Effectiveness of iron-fortified infant cereals on hemoglobin levels of children aged 12-24 months: A cross-sectional study from New Delhi, India.

INTRODUCTION: Iron deficiency anemia represents 3rd largest disease burden, with an estimated 6.9 billion disability-adjusted life years. Iron-fortified cereals (IFIC) can contribute substantially in preventing iron deficiency anemia and maintaining an adequate body iron status. The aim of this study is to assess the effectiveness of IFIC intake along with other complementary food/s on the hemoglobin (hb) level of children from 12 to 24 months of age. MATERIALS AND METHODS: A cross-sectional study was conducted from November 2015 to February 2016 in three pediatric outpatient clinics of New Delhi, India. A predesigned questionnaire was used to elicit information on socio-demography, complementary feeding, and intake of IFIC from 66 mother and child pairs. Child's anthropometric measurement and hb levels were recorded by the pediatrician. Chi-square and Student's t-tests were used to compare the key study variables between IFIC (minimum 1-2 serving/day) and non-IFIC groups. Multiple logistic regression analysis was applied to explore the independent correlates of anemia in the study groups. RESULTS: Out of 66 children, 60.6% (n = 40) of children were boys. The prevalence of anemia (hb% <11 g/dl) was 42.4% (95% confidence interval (CI): 30.5%-55.2%, n = 28). Multiple logistic regression analysis revealed that the children in IFIC group were unlikely to be anemic (adjusted odds ratio (OR): 0.007, 95% CI: 0.001-0.079, P < 0.001). On the contrary, boys (adjusted OR: 11.6, 95% CI: 1.23-108.9, P = 0.032) and children with low birth weight (adjusted OR: 11.7, 95% CI: 1.23-111.76, P = 0.032) were associated with anemic status. CONCLUSION: Intake of IFIC (minimum 1-2 serving/day) was associated with the lesser chance of anemia in children of 12-24 months. However, gender and low birth weight were also associated with anemia. IFIC may have a role in mass fortification programs. However, further larger and controlled studies are recommended to test this hypothesis.

Phosphorylation of the PCNA binding domain of the large subunit of replication factor C on Thr506 by cyclin-dependent kinases regulates binding to PCNA.

Replication factor C (RF-C) complex binds to DNA primers and loads PCNA onto DNA, thereby increasing the processivity of DNA polymerases. We have previously identified a distinct region, domain B, in the large subunit of human RF-C (RF-Cp145) which binds to PCNA. We show here that the functional interaction of RF-Cp145 with PCNA is regulated by cdk-cyclin kinases. Phosphorylation of either RF-Cp145 as a part of the RF-C complex or RF-Cp145 domain B by cdk-cyclin kinases inhibits their ability to bind PCNA. A cdk-cyclin phosphorylation site, Thr506 in RF-Cp145, identified by mass spectrometry, is also phosphorylated in vivo. A Thr506-->Ala RF-Cp145 domain B mutant is a poor in vitro substrate for cdk-cyclin kinase and, consequently, the ability of this mutant to bind PCNA was not suppressed by phosphorylation. By generating an antibody directed against phospho-Thr506 in RF-Cp145, we demonstrate that phosphorylation of endogenous RF-Cp145 at Thr506 is mediated by CDKs since it is abolished by treatment of cells with the cdk-cyclin inhibitor roscovitine. We have thus mapped an in vivo cdk-cyclin phosphorylation site within the PCNA binding domain of RF-Cp145.

Mechanisms of sulindac-induced apoptosis and cell cycle arrest.

The mechanism underlying the chemopreventive effects of the non-steroidal anti-inflammatory drug sulindac remains unclear. Its active metabolite, sulindac sulfide, induces cell cycle arrest as well as apoptosis in mammalian cell lines. We now show that in murine thymocytes, sulindac sulfide-induced cell death is p53, bax, Fas, and FasL independent. In contrast, bcl2 transgenic thymocytes are resistant to sulindac sulfide-induced apoptosis. In addition, we demonstrate that sulindac sulfide-induced cell cycle arrest in mouse embryonic fibroblasts (MEFs) is partly mediated by the retinoblastoma tumor suppressor protein (Rb) and the cyclin kinase inhibitor p21waf1/cip1. Furthermore, MEFs deficient in p21 or Rb are more susceptible to sulindac sulfide-induced cell death. These results suggest that sulindac may selectively target premalignant cells with cell cycle checkpoint deficits.

