Nonoptimal bacteria species induce neutrophil-driven inflammation and barrier disruption in the female genital tract.

Neutrophil recruitment and activation within the female genital tract are often associated with tissue inflammation, loss of vaginal epithelial barrier integrity, and increased risk for sexually transmitted infections, such as HIV-1. However, the direct role of neutrophils on vaginal epithelial barrier function during genital inflammation in vivo remains unclear. Using complementary proteome and immunological analyses, we show high neutrophil influx into the lower female genital tract in response to physiological surges in progesterone, stimulating distinct stromal, immunological, and metabolic signaling pathways. However, despite the release of extracellular matrix-modifying proteases and inflammatory mediators, neutrophils contributed little to physiological mucosal remodeling events such as epithelial shedding or re-epithelialization during transition from diestrus to estrus phase. In contrast, the presence of bacterial vaginosis-associated bacteria resulted in a rapid and sustained neutrophil recruitment, resulting in vaginal epithelial barrier leakage and decreased cell-cell junction protein expression in vivo. Thus, neutrophils are important mucosal sentinels that rapidly respond to various biological cues within the female genital tract, dictating the magnitude and duration of the ensuing inflammatory response at steady state and during disease processes.

Gene expression profiling identifies candidate biomarkers for new latent tuberculosis infections. A cohort study.

OBJECTIVE: To determine the gene expression profile in individuals with new latent tuberculosis infection (LTBI), and to compare them with people with active tuberculosis (TB) and those exposed to TB but not infected. DESIGN: A prospective cohort study. Recruitment and follow-up were conducted between September 2016 to December 2018. Gene expression and data processing and analysis from April 2019 to April 2021. SETTING: Two male Colombian prisons. PARTICIPANTS: 15 new tuberculin skin test (TST) converters (negative TST at baseline that became positive during follow-up), 11 people that continued with a negative TST after two years of follow-up, and 10 people with pulmonary ATB. MAIN OUTCOME MEASURES: Gene expression profile using RNA sequencing from PBMC samples. The differential expression was assessed using the DESeq2 package in Bioconductor. Genes with |logFC| >1.0 and an adjusted p-value < 0.1 were differentially expressed. We analyzed the differences in the enrichment of KEGG pathways in each group using InterMiner. RESULTS: The gene expression was affected by the time of incarceration. We identified group-specific differentially expressed genes between the groups: 289 genes in people with a new LTBI and short incarceration (less than three months of incarceration), 117 in those with LTBI and long incarceration (one or more years of incarceration), 26 in ATB, and 276 in the exposed but non-infected individuals. Four pathways encompassed the largest number of down and up-regulated genes among individuals with LTBI and short incarceration: cytokine signaling, signal transduction, neutrophil degranulation, and innate immune system. In individuals with LTBI and long incarceration, the only enriched pathway within up-regulated genes was Emi1 phosphorylation. CONCLUSIONS: Recent infection with MTB is associated with an identifiable RNA pattern related to innate immune system pathways that can be used to prioritize LTBI treatment for those at greatest risk for developing active TB.

Expansion of cytotoxic tissue-resident CD8+ T cells and CCR6+CD161+ CD4+ T cells in the nasal mucosa following mRNA COVID-19 vaccination.

Vaccines against SARS-CoV-2 have shown high efficacy in clinical trials, yet a full immunologic characterization of these vaccines, particularly within the human upper respiratory tract, is less well known. Here, we enumerate and phenotype T cells in nasal mucosa and blood using flow cytometry before and after vaccination with the Pfizer-BioNTech COVID-19 vaccine (n = 21). Tissue-resident memory (Trm) CD8+ T cells expressing CD69+CD103+ increase in number ~12 days following the first and second doses, by 0.31 and 0.43 log10 cells per swab respectively (p = 0.058 and p = 0.009 in adjusted linear mixed models). CD69+CD103+CD8+ T cells in the blood decrease post-vaccination. Similar increases in nasal CD8+CD69+CD103- T cells are observed, particularly following the second dose. CD4+ cells co-expressing CCR6 and CD161 are also increased in abundance following both doses. Stimulation of nasal CD8+ T cells with SARS-CoV-2 spike peptides elevates expression of CD107a at 2- and 6-months (p = 0.0096) post second vaccine dose, with a subset of donors also expressing increased cytokines. These data suggest that nasal T cells may be induced and contribute to the protective immunity afforded by this vaccine.

