An insulator is a DNA sequence that can act as a barrier to the influences of neighboring cis-acting elements, preventing gene activation, for example, when located between an enhancer and a promoter. We have identified a 42 bp fragment of the chicken beta-globin insulator that is both necessary and sufficient for enhancer blocking activity in human cells. We show that this sequence is the binding site for CTCF, a previously identified eleven-zinc finger DNA-binding protein that is highly conserved in vertebrates. CTCF sites are present in all of the vertebrate enhancer-blocking elements we have examined. We suggest that directional enhancer blocking by CTCF is a conserved component of gene regulation in vertebrates.

Naive lymphocytes continually enter and exit lymphoid organs in a recirculation process that is essential for immune surveillance. During immune responses, the egress process can be shut down transiently. When this occurs locally it increases lymphocyte numbers in the responding lymphoid organ; when it occurs systemically it can lead to immunosuppression as a result of the depletion of recirculating lymphocytes. Several mediators of the innate immune system are known to cause shutdown, including interferon alpha/beta (IFN-alpha/beta) and tumour necrosis factor, but the mechanism has been unclear. Here we show that treatment with the IFN-alpha/beta inducer polyinosine polycytidylic acid (hereafter 'poly(I:C)') inhibited egress by a mechanism that was partly lymphocyte-intrinsic. The transmembrane C-type lectin CD69 was rapidly induced and CD69-/- cells were poorly retained in lymphoid tissues after treatment with poly(I:C) or infection with lymphocytic choriomeningitis virus. Lymphocyte egress requires sphingosine 1-phosphate receptor-1 (S1P1), and IFN-alpha/beta was found to inhibit lymphocyte responsiveness to S1P. By contrast, CD69-/- cells retained S1P1 function after exposure to IFN-alpha/beta. In coexpression experiments, CD69 inhibited S1P1 chemotactic function and led to downmodulation of S1P1. In a reporter assay, S1P1 crosslinking led to co-crosslinking and activation of a CD69-CD3zeta chimaera. CD69 co-immunoprecipitated with S1P1 but not the related receptor, S1P3. These observations indicate that CD69 forms a complex with and negatively regulates S1P1 and that it functions downstream of IFN-alpha/beta, and possibly other activating stimuli, to promote lymphocyte retention in lymphoid organs.

The nuclear expression and action of the nuclear factor kappa B (NF-kappaB) transcription factor requires signal-coupled phosphorylation and degradation of the IkappaB inhibitors, which normally bind and sequester this pleiotropically active factor in the cytoplasm. The subsequent molecular events that regulate the termination of nuclear NF-kappaB action remain poorly defined, although the activation of de novo IkappaBalpha gene expression by NF-kappaB likely plays a key role. Our studies now demonstrate that the RelA subunit of NF-kappaB is subject to inducible acetylation and that acetylated forms of RelA interact weakly, if at all, with IkappaBalpha. Acetylated RelA is subsequently deacetylated through a specific interaction with histone deacetylase 3 (HDAC3). This deacetylation reaction promotes effective binding to IkappaBalpha and leads in turn to IkappaBalpha-dependent nuclear export of the complex through a chromosomal region maintenance-1 (CRM-1)-dependent pathway. Deacetylation of RelA by HDAC3 thus acts as an intranuclear molecular switch that both controls the duration of the NF-kappaB transcriptional response and contributes to the replenishment of the depleted cytoplasmic pool of latent NF-kappaB-IkappaBalpha complexes.

The promoter of the murine gene encoding inducible nitric oxide synthase (iNOS) contains an NF-kappa B site beginning 55 base pairs upstream of the TATA box, designated NF-kappa Bd. Reporter constructs containing truncated promoter regions, when transfected into macrophages, revealed that NF-kappa Bd is necessary to confer inducibility by bacterial lipopolysaccharide (LPS). Oligonucleotide probes containing NF-kappa Bd plus the downstream 9 or 47 base pairs bound proteins that rapidly appeared in the nuclei of LPS-treated macrophages. The nuclear proteins bound to both probes in an NF-kappa Bd-dependent manner, but binding was resistant to cycloheximide only for the shorter probe. The proteins binding both probes reacted with antibodies against p50 and c-rel but not RelB; those binding the shorter probe also reacted with anti-RelA (p65). Pyrrolidine dithiocarbamate, which acts as a specific inhibitor of NF-kappa B, blocked both the activation of the NF-kappa Bd-binding proteins and the production of NO in LPS-treated macrophages. Thus, activation of NF-kappa B/Rel is critical in the induction of iNOS by LPS. However, additional, newly synthesized proteins contribute to the NF-kappa Bd-dependent transcription factor complex on the iNOS promoter in LPS-treated mouse macrophages.

