Regulatory substances produced by lymphocytes. V. Production of inhibitor of DNA synthesis (IDS) by proliferating T lymphocytes.

The conditions neccessary for production of inhibitor of DNA synthesis (IDS) by rat lymphocytes were investigated.In concanavalin A (Con A)-stimulated lymph node cell (LNC) cultures, IDS production was not detected in the culture supernatant during the first 24 hr, and it increased gradually after that to reach a maximum at 3 to 4 days.When the cells were pretreated with mitomycin C, IDS was not produced, suggesting that DNA synthesis of LNC or a LNC subpopulation is necessary for IDS production. In contrast, Con A-stimulated spleen cells priduced a high level of IDS within 24 hr, and its production fell off sharply thereafter. Con A-stimulated rat thymocytes also produced IDS reaching a maximum at 2 to 3 dyas. However, thymus cells from rats treated with hydrocortisone 48 hr previously did not produce IDS. This finding implies that cortisol-sensitive (cortical) thymocytes are capable of producing IDS and cortisol-resistant (medullary) thymocytes are not. IDS production by lymphoblasts was proportional to cell number and unaffected eith by cell density (1 to 10 x 106/ml) or by the concomitant presence of normal cells from spleen, lymph node, or thymus. Thus Con A-stimulated cells, after becoming blasts, appear to produce IDS automatically wihtout affecting or being affected by other cells. Both spleen and thymus cells from rats injected with a large dose of antigen (ovalbumin, 100 mg, i.p.) 24 hr in advance produced substantial amounts of IDS in culture within 24 hr in the absence of mitogen or additional antigen, but not the cells from rats injected with an immunizing dose (1 mg) of the same antigen. The cells producing IDS in the spleen were shown to be adherent to glass wool, and those in the thymus were partially so. IDS production by antigen-stimulated spleen cells was abrogated by injecting rats with bromodexyuridine (BUdR) at 0 and 12 hr after the ovalbumin. These findings suggest that a subpopulation ofadherent spleen cells (possibly resembling cortical thymocytes), which begins to proliferate within a few hours after a large dose of systemic antigen, produces IDS. This may account for increased nonspecific suppressor activity observed at the same time.     

Modulatory effect of sesamol on DOCA-salt-induced oxidative stress in uninephrectomized hypertensive rats

The present study was undertaken to investigate the antihypertensive and antioxidant effects of sesamol on uninephrectomized deoxycorticosterone acetate (DOCA)-salt-induced hypertensive rats. Hypertension was induced in surgically single-kidney-removed (left) adult male albino Wistar rats, weighing 180–200 g, by injecting DOCA (25 mg/kg BW) subcutaneously twice a week for 6 weeks, with saline instead of tap water for drinking. Rats were treated with three different doses of sesamol (50, 100 and 200 mg/kg BW) post-orally by gavage daily for 6 weeks. Hypertension was revealed by increased systolic and diastolic blood pressure and the toxicity of DOCA-salt was determined using hepatic marker enzymes, aspartate aminotransferase, alanine aminotransferase, alkaline phospatase and gamma-glutamyl transpeptidase; and, lipid peroxidative markers, thiobarbituric acid reactive substances, lipid hydroperoxides and conjugated dienes were assayed. The activities of enzymatic antioxidants, superoxide dismutase, catalase and glutathione peroxidase and the levels of non-enzymatic antioxidants (vitamin C, vitamin E and reduced glutathione) were evaluated in erythrocytes, plasma and tissues. Post-oral administration of sesamol at the dosage of 50 mg/kg BW remarkably decreased systolic and diastolic blood pressure, hepatic marker enzyme activities and lipid peroxidation products and also enhanced the antioxidant activity. The biochemical observations were also supported by histopathological examinations of the rat liver, kidney and heart sections. These results suggest that sesamol possesses antihypertensive and antioxidant effects.     

Role of W181 in modulating kinetic properties of Plasmodium falciparum hypoxanthine guanine xanthine phosphoribosyltransferase

Hypoxanthine‐guanine‐xanthine phosphoribosyltransference (HGXPRT), a key enzyme in the purine salvage pathway of the malarial parasite, Plasmodium falciparum (Pf), catalyses the conversion of hypoxanthine, guanine, and xanthine to their corresponding mononucleotides; IMP, GMP, and XMP, respectively. Out of the five active site loops (I, II, III, III', and IV) in PfHGXPRT, loop III' facilitates the closure of the hood over the core domain which is the penultimate step during enzymatic catalysis. PfHGXPRT mutants were constructed wherein Trp 181 in loop III' was substituted with Ser, Thr, Tyr, and Phe. The mutants (W181S, W181Y and W181F), when examined for xanthine phosphoribosylation activity, showed an increase in K_m for PRPP by 2.1‐3.4 fold under unactivated condition and a decrease in catalytic efficiency by more than 5‐fold under activated condition as compared to that of the wild‐type enzyme. The W181T mutant showed 10‐fold reduced xanthine phosphoribosylation activity. Furthermore, molecular dynamics simulations of WT and in silico W181S/Y/F/T PfHGXPRT mutants bound to IMP.PPi.Mg²⁺ have been carried out to address the effect of the mutation of W181 on the overall dynamics of the systems and identify local changes in loop III'. Dynamic cross‐correlation analyses show a communication between loop III' and the substrate binding site. Differential cross‐correlation maps indicate altered communication among different regions in the mutants. Changes in the local contacts and hydrogen bonding between residue 181 with the nearby residues cause altered substrate affinity and catalytic efficiency of the mutant enzymes. Proteins 2016; 84:1658–1669. © 2016 Wiley Periodicals, Inc.     

