Purification and Characterization of Lectin from Lathyrus sativus

Lectin has been isolated and purified from Lathyrus sativus using ammonium sulphate precipitation followed by affinity chromatography. The molecular weight as determined by HPLC was found to be 42kD. The lectin is a tetramer, consisting of two types of subunits of which the heavier subunit consists of 2 polypeptides of mol wt of about 21 kD and 16 kD while the smaller subunits consists of two polypeptides of about 5kD as revealed by SDS-PAGE. The most potent sugar inhibitor of the Lathyrus lectin was found to be α-methyl D-mannoside. The N-terminal amino acid sequence was similar to that of pea lectin sequence.     

Potassium Fluxes in Chlamydomonas reinhardtii (I.Kinetics and Electrical Potentials)

Potassium influx and cellular [K+] were measured in the unicellular green alga Chlamydomonas reinhardtii after pretreatment in either 10 or 0 mM external K+ ([K]0). K+ (42K+ or 86Rb+) influx was mediated by a saturable, high-affinity transport system (HATS) at low [K+]0 and a linear, low-affinity transport system at high [K+]o. The HATS was typically more sensitive to metabolic inhibition (and darkness) than the low-affinity transport system. Membrane electrical potentials were determined by measuring the equilibrium distribution of tetraphenylphosphonium. These values, together with estimates of cytoplasmic [K+] (B. Malhotra and A.D.M. Glass [1995] Plant Physiol 108: 1537-1545), demonstrated that at 0.1 mM [K+]0 K+ uptake must be active. At higher [K+]0 (>0.3 mM) K+ influx appeared to be passive and possibly channel mediated. When cells were deprived of K+ for 24 h, the Vmax for the HATS increased from 50 x 10-6 to 85 x 10-6 nmol h-1 cell-1 and the Km value decreased from 0.25 to 0.162 mM. Meanwhile, cellular [K+] declined from 24 x 10-6 to 9 x 10-6 nmol cell-1. During this period influx increased exponentially, reaching its peak value after 18 h of K+ deprivation. This increase of K+ influx was not expressed when cells were exposed to inhibitors of protein synthesis. The use of 42K+ and 86Rb+ in parallel experiments demonstrated that Chlamydomonas discriminated in favor of K+ over Rb+, and this effect increased with the duration of K+ deprivation.     

Free and total thyroid hormones in humans at extreme altitude

Alterations in circulatory levels of total T₄ (TT₄), total T₃ (TT₃), free T₄ (FT₄), free T₃ (FT₃), thyrotropin (TSH) and T₃ uptake (T₃U) were studied in male and female sea-level residents (SLR) at sea level, in Armed forces personnel staying at high altitude (3750 m) for prolonged duration (acclimatized lowlanders, ALL) and in high-altitude natives (HAN). Identical studies were also performed on male ALL who trekked to an extreme altitude of 5080 m and stayed at an altitude of more than 6300 m for about 6 months. The total as well as free thyroid hormones were found to be significantly higher in ALL and HAN as compared to SLR values. Both male as well as female HAN had higher levels of thyroid hormones. The rise in hormone levels in different ALL ethnic groups drawn from amongst the southern and northern parts of the country was more or less identical. In both HAN and ALL a decline in FT₃ and FT₄ occurred when these subjects trekked at subzero temperatures to extreme altitude of 5080 m but the levels were found to be higher in ALL who stayed at 6300 m for a prolonged duration. Plasma TSH did not show any appreciable change at lower altitudes but was found to be decreased at extreme altitude. The increase in thyroid hormones at high altitude was not due to an increase in hormone binding proteins, since T₃U was found to be higher at high altitudes. A decline in TSH and hormone binding proteins and an increase in the free moiety of the hormones is indicative of a subtle degree of tissue hyperthyroidism which may be playing an important role in combating the extreme cold and hypoxic environment of high altitudes.     

Helical stacking in DNA three-way junctions containing two unpaired pyrimidines: proton NMR studies.