Transient Increases in Inflammation and Proapoptotic Potential Are Associated with the HESN Phenotype Observed in a Subgroup of Kenyan Female Sex Workers.

Interferon (IFN) -stimulated genes (ISGs) are critical effectors of IFN response to viral infection, but whether ISG expression is a correlate of protection against HIV infection remains elusive. A well-characterized subcohort of Kenyan female sex workers, who, despite being repeatedly exposed to HIV-1 remain seronegative (HESN), exhibit reduced baseline systemic and mucosal immune activation. This study tested the hypothesis that regulation of ISGs in the cells of HESN potentiates a robust antiviral response against HIV. Transcriptional profile of a panel of ISGs with antiviral function in PBMC and isolated CD4+ T cells from HESN and non-HESN sex worker controls were defined following exogenous IFN-stimulation using relative RT-qPCR. This study identified a unique profile of proinflammatory and proapoptotic ISGs with robust but transient responses to exogenous IFN-γ and IFN-α2 in HESN cells. In contrast, the non-HESN cells had a strong and prolonged proinflammatory ISG profile at baseline and following IFN challenge. Potential mechanisms may include augmented bystander apoptosis due to increased TRAIL expression (16-fold), in non-HESN cells. The study also identified two negative regulators of ISG induction associated with the HESN phenotype. Robust upregulation of SOCS-1 and IRF-1, in addition to HDM2, could contribute to the strict regulation of proinflammatory and proapoptotic ISGs in HESN cells. As reducing IRF-1 in the non-HESN cells resulted in the identified HESN ISG profile, and decreased HIV susceptibility, the unique HESN ISG profile could be a correlate of protection against HIV infection.

Endothelial Cells Promote Productive HIV Infection of Resting CD4+ T Cells by an Integrin-Mediated Cell Adhesion-Dependent Mechanism.

Resting CD4+ T cells are primary targets of early HIV infection events in vivo, but do not readily support HIV replication in vitro. This barrier to infection can be overcome by exposing resting CD4+ T cells to endothelial cells (ECs). ECs line blood vessels and direct T cell trafficking into inflamed tissues. Cell trafficking pathways have been shown to have overlapping roles in facilitating HIV replication, but their relevance to EC-mediated enhancement of HIV susceptibility in resting CD4+ T cells has not previously been examined. We characterized the phenotype of primary human resting CD4+ T cells that became productively infected with HIV when cocultured with primary human blood and lymphatic ECs. The infected CD4+ T cells were primarily central memory cells enriched for high expression of the integrins LFA-1 and VLA-4. ICAM-1 and VCAM-1, the cognate ligands for LFA-1 and VLA-4, respectively, were expressed by the ECs in the coculture. Blocking LFA-1 and VLA-4 on resting CD4+ T cells inhibited infection by 65.4%-96.9%, indicating that engagement of these integrins facilitates EC-mediated enhancement of productive HIV infection in resting CD4+ T cells. The demonstration that ECs influence cellular HIV susceptibility of resting memory CD4+ T cells through cell trafficking pathways engaged during the transmigration of T cells into tissues highlights the physiological relevance of these findings for HIV acquisition and opportunities for intervention.

The transcription factor scleraxis is a critical regulator of cardiac fibroblast phenotype.