Hepatocyte growth factor (HGF) is the most potent mitogen for mature parenchymal hepatocytes in primary culture, and seems to be a hepatotrophic factor that acts as a trigger for liver regeneration after partial hepatectomy and liver injury. The partial purification and characterization of HGF have been reported. We have demonstrated that pure HGF from rat platelets is a new growth factor effective at concentrations as low as 1 ng ml-1. The effects of HGF and epidermal growth factor (EGF) are additive. The activity of HGF is not species-specific, although it does not stimulate growth in Swiss 3T3 fibroblasts. HGF has a relative molecular mass (Mr) of 82,000 and is a heterodimer composed of a large alpha-subunit of Mr 69,000 and a small beta-subunit of Mr 34,000. Here we report the amino-acid sequence of human HGF determined by complementary DNA cloning and the expression of biologically active human HGF from COS-1 cells transfected with cloned cDNA. The nucleotide sequence of the human HGF cDNA reveals that both alpha- and beta-chains are contained in a single open reading frame coding for a pre-pro precursor protein of 728 amino acids.

The AMP-activated protein kinase (AMPK) system acts as a sensor of cellular energy status that is conserved in all eukaryotic cells. It is activated by increases in the cellular AMP:ATP ratio caused by metabolic stresses that either interfere with ATP production (eg, deprivation for glucose or oxygen) or that accelerate ATP consumption (eg, muscle contraction). Activation in response to increases in AMP involves phosphorylation by an upstream kinase, the tumor suppressor LKB1. In certain cells (eg, neurones, endothelial cells, and lymphocytes), AMPK can also be activated by a Ca(2+)-dependent and AMP-independent process involving phosphorylation by an alternate upstream kinase, CaMKKbeta. Once activated, AMPK switches on catabolic pathways that generate ATP, while switching off ATP-consuming processes such as biosynthesis and cell growth and proliferation. The AMPK complex contains 3 subunits, with the alpha subunit being catalytic, the beta subunit containing a glycogen-sensing domain, and the gamma subunits containing 2 regulatory sites that bind the activating and inhibitory nucleotides AMP and ATP. Although it may have evolved to respond to metabolic stress at the cellular level, hormones and cytokines such as insulin, leptin, and adiponectin can interact with the system, and it now appears to play a key role in maintaining energy balance at the whole body level. The AMPK system may be partly responsible for the health benefits of exercise and is the target for the antidiabetic drug metformin. It is a key player in the development of new treatments for obesity, type 2 diabetes, and the metabolic syndrome.

Small-molecule inhibitors of PARP are thought to mediate their antitumor effects as catalytic inhibitors that block repair of DNA single-strand breaks (SSB). However, the mechanism of action of PARP inhibitors with regard to their effects in cancer cells is not fully understood. In this study, we show that PARP inhibitors trap the PARP1 and PARP2 enzymes at damaged DNA. Trapped PARP-DNA complexes were more cytotoxic than unrepaired SSBs caused by PARP inactivation, arguing that PARP inhibitors act in part as poisons that trap PARP enzyme on DNA. Moreover, the potency in trapping PARP differed markedly among inhibitors with niraparib (MK-4827)>> olaparib (AZD-2281)>>) veliparib (ABT-888), a pattern not correlated with the catalytic inhibitory properties for each drug. We also analyzed repair pathways for PARP-DNA complexes using 30 genetically altered avian DT40 cell lines with preestablished deletions in specific DNA repair genes. This analysis revealed that, in addition to homologous recombination, postreplication repair, the Fanconi anemia pathway, polymerase β, and FEN1 are critical for repairing trapped PARP-DNA complexes. In summary, our study provides a new mechanistic foundation for the rational application of PARP inhibitors in cancer therapy.

