Effects of SRI-32743, a Novel Quinazoline Structure-Based Compound, on HIV-1 Tat and Cocaine Interaction with Norepinephrine Transporter.

Prolonged exposure to HIV-1 transactivator of transcription (Tat) protein dysregulates monoamine transmission, a physiological change implicated as a key factor in promoting neurocognitive disorders among people living with HIV. We have demonstrated that in vivo expression of Tat in Tat transgenic mice decreases dopamine uptake through both dopamine transporter (DAT) and norepinephrine transporter (NET) in the prefrontal cortex. Further, our novel allosteric inhibitor of monoamine transporters, SRI-32743, has been shown to attenuate Tat-inhibited dopamine transport through DAT and alleviates Tat-potentiated cognitive impairments. The current study reports the pharmacological profiles of SRI-32743 in basal and Tat-induced inhibition of human NET (hNET) function. SRI-32743 exhibited less affinity for hNET binding than desipramine, a classical NET inhibitor, but displayed similar potency for inhibiting hDAT and hNET activity. SRI-32743 concentration-dependently increased hNET affinity for [3H]DA uptake but preserved the Vmax of dopamine transport. SRI-32743 slowed the cocaine-mediated dissociation of [3H]Nisoxetine binding and reduced both [3H]DA and [3H]MPP+ efflux but did not affect d-amphetamine-mediated [3H]DA release through hNET. Finally, we determined that SRI-32743 attenuated a recombinant Tat1-86-induced decrease in [3H]DA uptake via hNET. Our findings demonstrated that SRI-32743 allosterically disrupts the recombinant Tat1-86-hNET interaction, suggesting a potential treatment for HIV-infected individuals with concurrent cocaine abuse.

Role of HIV-1 Tat Protein Interactions with Host Receptors in HIV Infection and Pathogenesis.

Each time the virus starts a new round of expression/replication, even under effective antiretroviral therapy (ART), the transactivator of viral transcription Tat is one of the first HIV-1 protein to be produced, as it is strictly required for HIV replication and spreading. At this stage, most of the Tat protein exits infected cells, accumulates in the extracellular matrix and exerts profound effects on both the virus and neighbor cells, mostly of the innate and adaptive immune systems. Through these effects, extracellular Tat contributes to the acquisition of infection, spreading and progression to AIDS in untreated patients, or to non-AIDS co-morbidities in ART-treated individuals, who experience inflammation and immune activation despite virus suppression. Here, we review the role of extracellular Tat in both the virus life cycle and on cells of the innate and adaptive immune system, and we provide epidemiological and experimental evidence of the importance of targeting Tat to block residual HIV expression and replication. Finally, we briefly review vaccine studies showing that a therapeutic Tat vaccine intensifies ART, while its inclusion in a preventative vaccine may blunt escape from neutralizing antibodies and block early events in HIV acquisition.

HIV gp120/Tat protein-induced epithelial-mesenchymal transition promotes the progression of cervical lesions.

BACKGROUND: Human immunodeficiency virus (HIV) infection is associated with an elevated incidence of cervical cancer, and accelerated disease progression, but the underlying mechanisms are not well understood. This study aimed to investigate the relationship between HIV infection and epithelial-mesenchymal transition (EMT) in cervical cancer. METHODS: Tissue samples from HIV-positive and negative patients with cervical intraepithelial neoplasia (CIN) and cervical cancer were analyzed for EMT-related proteins. Human cervical cancer SiHa cells were treated with HIV Tat and gp120 proteins to test their effects on EMT, migration, and invasion. RESULTS: HIV-positive patients had lower E-cadherin and cytokeratin, and higher N-cadherin and vimentin levels than HIV-negative patients. HIV Tat and gp120 proteins induced EMT, migration, and invasion in SiHa cells. Transcriptome sequencing analysis revealed that, compared to the control group, the protein-treated group showed upregulation of 22 genes and downregulation of 77 genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed the involvement of the Wnt signaling pathway in EMT. Further analysis of gene expression related to this pathway revealed upregulation of DVL1, TCF7, KRT17, and VMAC, while GSK3ß, SFRP2, and CDH1 were downregulated. Immunofluorescence assay demonstrated that HIVgp120 and Tat proteins treatment induced elevated ß-catenin expression with nuclear accumulation in SiHa cells. CONCLUSIONS: The treatment of SiHa cells with HIV Tat and gp120 proteins induces EMT and activates the Wnt/ß-catenin pathway, suggesting that the Wnt/ß-catenin pathway may play a crucial role in promoting EMT progression in cervical lesion tissues of HIV-infected patients.