Stage-specific profiling of Plasmodium falciparum proteases using an internally quenched multispecificity protease substrate

Novel internally quenched fluorescence peptide substrates containing sequence specific sites for cleavage by multiple proteases were designed and synthesized. The 28 and 29 residue peptides contain an N-terminal fluorescence acceptor group, 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL), and a C-terminal fluorescence donor group, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS). Efficient energy transfer between the donor and acceptor groups flanking the peptide sequence was achieved by incorporation of a central DPro-Gly segment, which serves as a conformation nucleating site, inducing hairpin formation. This multispecificity protease substrate was used to profile the proteolytic activities in the malarial parasite Plasmodium falciparum in a stage dependent manner using a combination of fluorescence and MALDI mass spectrometry. Cysteine protease activity was shown to be dominating at neutral pH, whereas aspartic protease activity contributed predominantly to the proteolytic repertoire at acidic pH. Maximum proteolysis was observed at the trophozoite stage followed by the schizonts and the rings.     

Tight Chk1 Levels Control Replication Cluster Activation in Xenopus

DNA replication in higher eukaryotes initiates at thousands of origins according to a spatio-temporal program. The ATR/Chk1 dependent replication checkpoint inhibits the activation of later firing origins. In the Xenopus in vitro system initiations are not sequence dependent and 2-5 origins are grouped in clusters that fire at different times despite a very short S phase. We have shown that the temporal program is stochastic at the level of single origins and replication clusters. It is unclear how the replication checkpoint inhibits late origins but permits origin activation in early clusters. Here, we analyze the role of Chk1 in the replication program in sperm nuclei replicating in Xenopus egg extracts by a combination of experimental and modelling approaches. After Chk1 inhibition or immunodepletion, we observed an increase of the replication extent and fork density in the presence or absence of external stress. However, overexpression of Chk1 in the absence of external replication stress inhibited DNA replication by decreasing fork densities due to lower Cdk2 kinase activity. Thus, Chk1 levels need to be tightly controlled in order to properly regulate the replication program even during normal S phase. DNA combing experiments showed that Chk1 inhibits origins outside, but not inside, already active clusters. Numerical simulations of initiation frequencies in the absence and presence of Chk1 activity are consistent with a global inhibition of origins by Chk1 at the level of clusters but need to be combined with a local repression of Chk1 action close to activated origins to fit our data.     

Reversible binding of zinc in Plasmodium falciparum Sir2: Structure and activity of the apoenzyme

Reversible zinc chelation via thiol groups of cysteines leading to modulation of activity in redox regulated proteins forms a basis for switching on–off of various biochemical processes. Silent information regulator 2 (Sir2), a NAD⁺ dependent deacetylase, contains a non-catalytic zinc ion coordinated by thiol groups of cysteines. Using Plasmodium falciparum Sir2 (PfSir2), we have examined the effect of zinc removal on the structure and activity of this enzyme. Our studies show that the enzyme with high affinity for zinc exhibits partial collapse of structure upon removal of the metal ion. Zinc reconstitution of apo PfSir2 led to recovery of both structure and activity highlighting the reversibility of the process.     

Substrate-induced conformational changes in Plasmodium falciparum guanosine monophosphate synthetase

GMP synthetase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the nucleotide xanthosine 5'-monophosphate (XMP) to form guanosine 5'-monophosphate (GMP). Functional coordination of domains in glutamine amidotransferases leads to upregulation of glutamine hydrolysis in the presence of acceptor substrates and is a common feature in this class of enzymes. We have shown earlier that binding of substrates to the acceptor domain of Plasmodium falciparum GMP synthetase (PfGMPS) leads to enhancement in both glutaminase activity and rate of glutaminase inactivation, by the irreversible inhibitors acivicin and diazo-oxonorleucine [Bhat JY et al. (2008) Biochem J409, 263-273], a process that must be driven by conformational alterations. In this paper, through the combined use of biochemical assays, optical spectroscopy and mass spectrometry, we demonstrate that PfGMPS undergoes conformational transitions upon binding of substrates to the acceptor domain. Limited proteolysis and hydrogen-deuterium exchange in conjunction with mass spectrometry unveil region-specific conformational changes in the ATP + XMP bound state of PfGMPS. Decreased accessibility of R294 and K428 residues to trypsin in the ATP pyrophosphatase domain and reduced deuterium incorporation in the 143-155 region, pertaining to the glutaminase domain, suggest that in PfGMPS ligand-induced conformational changes are not only local but also transmitted over a long range across the domains. Overall, these results provide a detailed understanding of the substrate-induced changes in PfGMPS that could be essential for the overall catalytic process.     

A non-active site mutation in human hypoxanthine guanine phosphoribosyltransferase expands substrate specificity

Human hypoxanthine guanine phosphoribosyltransferase (HGPRT) lacks the ability to phosphoribosylate xanthine, a property exhibited by HGPRTs from many parasitic protozoa. Using random mutagenesis we have obtained a mutant, F36L, of human HGPRT that phosphoribosylates xanthine. Examination of the structure indicates that F36 does not make direct contact with the purine, but long-range modulation via loop IV, a segment contacting purine at C2 position, could influence substrate specificity. Expanded substrate specificity to include xanthine probably arises from increased flexibility of loop IV as a consequence of mutation at F36. Mutation of the corresponding residue, L44 in Plasmodium falciparum HGPRT, also results in alteration of K(m) and k(cat) for xanthine, substantiating its role in affecting purine base affinity. Our studies show that mutation of this residue in the core of the protein also affects the stability of both enzymes.