Mechanism of regulation of phosphorylation-dephosphorylation of Ca2+, Mg2+ and Ca2+ -Atpases by modulator proteins isolated from rat brain cytosol

Two proteins of molecular mass 13 kDa, a specific inhibitor of Na+, K+ -ATPase and another of 12 kDa, which can distinguish between Ca2, Mg2+ and Ca2+ -ATPase activities have been obtained from the pooled fractions isolated from rat brain, using Sephadex G-100 chromatography. In order to determine the key step(s), which is affected by the modulators, we have designed an in vitro experiment of phosphorylation and dephosphorylation of these ATPases in the absence and presence of the modulators. The results suggest that the phosphorylation step of Mg2+ -independent Ca2+ -ATPase is inhibited, while in Mg2+ -dependent Ca2 -ATPase, the dephosphorylation step is stimulated by the modulators. The findings support our earlier observation that the modulators are able to distinguish between Mg2+ -independent and dependent Ca2+ -ATPases activities.     

Reduction in animal numbers by long-term implantation of intravenous and intra-arterial catheters in thyroparathyroidectomized rats

The thyroparathyroidectomized (TPTx) rat has been extensively used to study parathyroid hormone (PTH)-mediated bone resorption by measuring systemic Ca2+ concentrations. Animals have been traditionally used acutely; that is, they are often infused immediately after surgery and are sacrificed after a single use. To perform multiple experiments using a single group of animals we developed a system of long-term implanted intravenous/arterial catheters. Using calcitonin (CT) as a positive control, we successfully completed 12 separate controlled subexperiments documenting significant reductions in PTH-induced hypercalcemia in rats of the CT group. We then successfully completed two separate TPTx subexperiments, using a 3 x 3 Latin square experimental design. In both subexperiments, CT significantly inhibited the increase of blood Ca2+ concentration resulting from continuous PTH infusion. Our results indicate that, by combining the long-term use of catheters with the Latin square design, we can successfully reduce the number of animals used, increase the number of compounds screened, and improve the quality of the data. Although results of this study confirmed the acceptability of multiple infusions in anti-resorptive studies, investigations into the applicability of this set up to other areas of study requiring infusions and frequent blood sample collections seem appropriate.     

Gastric toxicity and mucosal ulceration induced by oxygen-derived reactive species: protection by melatonin

Uncontrolled hydrochloric acid secretion and ulceration of the stomach mucosa due to various factors are serious global problems. Although the mechanism of acid secretion from the parietal cell is now well understood, the processes involved in gastric ulceration are still not clear. Among various causes of gastric ulceration, lesions caused by stress, alcohol consumption, Helicobacter pylori infection and due to use of nonsteroidal antiinflammatory drugs have been shown to be mediated largely through the generation of reactive oxygen species, especially the hydroxyl radical. A number of excellent drugs have proven useful in controlling hyperacidity and ulceration but their long-term use is associated with disturbing side-effects. Hence, the search is still on to find a compound possessing antisecretory, antiulcer and antioxidant properties which will serve as a therapeutic agent to reduce gastric hyperacidity and ulcers. This article describes the role of reactive oxygen species in gastric ulceration, drugs controlling them with their merits and demerits and, the role of melatonin, a pineal secretory product, in protecting against gastric lesions. In experimental studies, melatonin has been shown to be effective in reducing mucosal breakdown and ulcer formation in a wide variety of situations. Additionally, the low toxicity of melatonin supports further investigation of this molecule as a gastroprotective agent. Finally, we include a commentary on how melatonin research with respect to gastric pathophysiology can move forward with a view of eventually using this indole as a therapeutic agent to control gastric ulceration in humans.     

The Arabidopsis Information Resource (TAIR): a comprehensive database and web-based information retrieval, analysis, and visualization system for a model plant

Arabidopsis thaliana, a small annual plant belonging to the mustard family, is the subject of study by an estimated 7000 researchers around the world. In addition to the large body of genetic, physiological and biochemical data gathered for this plant, it will be the first higher plant genome to be completely sequenced, with completion expected at the end of the year 2000. The sequencing effort has been coordinated by an international collaboration, the Arabidopsis Genome Initiative (AGI). The rationale for intensive investigation of Arabidopsis is that it is an excellent model for higher plants. In order to maximize use of the knowledge gained about this plant, there is a need for a comprehensive database and information retrieval and analysis system that will provide user-friendly access to Arabidopsis information. This paper describes the initial steps we have taken toward realizing these goals in a project called The Arabidopsis Information Resource (TAIR) (www.arabidopsis.org).     