Accurate Prediction of Transcriptional Activity of Single Missense Variants in HIV Tat with Deep Learning.

Tat is an essential gene for increasing the transcription of all HIV genes, and affects HIV replication, HIV exit from latency, and AIDS progression. The Tat gene frequently mutates in vivo and produces variants with diverse activities, contributing to HIV viral heterogeneity as well as drug-resistant clones. Thus, identifying the transcriptional activities of Tat variants will help to better understand AIDS pathology and treatment. We recently reported the missense mutation landscape of all single amino acid Tat variants. In these experiments, a fraction of double missense alleles exhibited intragenic epistasis. However, it is too time-consuming and costly to determine the effect of the variants for all double mutant alleles through experiments. Therefore, we propose a combined GigaAssay/deep learning approach. As a first step to determine activity landscapes for complex variants, we evaluated a deep learning framework using previously reported GigaAssay experiments to predict how transcription activity is affected by Tat variants with single missense substitutions. Our approach achieved a 0.94 Pearson correlation coefficient when comparing the predicted to experimental activities. This hybrid approach can be extensible to more complex Tat alleles for a better understanding of the genetic control of HIV genome transcription.

T-cell evasion and invasion during HIV-1 infection: The role of HIV-1 Tat protein.

T-cell-mediated immune responses play indispensable roles in the defense against infectious pathogens including human immunodeficiency virus type 1 (HIV-1) which can establish a persistent infection that leads to many alterations in T-cell-mediated immunity. The latter include T-cell hyperactivation and depletion, both of which are essential for disease progression. Determining the factors and mechanisms pathways that lead to such abnormalities in T-cell mediated immunity during HIV-1 infection and ascertaining how the virus is able to evade immune responses elicited by T cells are critical for understanding the pathophysiology of HIV-1 infection, which in turn, could lead to new insights that may accelerate the development of novel and effective therapeutic strategies. To this end, we addressed the roles played by HIV-1 Tat protein, one of the first proteins to be expressed, in the pathogenesis of HIV-1 infection, focusing on the pathological effects of this protein in the cellular adaptive immune response in which T cells are intimately involved.

Chronic Exposure to HIV-Derived Protein Tat Impairs Endothelial Function via Indirect Alteration in Fat Mass and Nox1-Mediated Mechanisms in Mice.

People living with human immunodeficiency virus (HIV) (PLWH) have increased risk for atherosclerosis-related cardiovascular disease (CVD), the main cause of death in this population. Notwithstanding, the mechanisms of HIV-associated vascular pathogenesis are not fully elucidated. Therefore, we sought to determine whether HIV-regulatory protein Tat mediates HIV-induced endothelial dysfunction via NADPH oxidase 1 (Nox1)-dependent mechanisms. Body weight, fat mass, leptin levels, expression of reactive oxygen species (ROS)-producing enzymes and vascular function were assessed in C57BL/6 male mice treated with Tat for 3 days and 4 weeks. Aortic rings and human endothelial cells were also treated with Tat for 2-24 h in ex vivo and in vitro settings. Chronic (4 weeks) but not acute (3 days and 2-24 h) treatment with Tat decreased body weight, fat mass, and leptin levels and increased the expression of Nox1 and its coactivator NADPH oxidase Activator 1 (NoxA1). This was associated with impaired endothelium-dependent vasorelaxation. Importantly, specific inhibition of Nox1 with GKT771 and chronic leptin infusion restored endothelial function in Tat-treated mice. These data rule out direct effects of HIV-Tat on endothelial function and imply the contribution of reductions in adipose mass and leptin production which likely explain upregulated expression of Nox1 and NoxA1. The Nox1 and leptin system may provide potential targets to improve vascular function in HIV infection-associated CVD.