The proton NMR spectra of DNA three-way junction complexes (TWJ) having unpaired pyrimidines, 5'-TT- and 5'-TC- on one strand at the junction site were assigned from 2D NOESY spectra acquired in H2O and D2O solvents and homonuclear 3D NOESY-TOCSY and 3D NOESY-NOESY in D2O solvent. TWJ are the simplest branched structures found in biologically active nucleic acids. Unpaired nucleotides are common features of such structures and have been shown to stabilize junction formation. The NMR data confirm that the component oligonucleotides assemble to form conformationally homogeneous TWJ complexes having three double-helical, B-form arms. Two of the helical arms stack upon each other. The unpaired pyrimidine bases lie in the minor groove of one of the helices and are partly exposed to solvent. The coaxial stacking arrangement deduced is different from that determined by Rosen and Patel (Rosen, M.A., and D.J. Patel. 1993. Biochemistry. 32:6576-6587) for a DNA three-way junction having two unpaired cytosines, but identical to that suggested by Welch et al. (Welch, J. B., D. R. Duckett, D. M. J. Lilley. 1993. Nucleic Acids Res. 21:4548-4555) on the basis of gel electrophoretic studies of DNA three-way junctions containing unpaired adenosines and thymidines.     

Intramolecular domain-domain interactions and intermolecular self-association in bovine prothrombin. A potentiometric and laser light-scattering study

The interaction of bovine prothrombin with Ca²⁺ and Mg²⁺ ions was investigated by following H⁺ release as a function of metal ion concentration at pH 6 and pH 7.4 at high and low ionic strength. Prothrombin Ca²⁺ and Mg²⁺ binding is characterized by high- and low-affinity sites. M²⁺ binding at these sites is associated with intramolecular conformational changes and also with intermolecular self-association. The pH dependence of H⁺ release by M²⁺ is bell shaped and consistent with controlling pK_a values of 4.8 and 6.5. At pH 6 and low ionic strength, both Ca²⁺ and Mg²⁺ titrations following H⁺ release clearly show independent low- and high-affinity binding sites. Laser light scattering reveals that at pH 7.4 and low ionic strength, and at pH 6.0 and high ionic strength, the prothrombin molecular weight is between 73 and 98 kD. At pH 7.4 and high ionic strength, prothrombin is monomeric in the absence of metal ions, but appears to dimerize in the presence of M²⁺. At pH 6.0 and low ionic strength prothrombin exists as a dimer in the absence of metal ions and is tetrameric in the presence of Ca²⁺ and remains dimeric in the presence of Mg²⁺. These results and those for metal ion-dependent H⁺ release indicate that H⁺ release occurs concomitantly with association processes involving prothrombin.Abbreviations GLA -carboxyglutamic acid; fragment 1. amino terminal residues 1–156 of bovine prothrombin - MES 2-(N-morpholino) ethanesulfonic acid - MOPS 3-(N-morpholino) propanesulfonic acid - PS/PC phosphatidylserine/phosphatidylcholine vesicles - ionic strength     

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.     

Ataxia with Vitamin E Deficiency: Refinement of Genetic Localization and Analysis of Linkage Disequilibrium by Using New Markers in 14 Families

Ataxia with vitamin E deficiency (AVED) is an autosomal recessive disease characterized clinically by neurological symptoms with often striking resemblance to those of Friedreich ataxia. This disorder has been reported previously as familial isolated vitamin E deficiency. We have mapped recently the AVED locus to a 5-cM confidence interval on chromosome 8q by homozygosity mapping in six Mediterranean families. We have now analyzed six new and two previously described families and demonstrate genetic homogeneity despite important clinical variability and wide geographic origins. Analysis of nine new tightly linked microsatellite markers, including four characterized in this study, revealed a predominant but not unique mutation in northern African populations, where this condition is more frequent. Haplotype analysis but also classical recombinations allowed us to refine the AVED position to a 1-cM interval. A YAC contig over this interval was constructed from marker STSs and YAC fingerprint data, in order to facilitate the search of the AVED gene.     

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.