The structure-specific nicking of small heteroduplexes by the RAG complex: implications for lymphoid chromosomal translocations

During V(D)J recombination, the RAG complex binds at recombination signal sequences and creates double-strand breaks. In addition to this sequence-specific recognition of the RSS, the RAG complex has been shown to be a structure-specific nuclease, cleaving 3' overhangs and 3' flaps, and, more recently, 10 nucleotides (nt) bubble (heteroduplex) structures. Here, we assess the smallest size heteroduplex that core and full-length RAGs can cleave. We also test whether bubbles adjacent to a partial RSS are nicked any differently or any more efficiently than bubbles that are surrounded by random sequence. These points are important in considering what types and what size of non-B DNA structure that the RAG complex can nick, and this helps assess the role of the RAG complex in mediating lymphoid chromosomal translocations. We find that the smallest bubble nicked by the RAG complex is 3nt, and proximity to a partial or full RSS sequence does not affect the nicking by RAGs. RAG nicking efficiency increases with the size of the heteroduplex and is only about two-fold less efficient than an RSS when the bubble is 6nt. We consider these findings in the context of RAG nicking at non-B DNA structures in lymphoid chromosomal translocations.     

Iron dependence and zinc inhibition of duodenal cytosolic aconitase of rat

The response of duodenal cytosolic aconitase (c-aconitase) to oral repletion of graded doses of iron (Fe) during Fe-deficiency was studied in rats (WNIN strain). In addition, in vitro effect of zinc (Zn) on the enzyme activity was studied using duodenal cytosol. Iron-depleted male rats were orally repleted with either 100 or 190 or 370 microg of Fe/day (n=6, each) for 2 weeks. Fe repletion was found to increase linearly the activity of duodenal c-aconitase along with the indicators of iron status. The correlation coefficient (r) between c-aconitase and haemoglobin and mucosal ferritin was 0.6453 and 0.8441, respectively. The effects of zinc (0-40 microM) in vitro on the kinetics of c-aconitase from iron-replete stock diet fed rats (n=4) showed that Zn competitively inhibited the enzyme with a Ki (app.) of 28 microM. These observations suggest that c-aconitase is a critical target involved in the assimilation of Fe and excess dietary Zn can result in negative interactions.     

Folate, homocysteine, endothelial function and cardiovascular disease

Evidence reported from numerous clinical studies over the past decade has revealed an association between increased plasma total homocysteine (tHcy) concentrations and cardiovascular disease (CVD). In addition, epidemiological studies have identified an inverse association between blood folate concentrations, folate intake and cardiovascular endpoints, that are independent of homocysteine. Folic acid supplementation can lower plasma tHcy concentrations safely and inexpensively. Furthermore, folic acid can reverse endothelial dysfunction observed in patients with CVD. This reversal in endothelial dysfunction with folic acid has been shown to be independent of plasma tHcy lowering, suggesting that folate has pleiotropic effects on the vasculature other than homocysteine lowering. In vitro evidence demonstrates that 5-methyltetrahydrofolate (5MeTHF) the main circulating metabolite of folate, can increase nitric oxide production and can directly scavenge superoxide radicals. The potential beneficial role of folic acid supplements on vascular disease are currently being tested in randomized placebo controlled studies.     

Expression of complement 3 and complement 5 in newt limb and lens regeneration

Some urodele amphibians possess the capacity to regenerate their body parts, including the limbs and the lens of the eye. The molecular pathway(s) involved in urodele regeneration are largely unknown. We have previously suggested that complement may participate in limb regeneration in axolotls. To further define its role in the regenerative process, we have examined the pattern of distribution and spatiotemporal expression of two key components, C3 and C5, during limb and lens regeneration in the newt Notophthalmus viridescens. First, we have cloned newt cDNAs encoding C3 and C5 and have generated Abs specifically recognizing these molecules. Using these newt-specific probes, we have found by in situ hybridization and immunohistochemical analysis that these molecules are expressed during both limb and lens regeneration, but not in the normal limb and lens. The C3 and C5 proteins were expressed in a complementary fashion during limb regeneration, with C3 being expressed mainly in the blastema and C5 exclusively in the wound epithelium. Similarly, during the process of lens regeneration, C3 was detected in the iris and cornea, while C5 was present in the regenerating lens vesicle as well as the cornea. The distinct expression profile of complement proteins in regenerative tissues of the urodele lens and limb supports a nonimmunologic function of complement in tissue regeneration and constitutes the first systematic effort to dissect its involvement in regenerative processes of lower vertebrate species.     