The principles of shared decision making are well documented but there is a lack of guidance about how to accomplish the approach in routine clinical practice. Our aim here is to translate existing conceptual descriptions into a three-step model that is practical, easy to remember, and can act as a guide to skill development. Achieving shared decision making depends on building a good relationship in the clinical encounter so that information is shared and patients are supported to deliberate and express their preferences and views during the decision making process. To accomplish these tasks, we propose a model of how to do shared decision making that is based on choice, option and decision talk. The model has three steps: a) introducing choice, b) describing options, often by integrating the use of patient decision support, and c) helping patients explore preferences and make decisions. This model rests on supporting a process of deliberation, and on understanding that decisions should be influenced by exploring and respecting "what matters most" to patients as individuals, and that this exploration in turn depends on them developing informed preferences.

The heterotrimeric guanine nucleotide-binding proteins (G proteins) act as switches that regulate information processing circuits connecting cell surface receptors to a variety of effectors. The G proteins are present in all eukaryotic cells, and they control metabolic, humoral, neural, and developmental functions. More than a hundred different kinds of receptors and many different effectors have been described. The G proteins that coordinate receptor-effector activity are derived from a large gene family. At present, the family is known to contain at least sixteen different genes that encode the alpha subunit of the heterotrimer, four that encode beta subunits, and multiple genes encoding gamma subunits. Specific transient interactions between these components generate the pathways that modulate cellular responses to complex chemical signals.

BACKGROUND

MicroRNAs (miRNAs), a class of short non-coding RNAs found in many plants and animals, often act post-transcriptionally to inhibit gene expression.

RESULTS

Here we report the analysis of miRNA expression in 93 primary human breast tumors, using a bead-based flow cytometric miRNA expression profiling method. Of 309 human miRNAs assayed, we identify 133 miRNAs expressed in human breast and breast tumors. We used mRNA expression profiling to classify the breast tumors as luminal A, luminal B, basal-like, HER2+ and normal-like. A number of miRNAs are differentially expressed between these molecular tumor subtypes and individual miRNAs are associated with clinicopathological factors. Furthermore, we find that miRNAs could classify basal versus luminal tumor subtypes in an independent data set. In some cases, changes in miRNA expression correlate with genomic loss or gain; in others, changes in miRNA expression are likely due to changes in primary transcription and or miRNA biogenesis. Finally, the expression of DICER1 and AGO2 is correlated with tumor subtype and may explain some of the changes in miRNA expression observed.

CONCLUSIONS

This study represents the first integrated analysis of miRNA expression, mRNA expression and genomic changes in human breast cancer and may serve as a basis for functional studies of the role of miRNAs in the etiology of breast cancer. Furthermore, we demonstrate that bead-based flow cytometric miRNA expression profiling might be a suitable platform to classify breast cancer into prognostic molecular subtypes.

Activated CD8+ T cells play a critical role in host defense against viruses, intracellular microbes, and tumors. It is not clear if a key regulatory transcription factor unites the effector functions of CD8+ T cells. We now show that Eomesodermin (Eomes), a paralogue of T-bet, is induced in effector CD8+ T cells in vitro and in vivo. Ectopic expression of Eomes was sufficient to invoke attributes of effector CD8+ T cells, including interferon-gamma (IFN-gamma), perforin, and granzyme B. Loss-of-function analysis suggests Eomes may also be necessary for full effector differentiation of CD8+ T cells. We suggest that Eomesodermin is likely to complement the actions of T-bet and act as a key regulatory gene in the development of cell-mediated immunity.