Tat basic domain: A "Swiss army knife" of HIV-1 Tat?

Tat (transactivator of transcription) regulates transcription from the HIV provirus. It plays a crucial role in disease progression, supporting efficient replication of the viral genome. Tat also modulates many functions in the host genome via its interaction with chromatin and proteins. Many of the functions of Tat are associated with its basic domain rich in arginine and lysine residues. It is still unknown why the basic domain exhibits so many diverse functions. However, the highly charged basic domain, coupled with the overall structural flexibility of Tat protein itself, makes the basic domain a key player in binding to or associating with cellular and viral components. In addition, the basic domain undergoes diverse posttranslational modifications, which further expand and modulate its functions. Here, we review the current knowledge of Tat basic domain and its versatile role in the interaction between the virus and the host cell.

Boosting Tat DNA vaccine with Tat protein stimulates strong cellular and humoral immune responses in mice.

The aim of the present study was to evaluate the efficacy of a novel DNA priming-protein boosting regimen in simultaneous enhancing humoral and cellular immunogenicity of the HIV-1-Tat-based candidate vaccines in mice. The experimental BALB/c mice were successfully immunized with the HIV-1-Tat DNA vaccine and boosted with the corresponding protein vaccine over a two-week interval and the elicitation of cellular and humoral immune responses were simultaneously assessed. The results showed that the prime-boost immunization has significantly given rise to lymphocyte proliferation and CTL responses, as well as the levels of both IgG and IgG antibodies compared to the other candidate vaccines. The results of the Th polarization also revealed that the Th1: Th2 indexes in the mice vaccinated with the HIV-1 Tat protein, Tat DNA, and the prime-boost vaccines were 1.03, 1.19, and 1.25, respectively. The results suggest that co-administration of the HIV-1-Tat DNA with the corresponding protein may serve as a potential formulation for enhancing of Tat vaccineinduced immunity and has measurable effects on shaping vaccines' induced Th polarization.

Effects of HIV on the Tumor Microenvironment.

Oncomodulatory viruses can affect the tumor microenvironment (TME) by triggering inflammation, suppressing apoptosis, initiating angiogenesis, altering tumor metabolism, and stimulating tumor cell signaling pathways, leading to tumor growth, proliferation, and invasion. The higher incidence of malignancies among people with HIV (PWH), despite the widespread use of antiretroviral therapy (ART), suggests a more complex relation than HIV-associated immune deregulation. Viral cooperation can have synergistic effect on tumorigenesis. The most relevant oncogenes involved in viral cooperation include the HIV-1-related Tat and Vpu genes, EBV LMP-1 and EBNA-2 genes, and Kaposi's sarcoma herpesvirus (KSHV) KIE2, Rta, and LANA genes. The TME in HIV-related malignancies is highly angiogenic and characterized by high microvessel density compared to sporadic cases. Tat protein, found in patients with HIV infection regardless of their immune status, has been widely implicated in the increased angiogenesis and has been a target of interest for therapeutic strategies. Similarly, HIV-1 matrix protein p17 can be detected in the plasma and tissues of PWH, including those treated with ART. Studies have found that p17 can cause dysregulation of the biological activity of different immune cells, is involved in aberrant angiogenesis, and exhibits an IL8 chemokine activity, activating multiple intracellular signaling pathways, promoting angiogenic responses in endothelial cells, and forming capillary like structures. In addition, several studies have demonstrated difference in the cellular immune components within the TME in patients with or without HIV infection, as well as cases in pre- and post-ART era. In this chapter, we review the existing literature about the role tumor microenvironment plays in the pathogenesis of HIV-related malignancies. Understanding the functions of each component of the TME and determining how these cellular and noncellular components contribute to tumorigenesis will impact the advancement of interventions and treatment in clinical oncology among PWH.

HIV-1 Tat Protein Enters Dysfunctional Endothelial Cells via Integrins and Renders Them Permissive to Virus Replication.