Physiological adaptations for nitrogen use efficiency in sorghum†

Known high nitrogen utilization efficiency (NUE₁, biomass per unit plant N) China lines of sorghum, China 17 and San Chi San, were compared with relatively low NUE₁ U.S. lines, CK60 and Tx623, for both their physiological and biochemical adaptations to tolerate an imposed N stress in the greenhouse. Assimilation efficiency indices (ACi) were significantly greater for the China lines than the U.S. lines at both low and high soil nitrogen levels by about two-fold. Chlorophyll levels in leaves of high NUE₁ lines were lower at both soil N treatments. Immunoblots of leaf extracts of sorghum subjected to N stress indicated reduced levels of both phosphoenolpyruvate carboxylase (PEPcase) and ribulose 1,5-bisphosphate carboxylase (Rubisco) while NADP-malic enzyme levels, in general, appear not to be affected. However, NUE₁ China line, China 17, retained a significantly greater PEPcase activity than the less-NUE₁ U.S. lines, and also the NUE₁ China line San Chi San, when grown under N stress conditions. This suggests that PEPcase and enzymes associated with phosphoenolpyruvate synthesis, perhaps, are significant factors in maintaining relatively high photosynthesis under N stress. Carbon isotope ratios of leaves from sorghum genotypes, as indicated by ¹³C values, became less negative when sorghum plants were grown under N stress, but a genotypic variation either at a low or high N was not observed.     

Combinatorial library of indinavir analogues: replacement for the aminoindanol at P2'

A 1X22X41 combinatorial library or 902 compounds of indinavir analogues was synthesized on the solid support to identify a replacement for the aminoindanol moiety at P2'. 2,6-Dimethyl-4-hydroxy phenol was discovered to be a good replacement for aminoindanol.     

Electrochemical and functional characterization of the proline dehydrogenase domain of the PutA flavoprotein from Escherichia coli

The multifunctional PutA flavoprotein from Escherichia coli is a peripherally membrane-bound enzyme that has both proline dehydrogenase (PDH) and Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities. In addition to its enzymatic functions, PutA displays DNA-binding activity and represses proline catabolism by binding to the control region DNA of the put regulon (put intergenic DNA). Presently, information on structure-function relationships for PutA is derived from primary structure analysis. To gain further insight into the functional organization of PutA, our objective is to dissect PutA into different domains and to characterize them separately. Here, we report the characterization of a bifunctional proline dehydrogenase (PutA(669)) that contains residues 1-669 of the PutA protein. PutA(669) purifies as a dimer and has a PDH specific activity that is 4-fold higher than that of PutA. As anticipated, PutA(669) lacks P5CDH activity. At pH 7.5, an E(m) (E-FAD/E-FADH(-)) of -0.091 V for the two-electron reduction of PutA(669)-bound FAD was determined by potentiometric titrations, which is 15 mV more negative than the E(m) for PutA-bound FAD. The pH behavior of the E(m) for PutA(669)-bound FAD was measured in the pH range 6.5-9.0 at 25 degrees C and exhibited a 0.03 V/pH unit slope. Analysis of the DNA and membrane-binding properties of PutA(669) shows that it binds specifically to the put intergenic control DNA with a binding affinity similar to that of PutA. In contrast, we did not observe functional association of PutA(669) with membrane vesicles. We conclude that PutA(669) has FAD-binding and DNA-binding properties comparable to those of PutA but lacks a membrane-binding domain necessary for stable association with the membrane.     

Green tea constituent epigallocatechin-3-gallate inhibits angiogenic differentiation of human endothelial cells

Several independent research studies have shown that consumption of green tea reduces the development of cancer in many animal models. Epidemiological observations, though inconclusive, are suggesting that green tea consumption may also reduce the risk of some cancers in humans. These anti-carcinogenic effects of green tea have been attributed to its constituent polyphenols. Angiogenesis is a crucial step in the growth and metastasis of cancers. We have investigated the effect of the major polyphenolic constituent of green tea, epigallocatechin-3-gallate (EGCG), on the tube formation of human umbilical vein endothelial cells (HUVEC) on matrigel. Tube formation was inhibited by treatment both prior to plating and after plating endothelial cells on matrigel. EGCG treatment also was found to reduce the migration of endothelial cells in matrigel plug model. The role of matrix metalloproteinases (MMP) has been shown to play an important role during angiogenesis. Zymography was performed to determine if EGCG had any effect on MMPs. Zymographs of EGCG-treated culture supernatants modulated the gelatinolytic activities of secreted proteinases indicating that EGCG may be exerting its inhibitory effect by regulating proteinases. These findings suggest that EGCG acts as an angiogenesis inhibitor by modulating protease activity during endothelial morphogenesis.     

Thermodynamic effects of replacements of Pro residues in helix interiors of maltose-binding protein

Introduction of Pro residues into helix interiors results in protein destabilization. It is currently unclear if the converse substitution (i.e., replacement of Pro residues that naturally occur in helix interiors would be stabilizing). Maltose-binding protein is a large 370-amino acid protein that contains 21 Pro residues. Of these, three nonconserved residues (P48, P133, and P159) occur at helix interiors. Each of the residues was replaced with Ala and Ser. Stabilities were characterized by differential scanning calorimetry (DSC) as a function of pH and by isothermal urea denaturation studies as a function of temperature. The P48S and P48A mutants were found to be marginally more stable than the wild-type protein. In the pH range of 5-9, there is an average increase in T(m) values of P48A and P48S of 0.4 degrees C and 0.2 degrees C, respectively, relative to the wild-type protein. The other mutants are less stable than the wild type. Analysis of the effects of such Pro substitutions in MBP and in three other proteins studied to date suggests that substitutions are more likely to be stabilizing if the carbonyl group i-3 or i-4 to the mutation site is not hydrogen bonded in the wild-type protein.