The dnaK and groESL operons of Bacillus subtilis are preceded by a potential sigma 43 promoter sequence (recognized by the vegetative sigma factor) and by an inverted repeat (IR) consisting of 9 bp separated by a 9-bp spacer. Since this IR has been found in many bacterial species, we suspected that it might be involved in heat shock regulation. In order to test this hypothesis, three different mutational alterations of three bases were introduced within the IR preceding the dnaK operon. These mutations were crossed into the chromosome of B. subtilis, and expression of the dnaK and of the unlinked groESL operons was studied. The dnaK operon exhibited increased expression at low temperature and a reduction in the stimulation after temperature upshift. Furthermore, these mutations reduced expression of the groESL operon at low temperature by 50% but did not interfere with stimulation after heat shock. These experiments show that the IR acts as a negative cis element of the dnaK operon. This conclusion was strengthened by the observation that the IR reduced expression of two different transcriptional fusions significantly after its insertion between the promoter and the reporter gene. Since this IR has been described in many bacterial species as preceding only genes of the dnaK and groESL operons, both encoding molecular chaperones (39 cases are documented so far), we designated this heat shock element CIRCE (controlling IR of chaperone expression). Furthermore, we suggest that this novel mechanism is more widespread among eubacteria than the regulation mechanism described for Escherichia coli and has a more ancient origin.

Defensins (molecular weight 3500 to 4000) act in the mammalian immune response by permeabilizing the plasma membranes of a broad spectrum of target organisms, including bacteria, fungi, and enveloped viruses. The high-resolution crystal structure of defensin HNP-3 (1.9 angstrom resolution, R factor 0.19) reveals a dimeric beta sheet that has an architecture very different from other lytic peptides. The dimeric assembly suggests mechanisms by which defensins might bind to and permeabilize the lipid bilayer.

Major histocompatibility complex (MHC) class I molecules are integral membrane proteins present on virtually all vertebrate cells and consist of a heterodimer between the highly polymorphic alpha-chain and the beta 2-microglobulin (beta 2-m) protein of relative molecular mass 12,000 (ref. 1). These cell-surface molecules play a pivotal part in the recognition of antigens, the cytotoxic response of T cells, and the induction of self tolerance. It is possible, however, that the function of MHC class I molecules is not restricted to the immune system, but extends to a wide variety of biological reactions including cell-cell interactions. For example, MHC class I molecules seem to be associated with various cell-surface proteins, including the receptors for insulin, epidermal growth factor, luteinizing hormone and the beta-adrenergic receptor. In mice, class I molecules are secreted in the urine and act as highly specific olfactory cues which influence mating preference. The beta 2-m protein has also been identified as the smaller component of the Fc receptor in neonatal intestinal cells, and it has been suggested that the protein induces collagenase in fibroblasts. Cells lacking beta 2-m are deficient in the expression of MHC class I molecules, indicating that the association with beta 2-m is crucial for the transport of MHC class I molecules to the cell surface. The most direct means of unravelling the many biological functions of beta 2-m is to create a mutant mouse with a defective beta 2-m gene. We have now used the technique of homologous recombination to disrupt the beta 2-m gene. We report here that introduction of a targeting vector into embryonic stem cells resulted in beta 2-m gene disruption with high frequency. Chimaeric mice derived from blastocysts injected with mutant embryonic stem cell clones transmit the mutant allele to their offspring.

Most examples of cell death in animals are controlled by a genetic program that is activated within the dying cell. The apoptotic process is further regulated by a set of genes that act as repressors of cell death. Of these, bcl-2 is expressed in a variety of embryonic and postnatal tissues which suggests a critical role for bcl-2 in organogenesis and tissue homeostasis. Surprisingly, mutant mice with targeted disruption of bcl-2 appear normal at birth and complete maturation of lymphoid tissues before succumbing to fulminant lymphopenia and polycystic renal disease by 2-5 weeks of age. This suggests that there may be genes other than bcl-2 that can regulate apoptosis during development. To begin to investigate this possibility, we have cloned and characterized the murine bcl-x gene, whose human counterpart displays striking homology to bcl-2. The predicted murine bcl-xL gene product exhibits a high level of amino acid identity (97%) to its human counterpart. Just like Bcl-2, the murine bcl-xL gene product can act as a dominant inhibitor of cell death upon growth factor withdrawal. In addition, the bulk of the bcl-xL product localizes to the periphery of mitochondria as assessed by a bcl-xL-tag expression system, suggesting that both Bcl-2 and Bcl-xL proteins prevent cell death by a similar mechanism. bcl-xL is the most abundant bcl-x mRNA species expressed in embryonic and adult tissues. The levels of bcl-xL mRNA appear higher than those of bcl-2 during embryonal development and in several adult organs including bone marrow, brain, kidney and thymus. In addition to bcl-xL, we have identified another form of bcl-x mRNA, bcl-x beta, that results from an unspliced bcl-x transcript. bcl-x beta mRNA is expressed in various embryonic and postnatal tissues. Surprisingly, the expression of bcl-xS (a negative regulator of programmed cell death) was undetectable by a sensitive S1-nuclease assay and polymerase chain reaction analysis of mouse tissues. Based on its tissue and developmental patterns of expression, it appears that bcl-x may play an important role in the regulation of cell death during development and tissue homeostasis.