Previous work has shown that the Tat protein of Human Immunodeficiency Virus (HIV)-1 is released by acutely infected cells in a biologically active form and enters dendritic cells upon the binding of its arginine-glycine-aspartic acid (RGD) domain to the α5ß1, αvß3, and αvß5 integrins. The up-regulation/activation of these integrins occurs in endothelial cells exposed to inflammatory cytokines that are increased in HIV-infected individuals, leading to endothelial cell dysfunction. Here, we show that inflammatory cytokine-activated endothelial cells selectively bind and rapidly take up nano-micromolar concentrations of Tat, as determined by flow cytometry. Protein oxidation and low temperatures reduce Tat entry, suggesting a conformation- and energy-dependent process. Consistently, Tat entry is competed out by RGD-Tat peptides or integrin natural ligands, and it is blocked by anti-α5ß1, -αvß3, and -αvß5 antibodies. Moreover, modelling-docking calculations identify a low-energy Tat-αvß3 integrin complex in which Tat makes contacts with both the αv and ß3 chains. It is noteworthy that internalized Tat induces HIV replication in inflammatory cytokine-treated, but not untreated, endothelial cells. Thus, endothelial cell dysfunction driven by inflammatory cytokines renders the vascular system a target of Tat, which makes endothelial cells permissive to HIV replication, adding a further layer of complexity to functionally cure and/or eradicate HIV infection.

The HIV-1 Tat Protein Enhances Splicing at the Major Splice Donor Site.

Transcription of the HIV-1 proviral DNA and subsequent processing of the primary transcript results in the production of a large set of unspliced and differentially spliced viral RNAs. The major splice donor site (5'ss) that is located in the untranslated leader of the HIV-1 transcript is used for the production of all spliced RNAs, and splicing at this site has to be tightly regulated to allow the balanced production of all viral RNAs and proteins. We demonstrate that the viral Tat protein, which is known to activate viral transcription, also stimulates splicing at the major 5'ss. As for the transcription effect, Tat requires the viral long terminal repeat promoter and the trans-acting responsive RNA hairpin for splicing regulation. These results indicate that HIV-1 transcription and splicing are tightly coupled processes through the coordinated action of the essential Tat protein.IMPORTANCE The HIV-1 proviral DNA encodes a single RNA transcript that is used as RNA genome and packaged into newly assembled virus particles. This full-length RNA is also used as mRNA for the production of structural and enzymatic proteins. Production of other essential viral proteins depends on alternative splicing of the primary transcript, which yields a large set of differentially spliced mRNAs. Optimal virus replication requires a balanced production of all viral RNAs, which means that the splicing process has to be strictly regulated. We show that the HIV-1 Tat protein, a factor that is well known for its transcription activating function, also stimulates splicing. Thus, Tat controls not only the level of the viral RNA but also the balance between spliced and unspliced RNAs.

HIV-1 Tat-induced diarrhea is improved by the PPARalpha agonist, palmitoylethanolamide, by suppressing the activation of enteric glia.

BACKGROUND: Diarrhea is a severe complication in HIV-1-infected patients with Trans-activator of transcription (HIV-1 Tat) protein being recognized as a major underlying cause. Beside its direct enterotoxic effects, Tat protein has been recently shown to affect enteric glial cell (EGC) activity. EGCs regulate intestinal inflammatory responses by secreting pro-inflammatory molecules; nonetheless, they might also release immune-regulatory factors, as palmytoilethanolamide (PEA), which exerts anti-inflammatory effects by activating PPARα receptors. We aimed at clarifying whether EGCs are involved in HIV-1 Tat-induced diarrhea and if PEA exerts antidiarrheal activity. METHODS: Diarrhea was induced by intracolonic administration of HIV-1 Tat protein in rats at day 1. PEA alone or in the presence of peroxisome proliferator-activated receptor (PPAR) antagonists was given intraperitoneally from day 2 to day 7. S100B, iNOS, NF-kappaB, TLR4 and GFAP expression were evaluated in submucosal plexi, while S100B and NO levels were measured in EGC submucosal plexi lysates, respectively. To verify whether PEA effects were PPARα-mediated, PPARα-/- mice were also used. After 7 days from diarrhea induction, endogenous PEA levels were measured in submucosal plexi homogenates deriving from rats and PPARα-/- mice. RESULTS: HIV-1 Tat protein induced rapid onset diarrhea alongside with a significant activation of EGCs. Tat administration significantly increased all hallmarks of neuroinflammation by triggering TLR4 and NF-kappaB activation and S100B and iNOS expression. Endogenous PEA levels were increased following HIV-1 Tat exposure in both wildtype and knockout animals. In PPARα-/- mice, PEA displayed no effects. In wildtype rats, PEA, via PPARα-dependent mechanism, resulted in a significant antidiarrheal activity in parallel with marked reduction of EGC-sustained neuroinflammation. CONCLUSIONS: EGCs mediate HIV-1 Tat-induced diarrhea by sustaining the intestinal neuroinflammatory response. These effects are regulated by PEA through a selective PPARα-dependent mechanism. PEA might be considered as an adjuvant therapy in HIV-1-induced diarrhea.