Dopamine plays an important role in the etiology of schizophrenia, and D2 class dopamine receptors are the best-established target of antipsychotic drugs. Here we show that D2 class-receptor-mediated Akt regulation involves the formation of signaling complexes containing beta-arrestin 2, PP2A, and Akt. beta-arrestin 2 deficiency in mice results in reduction of dopamine-dependent behaviors, loss of Akt regulation by dopamine in the striatum, and disruption of the dopamine-dependent interaction of Akt with its negative regulator, protein phosphatase 2A. Importantly, canonical cAMP-mediated dopamine-receptor signaling is not inhibited in the absence of beta-arrestin 2. These results demonstrate that, apart from its classical function in receptor desensitization, beta-arrestin 2 also acts as a signaling intermediate through a kinase/phosphatase scaffold. Furthermore, this function of beta-arrestin 2 is important for the expression of dopamine-associated behaviors, thus implicating beta-arrestin 2 as a positive mediator of dopaminergic synaptic transmission and a potential pharmacological target for dopamine-related psychiatric disorders.

BACKGROUND

Exposing eukaryotic cells to lithium ions (Li+) during development has marked effects on cell fate and organization. The phenotypic consequences of Li+ treatment on Xenopus embryos and sporulating Dictyostelium are similar to the effects of inhibition or disruption, respectively, of a highly conserved protein serine/threonine kinase, glycogen synthase kinase-3 (GSK-3). In Drosophila, the GSK-3 homologue is encoded by zw3sgg, a segment-polarity gene involved in embryogenesis that acts downstream of wg. In higher eukaryotes, GSK-3 has been implicated in signal transduction pathways downstream of phosphoinositide 3-kinase and mitogen-activated protein kinases.

RESULTS

We investigated the effect of Li+ on the activity of the GSK-3 family. At physiological doses, Li+ inhibits the activity of human GSK-3 beta and Drosophila Zw3Sgg, but has no effect on other protein kinases. The effect of Li+ on GSK-3 is reversible in vitro. Treatment of cells with Li+ inhibits GSK-3-dependent phosphorylation of the microtubule-associated protein Tau. Li+ treatment of Drosophila S2 cells and rat PC12 cells induces accumulation of cytoplasmic Armadillo/beta-catenin, demonstrating that Li+ can mimic Wingless signalling in intact cells, consistent with its inhibition of GSK-3.

CONCLUSIONS

Li+ acts as a specific inhibitor of the GSK-3 family of protein kinases in vitro and in intact cells, and mimics Wingless signalling. This reveals a possible molecular mechanism of Li+ action on development and differentiation.

Atherosclerosis is a chronic disease of the arterial wall where both innate and adaptive immunoinflammatory mechanisms are involved. Inflammation is central at all stages of atherosclerosis. It is implicated in the formation of early fatty streaks, when the endothelium is activated and expresses chemokines and adhesion molecules leading to monocyte/lymphocyte recruitment and infiltration into the subendothelium. It also acts at the onset of adverse clinical vascular events, when activated cells within the plaque secrete matrix proteases that degrade extracellular matrix proteins and weaken the fibrous cap, leading to rupture and thrombus formation. Cells involved in the atherosclerotic process secrete and are activated by soluble factors, known as cytokines. Important recent advances in the comprehension of the mechanisms of atherosclerosis provided evidence that the immunoinflammatory response in atherosclerosis is modulated by regulatory pathways, in which the two anti-inflammatory cytokines interleukin-10 and transforming growth factor-beta play a critical role. The purpose of this review is to bring together the current information concerning the role of cytokines in the development, progression, and complications of atherosclerosis. Specific emphasis is placed on the contribution of pro- and anti-inflammatory cytokines to pathogenic (innate and adaptive) and regulatory immunity in the context of atherosclerosis. Based on our current knowledge of the role of cytokines in atherosclerosis, we propose some novel therapeutic strategies to combat this disease. In addition, we discuss the potential of circulating cytokine levels as biomarkers of coronary artery disease.