We have recently shown that vascular endothelial growth factor (VEGF) is produced by human malignant glioma cells and acts on tumor endothelial cells, which express VEGF receptors, suggesting that VEGF is a regulator of tumor angiogenesis. To investigate the feasibility of antiangiogenic brain tumor therapy, we developed an intracerebral (i.c.) rat glioma model. We used two transplantable rat glioma cells lines, C6 and GS-9L, to analyze VEGF regulation in vitro and expression of VEGF and its high affinity tyrosine kinase receptors, flt-1 and flk-1, in vivo. Glioma cells were transplanted i.c. or s.c. into syngeneic rats. C6 gliomas exhibit morphological characteristics of human glioblastoma multiforme such as necroses with palisading cells. Immunocytochemistry with von Willebrand factor showed that C6 gliomas are highly vascularized and therefore show another prominent feature of human glioblastoma. GS-9L gliosarcomas were less vascularized. In situ hybridization showed that VEGF is expressed in vivo in rat glioma cells which reside along necrotic areas and therefore closely mimicks the expression pattern of VEGF observed in human glioblastoma. flt-1 and flk-1 are specifically expressed in endothelial cells in the tumor and at the border between tumor and normal brain but are absent from endothelial cells in the normal brain proper. The action of VEGF may therefore be restricted to tumor endothelium. Upregulation of VEGF, but not acid fibroblast growth factor, basic fibroblast growth factor, and platelet-derived growth factor B messenger RNA was observed in hypoxic C6 and GS-9L cells in vitro. These observations are consistent with a role for VEGF in tumor- and hypoxia-induced angiogenesis. Since the expression pattern of VEGF and its receptors in rat glioma appears to be indistinguishable from human glioblastoma multiforme, this model provides an excellent tool to study anti-angiogenic therapy.

Chronic exposure to nicotine elicits upregulation of high-affinity nicotinic receptors in the smoker's brain. To address the molecular mechanism of upregulation, we transfected HEK293 cells with human alpha4<em>beta</em>2 receptors and traced the subunits throughout their intracellular biosynthesis, using metabolic labeling and immunoprecipitation techniques. We show that high-mannose glycosylated subunits mature and assemble into pentamers in the endoplasmic reticulum and that only pentameric receptors reach the cell surface following carbohydrate processing. Nicotine is shown to <em>act</em> inside the cell and to increase the amount of <em>beta</em> subunits immunoprecipitated by the conformation-dependent mAb290, indicating that nicotine enhances a critical step in the intracellular maturation of these receptors. This effect, which also takes place at concentrations of nicotine found in the blood of smokers upon expression of alpha4<em>beta</em>2 in SH-SY5Y neuroblastoma cells, may play a crucial role in nicotine addiction and possibly implement a model of neural plasticity.