Chemical Composition of Essential Oils from Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis, and Their Effects on the HIV-1 Tat Protein Function.

New drugs would be beneficial to fight resistant HIV strains, in particular those capable of interfering with essential viral functions other than those targeted by highly active antiretroviral therapy drugs. Despite the central role played by Tat protein in HIV transcription, a search for vegetable extracts able to hamper this important viral function was never carried out. In this work, we evaluated the chemical composition and possible interference of essential oil from Thymus vulgaris, Cananga odorata, Cymbopogon citratus, and Rosmarinus officinalis with the Tat/TAR-RNA interaction and with Tat-induced HIV-1 LTR transcription. GC/MS Analysis demonstrated the biodiversity of herbal species translated into essential oils composed of different blends of terpenes. In all of them, 4 - 6 constituents represent from 81.63% to 95.19% of the total terpenes. Essential oils of Thymus vulgaris, Cymbopogon citratus, and Rosmarinus officinalis were active in interfering with Tat functions, encouraging further studies to identify single terpenes responsible for the antiviral activity. In view of the quite different composition of these essential oils, we concluded that their interference on Tat function depends on specific terpene or a characteristic blend.

Interaction of ARC and Daxx: A Novel Endogenous Target to Preserve Motor Function and Cell Loss after Focal Brain Ischemia in Mice.

UNLABELLED: The aim of this study was to explore the signaling and neuroprotective effect of transactivator of transcription (TAT) protein transduction of the apoptosis repressor with CARD (ARC) in in vitro and in vivo models of cerebral ischemia in mice. In mice, transient focal cerebral ischemia reduced endogenous ARC protein in neurons in the ischemic striatum at early reperfusion time points, and in primary neuronal cultures, RNA interference resulted in greater neuronal susceptibility to oxygen glucose deprivation (OGD). TAT.ARC protein delivery led to a dose-dependent better survival after OGD. Infarct sizes 72 h after 60 min middle cerebral artery occlusion (MCAo) were on average 30 ± 8% (mean ± SD; p = 0.005; T2-weighted MRI) smaller in TAT.ARC-treated mice (1 µg intraventricularly during MCAo) compared with controls. TAT.ARC-treated mice showed better performance in the pole test compared with TAT.ß-Gal-treated controls. Importantly, post-stroke treatment (3 h after MCAo) was still effective in affording reduced lesion volume by 20 ± 7% (mean ± SD; p < 0.05) and better functional outcome compared with controls. Delayed treatment in mice subjected to 30 min MCAo led to sustained neuroprotection and functional behavior benefits for at least 28 d. Functionally, TAT.ARC treatment inhibited DAXX-ASK1-JNK signaling in the ischemic brain. ARC interacts with DAXX in a CARD-dependent manner to block DAXX trafficking and ASK1-JNK activation. Our work identifies for the first time ARC-DAXX binding to block ASK1-JNK activation as an ARC-specific endogenous mechanism that interferes with neuronal cell death and ischemic brain injury. Delayed delivery of TAT.ARC may present a promising target for stroke therapy. SIGNIFICANCE STATEMENT: Up to now, the only successful pharmacological target of human ischemic stroke is thrombolysis. Neuroprotective pharmacological strategies are needed to accompany therapies aiming to achieve reperfusion. We describe that apoptosis repressor with CARD (ARC) interacts and inhibits DAXX and proximal signals of cell death. In a murine stroke model mimicking human malignant infarction in the territory of the middle cerebral artery, TAT.ARC salvages brain tissue when given during occlusion or 3 h delayed with sustained functional benefits (28 d). This is a promising novel therapeutic approach because it appears to be effective in a model producing severe injury by interfering with an array of proximal signals and effectors of the ischemic cascade, upstream of JNK, caspases, and BIM and BAX activation.