BACKGROUND: Resident fibroblasts synthesize the cardiac extracellular matrix, and can undergo phenotype conversion to myofibroblasts to augment matrix production, impairing function and contributing to organ failure. A significant gap in our understanding of the transcriptional regulation of these processes exists. Given the key role of this phenotype conversion in fibrotic disease, the identification of such novel transcriptional regulators may yield new targets for therapies for fibrosis. RESULTS: Using explanted primary cardiac fibroblasts in gain- and loss-of-function studies, we found that scleraxis critically controls cardiac fibroblast/myofibroblast phenotype by direct transcriptional regulation of myriad genes that effectively define these cells, including extracellular matrix components and α-smooth muscle actin. Scleraxis furthermore potentiated the TGFß/Smad3 signaling pathway, a key regulator of myofibroblast conversion, by facilitating transcription complex formation. While scleraxis promoted fibroblast to myofibroblast conversion, loss of scleraxis attenuated myofibroblast function and gene expression. These results were confirmed in scleraxis knockout mice, which were cardiac matrix-deficient and lost ~50% of their complement of cardiac fibroblasts, with evidence of impaired epithelial-to-mesenchymal transition (EMT). Scleraxis directly transactivated several EMT marker genes, and was sufficient to induce mesenchymal/fibroblast phenotype conversion of A549 epithelial cells. Conversely, loss of scleraxis attenuated TGFß-induced EMT marker expression. CONCLUSIONS: Our results demonstrate that scleraxis is a novel and potent regulator of cellular progression along the continuum culminating in the cardiac myofibroblast phenotype. Scleraxis was both sufficient to drive conversion, and required for full conversion to occur. Scleraxis fulfills this role by direct transcriptional regulation of key target genes, and by facilitating TGFß/Smad signaling. Given the key role of fibroblast to myofibroblast conversion in fibrotic diseases in the heart and other tissue types, scleraxis may be an important target for therapeutic development.

Microarray analysis of pancreatic gene expression during biotin repletion in biotin-deficient rats.

Biotin is a B vitamin involved in multiple metabolic pathways. In humans, biotin deficiency is relatively rare but can cause dermatitis, alopecia, and perosis. Low biotin levels occur in individuals with type-2 diabetes, and supplementation with biotin plus chromium may improve blood sugar control. The acute effect on pancreatic gene expression of biotin repletion following chronic deficiency is unclear, therefore we induced biotin deficiency in adult male rats by feeding them a 20% raw egg white diet for 6 weeks. Animals were then randomized into 2 groups: one group received a single biotin supplement and returned to normal chow lacking egg white, while the second group remained on the depletion diet. After 1 week, pancreata were removed from biotin-deficient (BD) and biotin-repleted (BR) animals and RNA was isolated for microarray analysis. Biotin depletion altered gene expression in a manner indicative of inflammation, fibrosis, and defective pancreatic function. Conversely, biotin repletion activated numerous repair and anti-inflammatory pathways, reduced fibrotic gene expression, and induced multiple genes involved in pancreatic endocrine and exocrine function. A subset of the results was confirmed by quantitative real-time PCR analysis, as well as by treatment of pancreatic AR42J cells with biotin. The results indicate that biotin repletion, even after lengthy deficiency, results in the rapid induction of repair processes in the pancreas.

Development of a high throughput luciferase reporter gene system for screening activators and repressors of human collagen Iα2 gene expression.

Fibrosis, which is characterized by the excessive production of matrix proteins, occurs in multiple tissues and is associated with increased morbidity and mortality. Despite its significant negative impact on patient outcomes, therapies targeted to treat fibrosis are currently lacking. Screening for inhibitors of the expression of collagen, the primary component of fibrotic lesions, represents an option for the identification of novel lead compounds for therapeutic development with potentially fewer off-target effects compared with the targeting of multifunctional cell signaling pathways. Here we report on the generation of a stable luciferase reporter system using a fibroblast cell line, which can be used for rapidly screening both activators and repressors of human collagen COL1A2 gene transcription in a high throughput setting. This in vitro screening tool was validated using known agonists (scleraxis, TGF-ß, angiotensin II, CTGF) and antagonists (TNF-α, pirfenidone) of COL1A2 gene expression. The COL1A2-luc NIH-3T3 fibroblast system provides a useful and effective screen for potential lead compounds with pro- or anti-fibrotic properties.

