Role of NAD+ and ADP-Ribosylation in the Maintenance of the Golgi Structure

We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114–1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065–7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD⁺ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD⁺-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD⁺ triggered Golgi disassembly by BFA. This effect of NAD⁺ was mimicked by the use of pre–ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200–14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre–ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50–enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.     

Molecular modeling studies on the ribosome

In the absence of a high resolution crystal structure for the ribosome, numerous research groups are carrying out low resolution structural studies using neutron diffraction, electron microscopy, fluorescence energy transfer, chemical crosslinking, chemical footprinting studies, and other methods. We have developed a computer-based refinement method for incorporating these data into low resolution three-dimensional models. The method is based on a molecular mechanics approach, with proteins represented by spherical particles of suitable diameter and the ribosomal RNA represented by a string of spherical pseudoatoms, one for each nucleotide. Experimental data are used to derive constraints that are introduced through a special force field (potential function). Models are refined by simulated annealing. Since every term in the force field is quadratic, any model that satisfies all of the input data has an energy of zero; higher energies indicate residual unsatisfied constraints. The residual energy provides a quantitative statement of model quality and can be used to identify conflicts in the experimental data. The method has been applied to the refinement of a low resolution model for the 30S subunit (the small subunit) of theE. coli ribosome. Since this is a very underdetermined system, the range of acceptable models has also been explored. This provides an estimate of the resolution of the structure, which is about 15 Å overall, with the uncertainty in position of individual nucleotides ranging from about 5 Å to 50 Å.     

Organogenesis in callus derived from stem and leaf tissues of apple and cherry rootstocks

Callus formation from stem internodes of the apple rootstocks M.9, M.25, M.26, M.27 and the cherry rootstock Colt, and from pith of Nicotiana tabacum cv. Wisconsin 38 was initiated on 4 -naphthaleneacetic acid (NAA)-based media (2.0–10.0 mg1^-1). Transfer of callus to corresponding media lacking NAA allowed regeneration of shoots from callus of M.25, M.27, Colt and tobacco but not of M.9 and M.26. With M.25 phloroglucinol (PG) depressed regeneration from 30 to 10% and no regeneration was observed in cultures grown in the presence of casein hydrolysate (CH) and glutathione (GSH).Organogenesis was also obtained from leaf discs of M.27 employing 6-benzyl-aminopurine (BAP) at 5.0mg 1^-1 and 2,4-dichlorophenoxy acetic acid (2,4-D) at 0.1 mg1^-1. The regenerated shoots have been multiplied and rooted.Organogenesis also occurred in M.26 from small (1–2mm), green, compact embryoid-like structures derived from stem and leaf surfaces of excised axillary shoots. These structures differentiated into shoots at a low frequency (< 1%) on media containing BAP (1.0mg1^-1) and indole-3-butyric acid (IBA) (0.1 mg1^-1) and could also be micropropagated by subsequent axillary shoot proliferation.     

Nucleotide sequence of a fragment of SV40 DNA that contains the origin of DNA replication and specifies the 5' ends of "early" and "late" viral RNA. III. Construction of the total sequence of EcoRII-G fragment of SV40 DNA.

Limited T1 RNase digestion of subfragments of the SV40 DNA restriction endonuclease fragment EcoRII-G were prepared and analyzed. The fragments were separately labeled with 32P at their 5' terminus and the terminal sequences analyzed with limited snake venom diesterase digestion. The data permitted us to deduce the nucleotide sequence for EcoRII-G. The sequence contains a stretch of 17 A-T base pairs preceding the DNA complementary to the 5' end of "early" message RNA, a stretch of 27 bases with a perfect 2-fold rotational symmetry near the origin of DNA replication and a perfect tandem repeat of 21 nucleotides.     

2′-p-Hydroxybenzoyl mussaenosidic acid,a new iridoid glucoside from Vitex negundo.

Ethanolic extract of the leaves of Vitex negundo yielded a new iridoid named 2′-p-hydroxybenzoyl mussaenosidic acid. The structure was established on the basis of chemical and spectral data.     

Sesquiterpene lactone from Salmalia malbarica

A new cadalane type sesquiterpenoid, 1, 6-dihydroxy-3-methyl-5-(1-methylethyl)-7-methoxy-8-carboxylic acid (8 → 1 lactone) has been isolated from a 50% aqueous methanolic extract of 6–9 months old cultivated roots of Salmalia malbarica (Bombax malbaricum).     

Cardiac contractile proteins and sarcoplasmic reticulum in hearts of rats trained by running

Rats were trained with two running protocols previously demonstrated to result in enhanced cardiac performance. Control groups included free-eating sedentary animals and food-restricted animals in which the body weights were the same as the runners. Calcium binding by isolated sarcoplasmic reticulum (SR) was slightly but significantly increased in SR from runners at low but not high calcium concentrations at 15 s and 1 min. Calcium uptake in the presence of 1 mM oxalate was increased in SR from runners. Actomyosin ATPase activity was increased by 10% (P less than 0.001) with one running protocol but not with the other. Myosin Ca2+ ATPase activity and actin-activated ATPase activity were also slightly increased in hearts of runners. In food-restricted cardiac actomyosin ATPase was significantly decreased. Actomyosin ATPase activity was found to be normal in hearts of sedentary animals subjected to water immersion without exercise. Therefore, physical training of rats by running, which produces a cardiac mechanical advantage similar to training by swimming, is not accompanied by cardiac biochemical changes of the same magnitude as in the hearts of swimmers.