Blood-brain barrier penetration of an Aß-targeted, arginine-rich, d-enantiomeric peptide.

The application of small peptides targeting amyloid beta (Aß) is one of many drug development strategies for the treatment of Alzheimer's disease (AD). We have previously identified several peptides consisting solely of D-enantiomeric amino acid residues obtained from mirror-image phage display selection, which bind to Aß in different assembly states and eliminate toxic Aß aggregates. Some of these D-peptides show both diagnostic and therapeutic potential in vitro and in vivo. Here we have analysed the similarity of the arginine-rich D-peptide D3 to the arginine-rich motif (ARM) of the human immunodeficiency virus type 1 transactivator of transcription (HIV-Tat) protein, and examined its in vivo blood-brain barrier (BBB) permeability using wild type mice and transgenic mouse models of Alzheimer's disease. We are able to demonstrate that D3 rapidly enters the brain where it can be found associated with amyloid plaques suggesting a direct penetration of BBB.

Neoflavonoids as Inhibitors of HIV-1 Replication by Targeting the Tat and NF-κB Pathways.

Twenty-eight neoflavonoids have been prepared and evaluated in vitro against HIV-1. Antiviral activity was assessed on MT-2 cells infected with viral clones carrying the luciferase reporter gene. Inhibition of HIV transcription and Tat function were tested on cells stably transfected with the HIV-LTR and Tat protein. Seven 4-phenylchromen-2-one derivatives showed HIV transcriptional inhibitory activity but only the phenylchrome-2-one 10 inhibited NF-κB and displayed anti-Tat activity simultaneously. Compounds 10, 14, and 25, inhibited HIV replication in both targets at concentrations <25 µM. The assays of these synthetic 4-phenylchromen-2-ones may aid in the investigation of some aspects of the anti-HIV activity of such compounds and could serve as a scaffold for designing better anti-HIV compounds, which may lead to a potential anti-HIV therapeutic drug.

HIV-Tat induces a decrease in IKr and IKsvia reduction in phosphatidylinositol-(4,5)-bisphosphate availability.

Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulates viral genes expression and is required for efficient HIV replication. Tat is actively secreted into the blood by infected T-cells and affects organs such as the heart. Tat has been shown to alter cardiac repolarization in animal models but how this is mediated and whether this is also the case in human cells is unknown. In the present study, we show that Tat transfection in heterologous expression systems led to a decrease in hERG (underlying cardiac IKr) and human KCNE1-KCNQ1 (underlying cardiac IKs) currents and to an acceleration of their deactivation. This is consistent with a decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP2). A mutant Tat, unable to bind PIP2, did not reproduce the observed effects. In addition, WT-Tat had no effect on a mutant KCNQ1 which is PIP2-insensitive, further confirming the hypothesis. Twenty-four-hour incubation of human induced pluripotent stem cells-derived cardiomyocytes with Wild-type Tat reduced IKr and accelerated its deactivation. Concordantly, this Tat incubation led to a prolongation of the action potential (AP) duration. Events of AP alternans were also recorded in the presence of Tat, and were exacerbated at a low pacing cycle length. Altogether, these data obtained on human K+ channels both in heterologous expression systems and in human cardiomyocytes suggest that Tat sequesters PIP2, leading to a reduction of IKr and IKs, and provide a molecular mechanism for QT prolongation in HIV-infected patients.

δPKC interaction with the d subunit of F1Fo ATP synthase impairs energetics and exacerbates ischemia/reperfusion injury in isolated rat hearts.