Natriuretic peptides (NPs), mainly produced in heart [atrial (ANP) and B-type (BNP)], brain (CNP), and kidney (urodilatin), decrease blood pressure and increase salt excretion. These functions are mediated by natriuretic peptide receptors A and B (NPRA and NPRB) having cytoplasmic guanylyl cyclase domains that are stimulated when the receptors bind ligand. A more abundantly expressed receptor (NPRC or C-type) has a short cytoplasmic domain without guanylyl cyclase activity. NPRC is thought to act as a clearance receptor, although it may have additional functions. To test how NPRC affects the cardiovascular and renal systems, we inactivated its gene (Npr3) in mice by homologous recombination. The half life of [125I]ANP in the circulation of homozygotes lacking NPRC is two-thirds longer than in the wild type, although plasma levels of ANP and BNP in heterozygotes and homozygotes are close to the wild type. Heterozygotes and homozygotes have a progressively reduced ability to concentrate urine, exhibit mild diuresis, and tend to be blood volume depleted. Blood pressure in the homozygotes is 8 mmHg (1 mmHg = 133 Pa) below normal. These results are consistent with the sole cardiovascular/renal function of NPRC being to clear natriuretic peptides, thereby modulating local effects of the natriuretic peptide system. Unexpectedly, Npr3 -/- homozygotes have skeletal deformities associated with a considerable increase in bone turnover. The phenotype is consistent with the bone function of NPRC being to clear locally synthesized CNP and modulate its effects. We conclude that NPRC modulates the availability of the natriuretic peptides at their target organs, thereby allowing the activity of the natriuretic peptide system to be tailored to specific local needs.

BACKGROUND

A significant number of patients treated with anthracyclines develop cardiotoxicity (anthracycline-induced cardiotoxicity [ACT]), mainly presenting as arrhythmias (acute ACT) or congestive heart failure (chronic ACT). There are no data on pharmacogenomic predictors of ACT.

RESULTS

We genotyped participants of the German non-Hodgkin lymphoma study (NHL-B) who were followed up for the development of heart failure for a median of >3 years. Single-nucleotide polymorphisms (SNPs) were selected from 82 genes with conceivable relevance to ACT. Of 1697 patients, 55 developed acute and 54 developed chronic ACT (cumulative incidence of either form, 3.2%). We detected 5 significant associations with polymorphisms of the NAD(P)H oxidase and doxorubicin efflux transporters. Chronic ACT was associated with a variant of the NAD(P)H oxidase subunit NCF4 (rs1883112, -212A->>G; symbols with right-pointing arrows, as edited?' odds ratio [OR], 2.5; 95% CI, 1.3 to 5.0). Acute ACT was associated with the His72Tyr polymorphism in the p22phox subunit (rs4673; OR, 2.0; 95% CI, 1.0 to 3.9) and with the variant 7508T->>A (rs13058338; OR, 2.6; 95% CI, 1.3 to 5.1) of the RAC2 subunit of the same enzyme. In agreement with these results, mice deficient in NAD(P)H oxidase activity, unlike wild-type mice, were resistant to chronic doxorubicin treatment. In addition, acute ACT was associated with the Gly671Val variant of the doxorubicin efflux transporter multidrug resistance protein 1 (MRP1) (OR, 3.6; 95% CI, 1.6 to 8.4) and with the Val1188Glu-Cys1515Tyr (rs8187694-rs8187710) haplotype of the functionally similar MRP2 (OR, 2.3; 95% CI, 1.0 to 5.4). Polymorphisms in adrenergic receptors previously demonstrated to be predictive of heart failure were not associated with ACT.

CONCLUSIONS

Genetic variants in doxorubicin transport and free radical metabolism may modulate the individual risk to develop ACT.

OBJECTIVE

Severe sepsis and septic shock is a consequence of a generalized inflammatory systemic response because of an invasive infection that may result in acute organ dysfunction. Mortality is high despite access to modern intensive care units. The nuclear DNA binding protein high mobility group 1 (HMG<em>B</em>1) protein has recently been suggested to <em>act</em> as a late mediator of septic shock via its function as a macrophage-derived pro-inflammatory cytokine (J Exp Med 2000; 192: 565, Science1999; 285: 248). We investigated the pro-inflammatory <em>act</em>ivities of the A-box and the <em>B</em>-box of HMG<em>B</em>1 on human umbilical venular endothelial cells (HUVEC).

METHODS

The HUVEC obtained from healthy donors were used for experiments. Recombinant human full-length HMGBB-box were cloned by polymerase chain reaction (PCR) amplification from a human brain quick-clone cDNA. The activation of HUVEC was studied regarding (i) upregulation of adhesion molecules, (ii) the release of cytokines and chemokines, (iii) the adhesion of neutrophils to HUVEC, (iv) the activation of signalling transduction pathways and (v) the involvement of the receptor for advanced glycation end-products (RAGE).

RESULTS

The full-length protein and the B-box of HMGBactivate HUVEC to upregulate adhesion molecules such as ICAM-1, VCAM-1 and E-selectin and to release IL-8 and G-CSF. The activation of HUVEC could be inhibited to 50% by antibodies directed towards the RAGE. HMGBBB.

