frequencies. This paper describes the isolation and partial characterization of second-site revertants of ptg19-80. In the course of their analysis, it was discovered that ptg19-80 is itself a double mutation consisting of a gene 23 mutation (ptg19-80c), which causes the morphogenetic defect, and a suppressor mutation located near the lysozyme gene. Phenotypic characterization of nine pseudo-wild-type revertants of this double-mutation revealed that these revertants all produced lower frequencies of abnormal capsids than did ptg19-80. Seven of these revertants were shown to contain two suppressor mutations, one mapping in or near gene 22 and done mapping in or near gene 24. Both mutations were required for suppression. These suppressors displayed no discernible phenotype in the absence of ptg19-80c.     

Common evolutionary origin of the central portions of the Ri TL-DNA of Agrobacterium rhizogenes and the Ti T-DNAs of Agrobacterium tumefaciens

Analysis of published sequences for Ri TL-DNA (root-inducing left-hand transferred DNA) of Agrobacterium rhizogenes revealed several unsuspected structural features. First, Ri TL-DNA genes are redundant. Using redundancy as a criterion, three regions (left, middle and right) were discerned. The left one, ORFs (open reading frames) 1–7, contains no detectable redundancy. In the middle region a highly diverged gene family was detected in ORFs 8, 11, 12, 13 and 14. The right region contains an apparently recent duplication (ORF 15 =18+17). We interpret the phenomenon of redundancy, particularly in the central region that encodes the transformed phenotype, to be an adaptation that ensures function in a variety of host species. Comparison of Ri TL-DNA and Ti T-DNAs from Agrobacterium tumefaciens revealed common structures, unpredicted by previous nucleic acid hybridization studies. Ri TL-DNA ORF 8 is a diverged Ti T-DNA tms1. Both Agrobacterium genes consist of a member of the diverged gene family detected in the central part of the Ri TL-DNA, but fused to a sequence similar to iaaM of Pseudomonas savastonoi. Other members of this gene family were found scattered throughout Ti T-DNA. We argue that the central region of Ri and the part of Ti T-DNA including ORFs 5–10 evolved from a common ancestor. We present the hypothesis that the gene family encodes functions that alter developmental plasticity in higher plants.     

Effects of some protein kinases, cyclic nucleotides and specific inhibitors of phosphorylation on the mitotic cycle of Physarum polycephalum

The true slime mould Physarum polycephalum was treated with various agents by spraying them upon the cell surface 4 hrs before the second synchronous mitosis. The onset of mitosis was considerably approximated after the plasmodium treatment with protein kinases from rat hepatoma or Ph. polycephalum at the late G2 phase. The catalytic and regulatory subunits of cAMP-dependent pig brain protein kinase caused retardation of mitosis, while the holoenzyme, casein kinase and alkaline phosphatase did not affect the timing of mitosis. The cyclic nucleotides and inhibitors of their metabolic enzymes were used to investigate the role of phosphorylation processes in the mitotic cycle.     

UVB radiation induced effects on cells studied by FTIR spectroscopy

We have made a preliminary analysis of the results about the effects on tumoral cell line (lymphoid T cell line Jurkat) induced by UVB radiation (dose of 310 mJ/cm²) with and without a vegetable mixture. In the present study, we have used two techniques: Fourier transform infrared spectroscopy (FTIR) and flow cytometry. FTIR spectroscopy has the potential to provide the identification of the vibrational modes of some of the major compounds (lipid, proteins and nucleic acids) without being invasive in the biomaterials. The second technique has allowed us to perform measurements of cytotoxicity and to assess the percentage of apoptosis. We already studied the induction of apoptotic process in the same cell line by UVB radiation; in particular, we looked for correspondences and correlations between FTIR spetroscopy and flow cytometry data finding three highly probable spectroscopic markers of apoptosis (Pozzi et al. in Radiat Res 168:698–705, 2007). In the present work, the results have shown significant changes in the absorbance and spectral pattern in the wavenumber protein and nucleic acids regions after the treatments.