Early growth response 2 (Egr-2) expression is triggered by NF-κB activation.

Transcription factors are known to play multiple roles in cellular function. Investigators report that factors such as early growth response (Egr) protein and nuclear factor kappa B (NF-κB) are activated in the brain during cancer, brain injury, inflammation, and/or memory. To explore NF-κB activity further, we investigated the transcriptomes of hippocampal slices following electrical stimulation of NF-κB p50 subunit knockout mice (p50-/-) versus their controls (p50+/+). We found that the early growth response gene Egr-2 was upregulated by NF-κB activation, but only in p50+/+ hippocampal slices. We then stimulated HeLa cells and primary cortical neurons with tumor necrosis factor alpha (TNFα) to activate NF-κB and increase the expression of Egr-2. The Egr-2 promoter sequence was analyzed for NF-κB binding sites and chromatin immunoprecipitation (ChIP) assays were performed to confirm promoter occupancy in vivo. We discovered that NF-κB specifically binds to an NF-κB consensus binding site within the proximal promoter region of Egr-2. Luciferase assay demonstrated that p50 was able to transactivate the Egr-2 promoter in vitro. Small interfering RNA (siRNA)-mediated p50 knockdown corroborated other Egr-2 expression studies. We show for the first time a novel link between NF-κB activation and Egr-2 expression with Egr-2 expression directly controlled by the transcriptional activity of NF-κB.

Early mechanisms of pathobiology are revealed by transcriptional temporal dynamics in hippocampal CA1 neurons of prion infected mice.

Prion diseases typically have long pre-clinical incubation periods during which time the infectious prion particle and infectivity steadily propagate in the brain. Abnormal neuritic sprouting and synaptic deficits are apparent during pre-clinical disease, however, gross neuronal loss is not detected until the onset of the clinical phase. The molecular events that accompany early neuronal damage and ultimately conclude with neuronal death remain obscure. In this study, we used laser capture microdissection to isolate hippocampal CA1 neurons and determined their pre-clinical transcriptional response during infection. We found that gene expression within these neurons is dynamic and characterized by distinct phases of activity. We found that a major cluster of genes is altered during pre-clinical disease after which expression either returns to basal levels, or alternatively undergoes a direct reversal during clinical disease. Strikingly, we show that this cluster contains a signature highly reminiscent of synaptic N-methyl-D-aspartic acid (NMDA) receptor signaling and the activation of neuroprotective pathways. Additionally, genes involved in neuronal projection and dendrite development were also altered throughout the disease, culminating in a general decline of gene expression for synaptic proteins. Similarly, deregulated miRNAs such as miR-132-3p, miR-124a-3p, miR-16-5p, miR-26a-5p, miR-29a-3p and miR-140-5p follow concomitant patterns of expression. This is the first in depth genomic study describing the pre-clinical response of hippocampal neurons to early prion replication. Our findings suggest that prion replication results in the persistent stimulation of a programmed response that is mediated, at least in part, by synaptic NMDA receptor activity that initially promotes cell survival and neurite remodelling. However, this response is terminated prior to the onset of clinical symptoms in the infected hippocampus, seemingly pointing to a critical juncture in the disease. Manipulation of these early neuroprotective pathways may redress the balance between degeneration and survival, providing a potential inroad for treatment.

SARS-CoV 9b protein diffuses into nucleus, undergoes active Crm1 mediated nucleocytoplasmic export and triggers apoptosis when retained in the nucleus.