The crystal structure of a ternary complex of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) heterodimer (p66/p51), a 19-base/18-base double-stranded DNA template-primer, and a monoclonal antibody Fab fragment has been determined at 3.0 A resolution. The four individual subdomains of RT that make up the polymerase domains of p66 and p51 are named fingers, palm, thumb, and connection [Kohlstaedt, L. A., Wang, J., Friedman, J. M., Rice, P. A. & Steitz, T. A. (1992) Science 256, 1783-1790]. The overall folding of the subdomains is similar in p66 and p51 but the spatial arrangements of the subdomains are dramatically different. The template-primer has A-form and B-form regions separated by a significant bend (40-45 degrees). The most numerous nucleic acid interactions with protein occur primarily along the sugar-phosphate backbone of the DNA and involve amino acid residues of the palm, thumb, and fingers of p66. Highly conserved regions are located in the p66 palm near the polymerase active site. These structural elements, together with two alpha-helices of the thumb of p66, act as a clamp to position the template-primer relative to the polymerase active site. The 3'-hydroxyl of the primer terminus is close to the catalytically essential Asp-110, Asp-185, and Asp-186 residues at the active site and is in a position for nucleophilic attack on the alpha-phosphate of an incoming nucleoside triphosphate. The structure of the HIV-1 RT/DNA/Fab complex should aid our understanding of general mechanisms of nucleic acid polymerization. AIDS therapies may be enhanced by a fuller understanding of drug inhibition and resistance emerging from these studies.

In addition to the canonical Wnt/beta-catenin signaling pathway, at least two noncanonical Wnt/Fz pathways have been described: the planar cell polarity (PCP) pathway in Drosophila [1] and the Wnt/calcium pathway in vertebrate embryos [2]. Recent work suggests that a vertebrate pathway homologous to the PCP pathway acts to regulate the convergent extension movements of gastrulation [3-7]. To further test this hypothesis, we have identified two zebrafish homologs of the Drosophila PCP gene prickle (pk) [8], both of which show discrete and dynamic expression patterns during gastrulation. Both gain and loss of pk1 function cause defects in convergent extension. Pk1 localizes to both the cytoplasm and the cell membrane, and its normal localization is partially dependent on its C-terminal prenylation motif. At the cell membrane, Pk1 is frequently localized asymmetrically around the cell and can colocalize with the signaling molecule Dishevelled (Dsh). In overexpression assays, Pk1 is able to activate AP-1-mediated transcription and inhibit activation of Wnt/beta-catenin signaling. Like noncanonical Wnts [9-10], overexpression of Pk1 increases the frequency of calcium transients in zebrafish blastulae. Our results support the idea that a vertebrate PCP pathway regulates gastrulation movements and suggest that there is overlap between the PCP and Wnt/calcium pathways.

Diabetes, a disease in which carbohydrate and lipid metabolism are regulated improperly by insulin, is a serious worldwide health issue. Insulin is secreted from pancreatic beta cells in response to elevated plasma glucose, with various factors modifying its secretion. Free fatty acids (FFAs) provide an important energy source as nutrients, and they also act as signalling molecules in various cellular processes, including insulin secretion. Although FFAs are thought to promote insulin secretion in an acute phase, this mechanism is not clearly understood. Here we show that a G-protein-coupled receptor, GPR40, which is abundantly expressed in the pancreas, functions as a receptor for long-chain FFAs. Furthermore, we show that long-chain FFAs amplify glucose-stimulated insulin secretion from pancreatic beta cells by activating GPR40. Our results indicate that GPR40 agonists and/or antagonists show potential for the development of new anti-diabetic drugs.

Diabetes, a disease in which the body does not produce or use insulin properly, is a serious global health problem. Gut polypeptides secreted in response to food intake, such as glucagon-like peptide-1 (GLP-1), are potent incretin hormones that enhance the glucose-dependent secretion of insulin from pancreatic beta cells. Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules in various cellular processes, including the secretion of gut incretin peptides. Here we show that a G-protein-coupled receptor, GPR120, which is abundantly expressed in intestine, functions as a receptor for unsaturated long-chain FFAs. Furthermore, we show that the stimulation of GPR120 by FFAs promotes the secretion of GLP-1 in vitro and in vivo, and increases circulating insulin. Because GLP-1 is the most potent insulinotropic incretin, our results indicate that GPR120-mediated GLP-1 secretion induced by dietary FFAs is important in the treatment of diabetes.