Previously, we demonstrated protection against hypoxic injury in neonatal cardiac myocytes and reduced release of cardiac troponin I from perfused rat hearts by a novel peptide inhibitor [NH2-YGRKKRRQRRRMLATRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK-] of the delta protein kinase C (δPKC) interaction with the "d" subunit of mitochondrial F1Fo ATP synthase (dF1Fo). This peptide was developed in our laboratory and contains: an HIV-Tat protein transduction domain; a mitochondrial targeting motif; the δPKC-dF1Fo inhibitor sequence; and a FLAG epitope. In the present study the δPKC-dF1Fo inhibitor attenuated co-immunoprecipitation of δPKC with dF1Fo, improved recovery of contractility, diminished levels of tissue t-carbonyls and 4-hydroxy-2-nonenal (HNE), and reduced 2,3,5-triphenyltetrazolium chloride-monitored infarct size following simulated global ischemia/reperfusion (IR) exposures. Perfusion of hearts with this peptide prior to IR enhanced ATP levels 2.1-fold, improved ADP (state 3)- and FCCP (maximal)-stimulated respiration in mitochondrial oxygen consumption assays, and attenuated Ca(++)-induced mitochondrial swelling following ischemic injury. Mitochondrial membrane potential (assessed by JC-1) was also improved 1.6-fold by the inhibitor in hearts subsequently exposed to IR injury. Brief IR exposures did not cause mitochondrial loss of cytochrome c in the presence or absence of the inhibitor. Additionally, the inhibitor did not modify accumulation of the autophagy marker LC3II after brief IR injury. Our results support the potential for this first-in-class peptide as a translational agent for combating cardiac IR injury.

HIV Tat protein: Is Tat-C much trickier than Tat-B?

Out of various subtypes of human immunodeficiency virus type 1 (HIV-1), subtype B and C cause most of the infections worldwide. Clade specific differences have been reported in differences in clinical picture of HIV pathogenesis. Transcription of the HIV-1 genome is regulated by the interaction of HIV Tat protein to the trans-activation response (TAR) element. The differential binding of clade B and C Tat proteins to TAR and differences in activation of NF-κB cascade leading to differential transactivation capacity and cytokine expression has been examined in this study. More stable Tat-TAR complex formation by Tat-C revealed by EMSA and higher TNF-α expression shown by Tat-C compared to Tat-B leads to higher NF-κB activation, which may be plausible cause for higher transactivation by Tat-C as obtained by FACS analysis. This comparative study would be helpful in understanding the basic mechanism of clade specific Tat protein differences and their functional relationships.

Neuroglobin and neuronal cell survival.

The balance between neuronal apoptosis and survival sculpts the developing brain and has an important role in neurodegenerative diseases. Thus, the individuation of signals that could modulate the cell death machinery as well as enhance survival in neurons promises to provide multiple points of therapeutic intervention in neurodegenerative diseases. Neuroglobin (NGB), the first nerve globin identified in neuronal tissues of humans, seems to possess a protective role in the brain only after up-regulation. Here, the NGB physiological role in the control of neuronal survival is reviewed. In vitro studies suggested that cytosolic NGB could react very rapidly with cytochrome c released from mitochondria, thus interfering with the intrinsic pathway of apoptosis. Although very suggestive, these data do not explain either the role of NGB up-regulation in neuroprotection or the recently reported NGB localization into mitochondria. Recently, we identified the steroid hormone 17ß-estradiol (E2) as an endogenous modulator of NGB levels in neuroblastoma SK-N-BE cell line. Upon E2 stimulation, NGB reallocates mainly into mitochondria where the association with the mitochondrial cytochrome c occurs. Remarkably, E2 treatment before an apoptotic stimulus strongly enhances the NGB:cytochrome c association reducing cytochrome c release into the cytosol. As a consequence, a decrease of caspase-3 activation and, in turn, of the apoptotic cascade activation take place. Besides E2, other compounds have been reported to up-regulate the NGB expression highlighting the possibility to develop NGB-mediated therapeutic strategies against stroke damage and neurodegenerative diseases. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.