BACKGROUND: 9b is an accessory protein of the SARS-CoV. It is a small protein of 98 amino acids and its structure has been solved recently. 9b is known to localize in the extra-nuclear region and has been postulated to possess a nuclear export signal (NES), however the role of NES in 9b functioning is not well understood. PRINCIPAL FINDINGS/METHODOLOGY: In this report, we demonstrate that 9b in the absence of any nuclear localization signal (NLS) enters the nucleus by passive transport. Using various cell cycle inhibitors, we have shown that the nuclear entry of 9b is independent of the cell cycle. Further, we found that 9b interacts with the cellular protein Crm1 and gets exported out of the nucleus using an active NES. We have also revealed that this NES activity influences the half-life of 9b and affects host cell death. We found that an export signal deficient SARS-CoV 9b protein induces apoptosis in transiently transfected cells and showed elevated caspase-3 activity. CONCLUSION/SIGNIFICANCE: Here, we showed that nuclear shuttling of 9b and its interaction with Crm1 are essential for the proper degradation of 9b and blocking the nuclear export of this protein induces apoptosis. This phenomenon may be critical in providing a novel role to the 9b accessory protein of SARS-CoV.

Extensive autolytic fragmentation of membranous versus cytosolic calpain following myocardial ischemia-reperfusion.

We investigated calpain activation in the heart during ischemia-reperfusion (I-R) by immunologically mapping the fragmentation patterns of calpain and selected calpain substrates. Western blots showed the intact 78 kDa large subunit of membrane-associated calpain was autolytically fragmented to 56 and 43 kDa signature immunopeptides following I-R. Under these conditions, the 78 kDa calpain large subunit from crude cytosolic fractions was markedly less fragmented, with only weakly stained autolytic peptides detected at higher molecular weights (70 and 64 kDa). Western blots also showed corresponding calpain-like degradation products (150 and 145 kDa) of membrane-associated alpha-fodrin (240 kDa) following I-R, but in crude myofibrils alpha-fodrin degradation occurred in a manner uncharacteristic of calpain. For control hearts perfused in the absence of ischemia, autolytic fragmentation of calpain and calpain-like alpha-fodrin degradation were completely absent from most subcellular fractions. The exception was sarcolemma-enriched membranes, where significant calpain autolysis and calpain-like alpha-fodrin degradation were detected. In purified sarcoplasmic reticulum membranes, RyR2 and SERCA2 proteins were also highly degraded, but for RyR2 this did not occur in a manner characteristic of calpain. When I-R-treated hearts were perfused with peptidyl calpain inhibitors (ALLN or ALLM; 25 micromol/L), calpain autolysis and calpain-like degradation of alpha-fodrin were equally attenuated by each inhibitor. However, only ALLN protected against early loss of developed pressure in hearts following I-R, with no functionally protective effect of ALLM observed. Our studies suggest calpain is preferentially activated at membranes following I-R, possibly contributing to impaired ion channel function implicated by others in I-R injury.

Regulation of cardiomyocyte Glut4 expression by ZAC1.

The transcription factor ZAC1 is expressed in a variety of tissues including the developing heart, but its physiological role is unclear. We examined the role of ZAC1 in regulating expression of the insulin-responsive glucose transporter GLUT4 and whether ZAC1 expression is altered in cardiomyocyte hypertrophy. We demonstrated expression of Zac1 mRNA and protein in rat cardiomyocytes by PCR and Western blotting, respectively. Using a combination of chromatin immunoprecipitation and luciferase assays, we showed that ZAC1 regulates Glut4 expression via a specific binding site in the Glut4 promoter. Overexpression of ZAC1 increased Glut4 mRNA and protein expression and resulted in increased glucose uptake in cardiomyocytes as determined by a fluorescent analog uptake assay. Induction of hypertrophy by phenylephrine or isoproterenol resulted in increased Zac1 expression. We identified a novel putative promoter in the Zac1 gene and demonstrated increased binding of MEF2 to this promoter in response to hypertrophic stimulation. MEF2 regulated transactivation of the Zac1 promoter and ZAC1 protein expression. This work identifies ZAC1 as a novel and previously unknown regulator of cardiomyocyte Glut4 expression and glucose uptake. Our results also implicate MEF2 as a regulator of ZAC1 expression in response to induction of hypertrophy.