FoxO Forkhead transcription factors are shown here to act as signal transducers at the confluence of Smad, PI3K, and FoxG1 pathways. Smad proteins activated by TGF-beta form a complex with FoxO proteins to turn on the growth inhibitory gene p21Cip1. This process is negatively controlled by the PI3K pathway, a known inhibitor of FoxO localization in the nucleus, and by the telencephalic development factor FoxG1, which we show binds to FoxO-Smad complexes and blocks p21Cip1 expression. We suggest that the activity of this network confers resistance to TGF-beta-mediated cytostasis during the development of the telencephalic neuroepithelium and in glioblastoma brain tumor cells.

A single entity, the AMP-activated protein kinase (AMPK), phosphorylates and regulates in vivo hydroxymethylglutaryl-CoA reductase and acetyl-CoA carboxylase (key regulatory enzymes of sterol synthesis and fatty acid synthesis, respectively), and probably many additional targets. The kinase is activated by high AMP and low ATP via a complex mechanism, which involves allosteric regulation, promotion of phosphorylation by an upstream protein kinase (AMPK kinase), and inhibition of dephosphorylation. This protein-kinase cascade represents a sensitive system, which is activated by cellular stresses that deplete ATP, and thus acts like a cellular fuel gauge. Our central hypothesis is that, when it detects a 'low-fuel' situation, it protects the cell by switching off ATP-consuming pathways (e.g. fatty acid synthesis and sterol synthesis) and switching on alternative pathways for ATP generation (e.g. fatty acid oxidation). Native AMP-activated protein kinase is a heterotrimer consisting of a catalytic alpha subunit, and beta and gamma subunits, which are also essential for activity. All three subunits have homologues in budding yeast, which are components of the SNF1 protein-kinase complex. SNF1 is activated by glucose starvation (which in yeast leads to ATP depletion) and genetic studies have shown that it is involved in derepression of glucose-repressed genes. This raises the intriguing possibility that AMPK may regulate gene expression in mammals. AMPK/SNF1 homologues are found in higher plants, and this protein-kinase cascade appears to be an ancient system which evolved to protect cells against the effects of nutritional or environmental stress.

Several specific conclusions can be drawn from these studies: 1. IL-4 is required for the generation of both primary polyclonal and secondary antigen-specific IgE responses in vivo. 2. IL-4 is required to maintain established, ongoing, antigen-specific and polyclonal IgE responses. 3. Most, but not all, polyclonal IgE production during a secondary immune response is IL-4-dependent. Memory B cells that have already switched to IgE at the DNA level may no longer require stimulation with IL-4 to be induced to secrete IgE. 4. The generation of a secondary IgE response is not dependent upon the presence of IL-4 during primary immunization. However, if IL-4 is not present during primary immunization, it is required during secondary immunization for the generation of an IgE response. 5. IL-4 does not appear to be required for the generation of in vivo IgG1 responses, and in at least some instances, does not contribute significantly to the generation of IgG1 responses in vivo. 6. A late-acting form of T-cell help other than IL-4 appears to be required for the generation of an IgE, but not an IgG1 response. 7. An antibody that inhibits IL-4 binding to IL-4 receptors affects Ig isotype selection in the same way as an antibody that neutralizes IL-4. 8. IFN-gamma can act in both spontaneous and induced immune responses to suppress IgE production. 9. IFN-gamma can also suppress IgG1 production and stimulate IgG2a production. However, IFN-gamma appears to suppress polyclonal IgG1 responses more than antigen-specific IgG1 responses, and it enhances, but is not required for, the generation of IgG2a responses. 10. IFN-alpha appears to resemble IFN-gamma in its ability to inhibit IgE and enhance IgG2a responses in GaM delta-injected mice, but it requires the presence of IFN-gamma to suppress IgG1 production in these mice. 11. Both IFN-alpha and IFN-gamma appear to be able to decrease IgE production in some human patients. 12. There is no direct evidence that IL-5 contributes to the generation of in vivo antibody responses. Two general conclusions may also be drawn.(ABSTRACT TRUNCATED AT 400 WORDS)