CONCLUSIONS

The HMGBacts as a potent pro-inflammatory cytokine on HUVEC and the activity is mainly mediated through the B-box of the protein. HMGBactor mediating part of the pro-inflammatory response occurring in septic shock and severe inflammation.

Xyloglucans are the principal glycans that interlace cellulose microfibrils in most flowering plants. The mur3 mutant of Arabidopsis contains a severely altered structure of this polysaccharide because of the absence of a conserved alpha-L-fucosyl-(1-->2)-beta-D-galactosyl side chain and excessive galactosylation at an alternative xylose residue. Despite this severe structural alteration, mur3 plants were phenotypically normal and exhibited tensile strength in their inflorescence stems comparable to that of wild-type plants. The MUR3 gene was cloned positionally and shown to encode a xyloglucan galactosyltransferase that acts specifically on the third xylose residue within the XXXG core structure of xyloglucan. MUR3 belongs to a large family of type-II membrane proteins that is evolutionarily conserved among higher plants. The enzyme shows sequence similarities to the glucuronosyltransferase domain of exostosins, a class of animal glycosyltransferases that catalyze the synthesis of heparan sulfate, a glycosaminoglycan with numerous roles in cell differentiation and development. This finding suggests that components of the plant cell wall and of the animal extracellular matrix are synthesized by evolutionarily related enzymes even though the structures of the corresponding polysaccharides are entirely different from each other.

Deletion of the fission yeast mitotic B-type cyclin gene cdc13 causes cells to undergo successive rounds of DNA replication. We have used a strain which expresses cdc13 conditionally to investigate re-replication. Activity of Start genes cdc2 and cdc10 is necessary and p34cdc2 kinase is active in re-replicating cells. We tested to see whether other cyclins were required for re-replication using cdc13delta. Further deletion of cig1 and puc1 had no effect, but deletion of cig2/cyc17 caused a severe delay in re-replication. Deletion of cig1 and cig2/cyc17 together abolished re-replication completely and cells arrested in G1. This, and analysis of the temperature sensitive cdc13-117 mutant, suggests that cdc13 can effectively substitute for the G1 cyclin activity of cig2/cyc17. We have characterized p56cdc13 activity and find evidence that in the absence of G1 cyclins, S-phase is delayed until the mitotic p34cdc2-p56cdc13 kinase is sufficiently active. These data suggest that a single oscillation of p34cdc2 kinase activity provided by a single B-type cyclin can promote ordered progression into both DNA replication and mitosis, and that the level of cyclin-dependent kinase activity may act as a master regulator dictating whether cells undergo S-phase or mitosis.

OBJECTIVE

We sought to determine whether exosome-like vesicles (ELVs) released from adipose tissue play a role in activation of macrophages and subsequent development of insulin resistance in a mouse model.

METHODS

ELVs released from adipose tissue were purified by sucrose gradient centrifugation and labeled with green fluorescent dye and then intravenously injected into B6 ob/ob mice (obese model) or B6 mice fed a high-fat diet. The effects of injected ELVs on the activation of macrophages were determined through analysis of activation markers by fluorescence-activated cell sorter and induction of inflammatory cytokines using an ELISA. Glucose tolerance and insulin tolerance were also evaluated. Similarly, B6 mice with different gene knockouts including TLR2, TLR4, MyD88, and Toll-interleukin-1 receptor (TIR) domain-containing adaptor protein inducing interferon-beta (TRIF) were also used for testing their responses to the injected ELVs.

RESULTS

ELVs are taken up by peripheral blood monocytes, which then differentiate into activated macrophages with increased secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Injection of obELVs into wild-type C57BL/6 mice results in the development of insulin resistance. When the obELVs were intravenously injected into TLR4 knockout B6 mice, the levels of glucose intolerance and insulin resistance were much lower. RBP4 is enriched in the obELVs. Bone marrow-derived macrophages preincubated with recombinant RBP4 led to attenuation of obELV-mediated induction of IL-6 and TNF-alpha.

CONCLUSIONS

ELVs released by adipose tissue can act as a mode of communication between adipose tissues and macrophages. The obELV-mediated induction of TNF-alpha and IL-6 in macrophages and insulin resistance requires the TLR4/TRIF pathway.