Role of p21WAF1 in the cellular response to UV.

UV or g irradiation mediated DNA damage activates p53 and induces cell cycle arrest. Induction of cyclin dependent kinase inhibitor p21WAF1 by p53 after DNA damage plays an important role in cell cycle arrest after gamma irradiation. The p53 mediated cell cycle arrest has been postulated to allow cells to repair the DNA damage. Repair of UV damaged DNA occurs primarily by the nucleotide excision pathway (NER). It is known that p21WAF1 binds PCNA and inhibits PCNA function in DNA replication. PCNA is also required for repair by NER but there have been conflicting reports on whether p21WAF1 can inhibit PCNA function in NER. It has therefore been difficult to integrate the UV induced cell cycle arrest by p21 in the context of repair of UV damaged DNA. A recent study reported that p21WAF1 protein is degraded after low but not high doses of UV irradiation, that cell cycle arrest after UV is p21 independent, and that at low dose UV irradiation p21WAF1 degradation is essential for optimal DNA repair. These findings shed new light on the role of p21 in the cellular response to UV and clarify some outstanding issues concerning p21WAF1 function.

WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility.

We previously identified Waf1 Cip1 stabilizing protein 39 (WISp39) as a binding partner for heat shock protein 90 (Hsp90). We now report that WISp39 has an essential function in the control of directed cell migration, which requires WISp39 interaction with Hsp90. WISp39 knockdown (KD) resulted in the loss of directional motility of mammalian cells and profound changes in cell morphology, including the loss of a single leading edge. WISp39 binds Coronin 1B, known to regulate the Arp2/3 complex and Cofilin at the leading edge. WISp39 preferentially interacts with phosphorylated Coronin 1B, allowing it to complex with Slingshot phosphatase (SSH) to dephosphorylate and activate Cofilin. WISp39 also regulates Arp2/3 complex localization at the leading edge. WISp39 KD-induced morphological changes could be rescued by overexpression of Coronin 1B together with a constitutively active Cofilin mutant. We conclude that WISp39 associates with Hsp90, Coronin 1B, and SSH to regulate Cofilin activation and Arp2/3 complex localization at the leading edge.

Distinct p21 requirements for regulating normal and self-reactive T cells through IFN-γ production.

Self/non-self discrimination characterizes immunity and allows responses against pathogens but not self-antigens. Understanding the principles that govern this process is essential for designing autoimmunity treatments. p21 is thought to attenuate autoreactivity by limiting T cell expansion. Here, we provide direct evidence for a p21 role in controlling autoimmune T cell autoreactivity without affecting normal T cell responses. We studied C57BL/6, C57BL/6/lpr and MRL/lpr mice overexpressing p21 in T cells, and showed reduced autoreactivity and lymphadenopathy in C57BL/6/lpr, and reduced mortality in MRL/lpr mice. p21 inhibited effector/memory CD4(+) CD8(+) and CD4(-)CD8(-) lpr T cell accumulation without altering defective lpr apoptosis. This was mediated by a previously non-described p21 function in limiting T cell overactivation and overproduction of IFN-γ, a key lupus cytokine. p21 did not affect normal T cell responses, revealing differential p21 requirements for autoreactive and normal T cell activity regulation. The underlying concept of these findings suggests potential treatments for lupus and autoimmune lymphoproliferative syndrome, without compromising normal immunity.

Functional analysis of CDK inhibitor p21WAF1.

p21WAF1 was originally identified as a protein that binds and inhibits cyclin-dependent kinases (CDKs). p21WAF1 is recognized to have at least two separate roles-first as a CDK inhibitor, and second as an inhibitor of PCNA, an accessory protein of DNA polymerase delta. p21WAF1 plays a critical role in the cellular response to DNA damage. Additionally, p21WAF1 plays a role in DNA repair, apoptosis, cellular senescence, terminal differentiation, and cell cycle arrest upon extracellular signaling. p21WAF1 protein levels are regulated both by transcriptional control by p53 and by factors other than p53, as well as by posttranscriptional regulation. Although the role of p21WAF1 has been explained so far only by its interaction with CDKs and with PCNA, it has several other binding partners. The ability of p21WAF1 to participate in several cellular functions has been widely studied by transfection of cells with p21WAF1 vectors. We describe here procedures for analysis of p21WAF1 function in mammalian cells after transfection of p21 plasmids. The procedures include inhibition of DNA synthesis, cellular localization, association with binding partners, and half-life measurements.