Nuclear factor kappa B (NF-kappa B), which was first detected by its binding to the kappa B site in the immunoglobulin kappa-gene enhancer, is important for the regulated expression of the kappa-gene and is partly responsible for the induction in appropriate cells of interleukin-2 (IL-2), IL-2 alpha receptor, beta-interferon and serum amyloid A protein. NF-kappa B is present as a nuclear DNA-binding protein in B lymphocytes and mature macrophages, but is found in the cytoplasm of many cells in a form unable to bind to DNA. The cytoplasmic form is bound to an inhibitor protein, I kappa B, from which it can be released in vitro by deoxycholate and other agents. Activation of cells by various agents, notably the phorbol esters that stimulate protein kinase C (PKC), leads to dissociation in vivo of the NF-kappa B/I kappa B complex and migration of NF-kappa B to the nucleus. Therefore, it acts as a second messenger system, transducing activation signals from the cytoplasm to the nucleus. To elucidate the mechanism of signal transfer, we have used an in vitro system in which addition of purified protein kinases to a partially purified NF-kappa B/I kappa B complex leads to the activation of the DNA-binding activity of NF-kappa B. Using gel retardation assays we found that PKC, cyclic AMP-dependent protein kinase (PKA) and a haem-regulated eIF-2 kinase (HRI) could activate NF-kappa B in vitro, whereas casein kinase II was ineffective. To determine the target for the protein kinases we purified and characterized both NF-kappa B and I kappa B and found that I kappa B is phosphorylated and inactivated in the presence of PKC and HRI but not PKA.

Naive CD4+ T cells when stimulated produce IL-2 as their major lymphokine. Upon priming, these cells develop into cells that produce either IFN gamma, TNF beta, and IL-2 or IL-4 and its congeners. The former cells are designated TH1-like, and the latter TH2-like. Here we review the regulation of the differentiation of naive CD4 cells into IFN gamma- or IL-4-producers. The dominant factors that determine such differentiation are lymphokines and other cytokines. IL-2 itself appears to be required for naive cells to develop into TH1- or TH2-like cells but is not deterministic of their differentiation fate. If IL-4 is also present during the priming period, the resultant CD4+ T cells produce IL-4 upon restimulation; the development of IFN gamma-producing cells is strikingly inhibited by IL-4. In the absence of IL-4, priming for IFN gamma-production occurs, but this is markedly enhanced by IL-12. The role of IFN gamma in enhancing priming for IFN gamma-production is not fully resolved. In some in vitro systems, it appears to act together with IL-12 to enhance such production. Anti-IFN gamma diminishes priming for IFN gamma production in vivo. Lymphokines also exert a "cross-regulatory" or inhibitory effect. As noted above, IL-4 strikingly diminishes priming for IFN gamma production, although this inhibitory effect is blunted in the presence of IL-12. IFN gamma similarly diminishes priming for IL-4 production; this effect is principally observed when low concentrations of IL-4 are used in the priming culture. Although other factors may play a role in the determination of lymphokine-producing phenotype, such as antigen dose, type of antigen-presenting cell, and expression of accessory molecules and hormones, these effects appear to be secondary to the dominant role of the lymphokines and cytokines.

The lipocalin protein family is a large group of small extracellular proteins. The family demonstrates great diversity at the sequence level; however, most lipocalins share three characteristic conserved sequence motifs, the kernel lipocalins, while a group of more divergent family members, the outlier lipocalins, share only one. Belying this sequence dissimilarity, lipocalin crystal structures are highly conserved and comprise a single eight-stranded continuously hydrogen-bonded antiparallel beta-barrel, which encloses an internal ligand-binding site. Together with two other families of ligand-binding proteins, the fatty-acid-binding proteins (FABPs) and the avidins, the lipocalins form part of an overall structural superfamily: the calycins. Members of the lipocalin family are characterized by several common molecular-recognition properties: the ability to bind a range of small hydrophobic molecules, binding to specific cell-surface receptors and the formation of complexes with soluble macromolecules. The varied biological functions of the lipocalins are mediated by one or more of these properties. In the past, the lipocalins have been classified as transport proteins; however, it is now clear that the lipocalins exhibit great functional diversity, with roles in retinol transport, invertebrate cryptic coloration, olfaction and pheromone transport, and prostaglandin synthesis. The lipocalins have also been implicated in the regulation of cell homoeostasis and the modulation of the immune response, and, as carrier proteins, to act in the general clearance of endogenous and exogenous compounds.