The A-kinase anchor protein AKAP121 is a negative regulator of cardiomyocyte hypertrophy.

Pathologic cardiac hypertrophy imposes a significant clinical burden on patients, yet the precise intracellular mechanisms responsible for its induction are only partially understood. We examined a potential role for AKAP121 to regulate cardiomyocyte hypertrophy, since recent reports have implicated other AKAPs in this process. We report here that knockdown of AKAP121 expression in isolated neonatal rat cardiomyocytes results in pronounced cellular hypertrophy. Loss of AKAP121 expression is associated with dephosphorylation and nuclear localization of NFATc3, a downstream effector of the hypertrophic phosphatase calcineurin. We also demonstrate that over-expression of AKAP121 in cardiac myocytes reduces basal cell size, and blocks hypertrophy induced by isoproterenol, indicating that AKAP121 negatively regulates the hypertrophic process. Co-immunoprecipitation data indicates that AKAP121 and calcineurin directly interact. Our findings are consistent with a model in which loss of AKAP121 expression leads to the release of an active pool of calcineurin, in turn causing nuclear translocation of NFATc3 and activation of the hypertrophic gene program. These results are the first to identify AKAP121 as a negative regulator of cardiomyocyte hypertrophy, and highlight AKAP121 as a potential target for therapeutic exploitation.

The role of sex in cardiac function and disease.

In the past decade, increasing attention has been paid to the importance of sex in the etiology of cardiac dysfunction. While focus has been primarily on how sex modulates atherogenesis, it is becoming clear that sex is both a predictor of outcome and an independent risk factor for a number of other cardiac diseases. Animal models and human studies have begun to shed light on the mechanisms by which sex influences the function of cardiomyocytes in health and disease. This review will survey the current literature on cardiac diseases that are influenced by sex and discuss the intracellular mechanisms by which steroid sex hormones affect heart function. A theory on how sex may regulate myocardial energy metabolism to affect disease susceptibility and progression will be presented, as well as a discussion of how sex may influence outcomes of experiments on isolated cardiomyocytes by epigenetic marking.

RyR1/SERCA1 cross-talk regulation of calcium transport in heavy sarcoplasmic reticulum vesicles.

We investigated the functional interdependence of sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1 in heavy sarcoplasmic reticulum membranes by synchronous fluorescence determination of extravesicular Ca2+ transients and catalytic activity. Under conditions of dynamic Ca2+ exchange ATPase catalytic activity was well coordinated to ryanodine receptor activation/inactivation states. Ryanodine-induced activation of Ca2+ release channel leaks also produced marked ATPase activation in the absence of measurable increases in bulk free extravesicular Ca2+. This suggested that Ca2+ pumps are highly sensitive to Ca2+ release channel leak status and potently buffer Ca2+ ions exiting cytoplasmic openings of ryanodine receptors. Conversely, ryanodine receptor activation was dependent on Ca2+-ATPase pump activity. Ryanodine receptor activation by cytosolic Ca2+ was (i) inversely proportional to luminal Ca2+ load and (ii) dependent upon the rate of presentation of cytosolic Ca2+. Progressive Ca2+ filling coincided with progressive loss of Ca2+ sequestration rates and at a threshold loading, ryanodine-induced Ca2+ release produced small transient reversals of catalytic activity. These data indicate that attainment of threshold luminal Ca2+ loads coordinates sensitization of Ca2+ release channels with autogenic inhibition of Ca2+ pumping. This suggests that Ca2+-dependent control of Ca2+ release in intact heavy sarcoplasmic reticulum membranes involves a Ca2+-mediated "cross-talk" between sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1.

Nucleoplasmic calcium regulation in rabbit aortic vascular smooth muscle cells.