Junctional diversity prevents negative selection of an antigen-specific T cell repertoire.

Endogenous mouse mammary tumor proviruses (MMTV; Mtv loci) deletes Vbeta6 expressing T cells in the thymus of Mtv-7(+) DBA/2 (H2(d)) mice through negative selection. We found that in Mtv-7(-) BALB/c (H2(d)) mice, Vbeta6 is a dominant V gene used in T cell responses to an 18 amino acid long peptide antigen: EYKEYAEYAEYAEYAEYA [abbreviated as K5 or EYK(EYA)(5)]. It was therefore surprising to find that despite the deletion of Vbeta6+ T cells, vigorous K5 specific T cell responses that use Vbeta6 can be raised in DBA/2 mice. Sequence analysis of Vbeta6 junctional diversity in K5 specific T cell lines revealed that the DBA/2 K5 repertoire compensates for the loss of most Vbeta6 T cells by overusing and amplifying Vbeta6+ T cells escaping central deletion and peripheral tolerization. In order to address the inability of some Vbeta6 T cells to recognize Mtv-7(+) we analyzed a panel of BALB/c Vbeta6 expressing T cell hybridomas. This data supported the argument that certain Vbeta6 junctional sequences preclude Mtv recognition and allows their escape from central deletion in DBA/2 mice. These cells are not anergic and can be activated with cognate peptide antigen in periphery. We suggest that junctional diversity at the V region of some of the T cell receptors does not allow these cells to recognize self-superantigens with high enough affinity and thus they escape negative selection in the thymus. These results for the first time provide a molecular explanation of how the immune system compensates for "hole in the repertoire" caused by deletion of the majority of T cells carrying certain V region segments.

DNA damage triggers p21WAF1-dependent Emi1 down-regulation that maintains G2 arrest.

Several regulatory proteins control cell cycle progression. These include Emi1, an anaphase-promoting complex (APC) inhibitor whose destruction controls progression through mitosis to G1, and p21(WAF1), a cyclin-dependent kinase (CDK) inhibitor activated by DNA damage. We have analyzed the role of p21(WAF1) in G2-M phase checkpoint control and in prevention of polyploidy after DNA damage. After DNA damage, p21(+/+) cells stably arrest in G2, whereas p21(-/-) cells ultimately progress into mitosis. We report that p21 down-regulates Emi1 in cells arrested in G2 by DNA damage. This down-regulation contributes to APC activation and results in the degradation of key mitotic proteins including cyclins A2 and B1 in p21(+/+) cells. Inactivation of APC in irradiated p21(+/+) cells can overcome the G2 arrest. siRNA-mediated Emi1 down-regulation prevents irradiated p21(-/-) cells from entering mitosis, whereas concomitant down-regulation of APC activity counteracts this effect. Our results demonstrate that Emi1 down-regulation and APC activation leads to stable p21-dependent G2 arrest after DNA damage. This is the first demonstration that Emi1 regulation plays a role in the G2 DNA damage checkpoint. Further, our work identifies a new p21-dependent mechanism to maintain G2 arrest after DNA damage.

Regulation of p21(WAF1/CIP1) stability by WISp39, a Hsp90 binding TPR protein.

p21(WAF1/CIP1), a cyclin-dependent kinase inhibitor and a critical regulator of cell cycle, is controlled transcriptionally by p53-dependent and -independent mechanisms and posttranslationally by the proteasome. We have identified WISp39, a tetratricopeptide repeat (TPR) protein that binds p21. WISp39 stabilizes newly synthesized p21 protein by preventing its proteasomal degradation. WISp39, p21, and hsp90 form a trimeric complex in vivo. The interaction of WISp39 with Hsp90 is abolished by point mutations within the C-terminal TPR domain of WISp39. Although this WISp39 TPR mutant binds p21 in vivo, it fails to stabilize p21. Our results suggest that WISp39 recruits Hsp90 to regulate p21 protein stability. WISp39 downregulation by siRNA prevents the accumulation of p21 and cell cycle arrest after ionizing radiation. The results demonstrate the importance of posttranslational stabilization of p21 protein by WISp39 in regulating cellular p21 activity.