Structural basis of DNA flexibility

It has been recently shown by us, on the basis of crystal structure database that the flexibility of B-DNA double helices depends significantly on their base sequence. Our model building studies further indicated that the existence of bifurcated cross-strand hydrogen bonds between successive base pairs is possibly the main factor behind the sequence directed DNA flexibility. These cross-strand hydrogen bonds are, of course, weaker than the usual Watson-Crick hydrogen bonds and their bond geometry is characterized by relatively larger bond lengths and smaller bond angles. We have tried to improve our model structures by incorporating non-planarity of the amino groups in DNA bases due to the presence of lone pair electrons at the nitrogen atoms. Energy minimization studies have been carried out by using different quantum chemical methods, whereby it is found that in all cases of N-H....O type cross-strand hydrogen bonds, the bond geometry improves significantly. In the cases of N-H....N type hydrogen bonds, however, no such consistent improvements can be noticed. Perhaps the true picture would emerge only if all the other interactions present in the DNA macromolecule could be appropriately taken into account.     

Interaction of chlorpromazine with milk proteins

The mode and nature of the binding of chlorpromazine (CPZ), a psychotropic drug, with milk proteins – -lactalbumin (with substantial amounts of -helix, -sheet and random coil), -lactoglobulin (a major -sheeted protein) and _s-casein (a random coiled protein) have been studied spectrofluorometrically and spectropolarimetrically. The binding affinity of CPZ for unfolded proteins is comparatively less than that of folded proteins although the number of binding sites is smaller in the latter case, due to the greater extent of binding of CPZ for folded proteins. Thermodynamic analysis reveals that CPZ binds to -lactalbumin and _s-casein in an endothermic (H^o is positive) and hydrophobic manner but with -lactoglobulin in an exothermic (H^o is negative) manner. Far UV Circular dichroic studies reveal that CPZ increases the secondary structure of the major -sheeted protein, -lactoglobulin possibly by increasing the relative contact orders (non-local contacts) within the residues. On the other hand, for proteins possessing random coil, it increases the unfolded state of the protein. CPZ does not affect local contacts in a-helix when its interaction is compared with a major -helical protein, myoglobin.     

DnaB helicase affects the initiation specificity of Escherichia coli primase on single-stranded DNA templates

The effect of DnaB helicase on the initiation specificity of primase was studied biochemically using a series of single-stranded DNA templates in which each nucleotide of the trinucleotide d(CTG) initiation sequence was systematically varied. DnaB helicase accelerated the rate of primer syntheisis, prevented "overlong" primers from forming and decreased the initiation specificity of primase. In the presence of DnaB helicase, all trinucleotides could serve as the primer initiation site although there was a distinct preference for d(CAG). These data may explain the high chromosomal prevalence of octanucleotides containing CTG on the leading strand and its complement CAG on the lagging strand. The specificity of DnaB helicase places it on the lagging strand template where it stimulates the initiation of Okazaki fragment synthesis. In the absence of DnaB helicase, primase preferentially primed the d(CTG) template. In the presence of DnaB helicase, the initiation preference was not only altered but also the preferred initiating nucleotide was found to be GTP rather than ATP, for both the d(CTG) and the d(CAG) templates. This suggested that the specificity of primase for the d(CTG) initiation trinucleotide was predominantly unaffected in the absence of DnaB helicase on short ssDNA templates, whereas in conjunction with DnaB helicase, the specificity was altered and this alteration has significant implications in the replication of Escherichia coli chromosome in vivo.     

NBD-isocolcemid-tubulin interaction: a novel one-step reaction involving no conformational adjustment of reactants

Isocolcemid, a colcemid analogue in which the positions of the C-ring methoxy and carbonyl are exchanged, is virtually inactive in binding to tubulin and inhibiting the formation of microtubule assembly. We have found that the substitution of a NBD group in the side chain of the B-ring of isocolcemid can reverse the effect of these structural alterations (at the C-ring) and the newly synthesized NBD-isocolcemid restores the lost biological activity. It inhibits microtubule assembly with an IC(50) of 12 microM and competes efficiently with [(3)H]colchicine, for binding to tubulin. NBD-isocolcemid has two binding sites on tubulin; one is characterized by fast binding, whereas the binding to the other site is slow. These two sites are independent and unrelated to each other. Colchicine and its analogues compete with NBD-isocolcemid for the slow site. Association and dissociation rate constants for the fast site, obtained from the stopped-flow measurements, are (7.37 +/- 0. 70) x 10(5) M(-1) s(-1) and 7.82 +/- 2.74 s(-1), respectively. While the interaction of colchicine and its analogues with tubulin involves two steps, NBD-isocolcemid binding to tubulin at the slow site has been found to be a one-step reaction. This is evident from the linear dependence of the observed rate constant (k(obs)) with both NBD-isocolcemid and tubulin concentrations. The interaction of NBD-isocolcemid with tubulin does not involve the conformational change of NBD-isocolcemid, as is evident from the unchanged CD spectra of the drug. The absence of enhanced GTPase activity of tubulin and the native-like protease cleavage pattern of the NBD-isocolcemid-tubulin complex suggest an unaltered conformation of tubulin upon NBD-isocolcemid binding to it as well. Implications of this on the mechanism of polymerization inhibition have been discussed.