In this study, we investigated whether nucleoplasmic free Ca2+ in aortic vascular smooth muscle cells (VSMCs) might be independently regulated from cytosolic free Ca2+. Understanding mechanisms and pathways responsible for this regulation is especially relevant given the role of a numerous intranuclear Ca2+-sensitive proteins in transcriptional regulation, apoptosis and cell division. The question of an independent regulatory mechanism remains largely unsettled because the previous use of intensitometric fluorophores (e.g., Fluo-3) has been criticized on technical grounds. To circumvent the potential problem of fluorescence artifact, we utilized confocal laser scanning microscopy to image intracellular Ca2+ movements with the ratiometric fluorophore Indo-1. In cultured rabbit VSMCs, we found sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) pumps and ryanodine receptor (RyR) Ca2+ channel proteins to be discretely arranged within a perinuclear locus, as determined by fluorescent staining patterns of BODIPY FL thapsigargin and BODIPY FL-X Ry. When intracellular Ca2+ stores were mobilized by addition of thapsigargin (5 microM) and activatory concentrations of ryanodine (1 microM), Indo-1 ratiometric signals were largely restricted to the nucleoplasm. Cytosolic signals, by comparison, were relatively small and even then its spatial distribution was largely perinuclear rather homogeneous. These observations indicate perinuclear RyR and SERCA proteins are intimately involved in regulating VSMC nucleoplasmic Ca2+ concentrations. We also observed a similar pattern of largely nucleoplasmic Ca2+ mobilization upon exposure of cells to the immunosuppressant drug FK506 (tacrolimus), which binds to the RyR-associated immunophillin-binding proteins FKBP12 and FKBP12.6. However, initial FK506-induced nucleoplasmic Ca2+ mobilization was followed by marked reduction of Indo-1 signal intensity close to pretreatment levels. This suggested FK506 exerts both activatory and inhibitory effects upon RyR channels. The latter was reinforced by observed effects of FK506 to only reduce nucleoplasmic Indo-1 signal intensity when added following pretreatment with both activatory and inhibitory concentrations of ryanodine. These latter observations raise the possibility that VSMC nuclei represent an important sink of intracellular Ca2+ and may help explain vasodilatory actions of FK506 observed by others.

Nucleolin is a calcium-binding protein.

We have purified a prominent 110-kDa protein (p110) from 1.6 M NaCl extracts of rat liver nuclei that appears to bind Ca2+. p110 was originally identified by prominent blue staining with 'Stains-All' in sodium dodecyl sulfate-polyacrylamide gels and was observed to specifically bind ruthenium red and 45Ca2+ in nitrocellulose blot overlays. In spin-dialysis studies, purified p110 saturably bound approximately 75 nmol Ca2+/mg protein at a concentration of 1 mM total Ca2+ with half-maximal binding observed at 105 microM Ca2+. With purification, p110 became increasingly susceptible to proteolytic (likely autolytic) fragmentation, although most intermediary peptides between 40 and 90 kDa retained "Stains-All", ruthenium red, and 45Ca2+ binding. N-terminal sequencing of intact p110 and a 70-kDa autolytic peptide fragment revealed a strong homology to nucleolin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/IEF revealed autolysis produced increasingly acidic peptide fragments ranging in apparent pI's from 5.5 for intact p110 to 3.5 for a 40 kDa peptide fragment. Intact p110 and several peptide fragments were immunostained with a highly specific anti-nucleolin antibody, R2D2, thus confirming the identity of this protein with nucleolin. These annexin-like Ca2+-binding characteristics of nucleolin are likely contributed by its highly acidic argyrophilic N-terminus with autolysis apparently resulting in largely selective removal of its basic C-terminal domain. Although the Ca2+-dependent functions of nucleolin are unknown, we discuss the possibility that like the structurally analogous HMG-1, its Ca2+-dependent actions may regulate chromatin structure, possibly during apoptosis.