Rb inhibits E2F-1-induced cell death in a LXCXE-dependent manner by active repression.

Rb (retinoblastoma protein) inhibits E2F-1-induced cell death. We now show that the ability of Rb to inhibit E2F-1-induced cell death is dependent on a functional LXCXE-binding site in Rb, thereby suggesting that proteins that bind the LXCXE-binding site in Rb may regulate the anti-apoptotic activity of Rb. HDAC1, an LXCXE protein that plays a critical role in Rb-mediated transcription repression, abrogates the effect of Rb on E2F-1-induced cell death. In contrast, RF-Cp145, another LXCXE protein, cooperates with Rb to inhibit E2F-1-induced cell death. Both proteins exert their effect in an LXCXE-dependent manner. Rb regulates E2F-induced cell death by acting upstream of p73. Rb represses the p73 promoter. Our results further suggest a model in which Rb-E2F-1 complexes mediate the anti-apoptotic activity of Rb through active repression of target genes without recruiting HDAC1.

UV irradiation triggers ubiquitin-dependent degradation of p21(WAF1) to promote DNA repair.

p53-mediated increase in cyclin-dependent kinase inhibitor p21(WAF1) protein is thought to be the major mediator of cell cycle arrest after DNA damage. Previously p21 protein levels have been reported to increase or to decrease after UV irradiation. We show that p21 protein is degraded after irradiation of a variety of cell types with low but not high doses of UV. Cell cycle arrest occurs despite p21 degradation via Tyr(15) inhibitory phosphorylation of cdk2 and differs from the classical p21-dependent checkpoint elicited by ionizing radiation. In contrast to the basal turnover of p21, degradation of p21 switches to ubiquitin/Skp2-dependent proteasome pathway following UV irradiation. ATR activation after UV irradiation is essential for signaling p21 degradation. Finally, UV-induced p21 degradation is essential for optimal DNA repair. These results provide novel insight into regulation of p21 protein and its role in the cellular response to DNA damage.

Loss of p53 compensates for alpha v-integrin function in retinal neovascularization.

alpha(v)-Integrin antagonists block neovascularization in various species, whereas 20% of alpha(v)-integrin null mice are born with many normal looking blood vessels. Given that blockade of alpha(v)-integrins during angiogenesis induces p53 activity, we utilized p53 null mice to elucidate whether loss of p53 can compensate for alpha(v)-integrin function in neovascularization of the retina. Murine retinal vascularization was inhibited by systemic administration of an alpha(v)-integrin antagonist. In contrast, mice lacking p53 were refractory to this treatment, indicating that neovascularization in normal mice depends on alpha(v)-integrin-mediated suppression of p53. Blockade of alpha(v)-integrins during neovascularization resulted in an induction of p21(CIP1) in wild type and, surprisingly, in p53 null retinas, indicating that alpha(v)-integrin ligation regulates p21(CIP1) levels in a p53-independent manner. In conclusion, we demonstrate for the first time an in vivo intracellular mechanism for compensation of integrin function and that p53 and alpha(v)-integrins act in concert during retinal neovascularization.

Cell cycle-dependent phosphorylation of the large subunit of replication factor C (RF-C) leads to its dissociation from the RF-C complex.

The five subunit replication factor C (RF-C) complex plays a critical role in DNA elongation. We find that the large subunit of RF-C (RF-Cp145) is phosphorylated in vivo whereas the smaller RF-C subunits are not phosphorylated. The phosphorylation of endogenous RFCp145 is modulated in a cell cycle-dependent manner. Phosphorylation is maximal in G2/M and is inhibited by an inhibitor of cyclin-dependent kinases. Phosphorylation of purified recombinant RF-C complex in vitro reveals that RF-Cp145 is preferentially phosphorylated by cdc2-cyclin B but not by cdk2-cyclin A or cdk2-cyclin E. In vitro phosphorylation of RF-C complex by cdc2-cyclin B kinases leads to dissociation of phosphorylated RFCp145 from the RF-C complex. Using different approaches we demonstrate that phosphorylated RFCp145 is indeed dissociated from RF-Cp40 and RF-Cp37 in vivo. These results suggest that destabilization of the RF-C complex by CDKs may inactivate the RF-C complex at the end of S phase.