Embryonic stem (ES) cells rely on growth factors provided by feeder cells or exogenously to maintain their pluripotency. In order to identify such factors, we have established sub-lines of STO feeder cells which exhibit variable ability in supporting ES cell self-renewal. Functional screening identifies WNT5A and WNT6 as STO cell-produced factors that potently inhibit ES cell differentiation in a serum-dependent manner. Furthermore, direct activation of beta-catenin without disturbing the upstream components of the WNT/beta-catenin pathway fully recapitulates the effect of WNTs on ES cells. Importantly, the WNT/beta-catenin pathway up-regulates the mRNA for Stat3, a known regulator of ES cell self-renewal in the mouse. Finally, LIF is able to mimic the serum effect to act synergistically with WNT proteins to inhibit ES cell differentiation. Therefore, our study reveals part of the molecular mechanisms by which the WNT/beta-catenin pathway acts to prevent ES cell differentiation through convergence on the LIF/JAK-STAT pathway at the level of STAT3.

Anti-cytokine antibodies that block interactions between cytokines and cytokine receptors have been used to inhibit endogenous cytokine function. However, injection of mice with mixtures of IL-4 and either of two neutralizing anti-IL-4 mAb, at a cytokine/anti-cytokine mAb molar ratio of approximately 2:1, enhances and prolongs in vivo IL-4 activity, as measured by induction of increased spleen cell Ia expression. Although splenocyte Ia expression returns to baseline two days after mice are injected with free IL-4, soluble IL-4-anti-IL-4 mAb complexes still induce several-fold increases in Ia expression 3 days after injection. Complexes that contain as little as 400 ng of IL-4 have considerable in vivo stimulatory activity, and a maximal effect on splenocyte Ia expression is induced by injection of 2 micrograms of complexed IL-4. The stimulatory effect of IL-4-containing complexes on splenocyte Ia expression can be blocked by increasing the ratio of anti-IL-4 mAb to IL-4, by injection of anti-IL-4R mAb, and by in vivo aggregation of the complexes. Complexes of IL-4 with a non-neutralizing anti-IL-4 mAb do not have increased IL-4 agonist activity in vivo. These observations are most consistent with the possibility that neutralizing anti-IL-4 mAb act as carrier proteins that increase the in vivo half-life of IL-4 by preventing its excretion, and possibly, by preventing modification of its active site. The enhanced agonist effect of IL-4-anti-IL-4 mAb complexes is not unique; complexes of IL-3 with a neutralizing anti-IL-3 mAb have a greatly increased ability, compared with free IL-3, to stimulate mucosal mastocytosis, and complexes of IL-7 with a neutralizing anti-IL-7 mAb have a greatly increased ability, compared with free IL-7 or IL-7 complexed with a non-neutralizing anti-IL-7 mAb, to stimulate an increase in pre-B cell number. These observations suggest that complexes of cytokines and neutralizing anti-cytokine mAb may provide a generally useful way to increase the magnitude and duration of cytokine effects in vivo.

Genetic and pharmacological studies of indoleamine 2,3-dioxygenase (IDO) have established this tryptophan catabolic enzyme as a central driver of malignant development and progression. IDO acts in tumor, stromal and immune cells to support pathogenic inflammatory processes that engender immune tolerance to tumor antigens. The multifaceted effects of IDO activation in cancer include the suppression of T and NK cells, the generation and activation of T regulatory cells and myeloid-derived suppressor cells, and the promotion of tumor angiogenesis. Mechanistic investigations have defined the aryl hydrocarbon receptor, the master metabolic regulator mTORC1 and the stress kinase Gcn2 as key effector signaling elements for IDO, which also exerts a non-catalytic role in TGF-β signaling. Small-molecule inhibitors of IDO exhibit anticancer activity and cooperate with immunotherapy, radiotherapy or chemotherapy to trigger rapid regression of aggressive tumors otherwise resistant to treatment. Notably, the dramatic antitumor activity of certain targeted therapeutics such as imatinib (Gleevec) in gastrointestinal stromal tumors has been traced in part to IDO downregulation. Further, antitumor responses to immune checkpoint inhibitors can be heightened safely by a clinical lead inhibitor of the IDO pathway that relieves IDO-mediated suppression of mTORC1 in T cells. In this personal perspective on IDO as a nodal mediator of pathogenic inflammation and immune escape in cancer, we provide a conceptual foundation for the clinical development of IDO inhibitors as a novel class of immunomodulators with broad application in the treatment of advanced human cancer.

Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic lung infections in cystic fibrosis patients and is a major source of nosocomial infections. This bacterium controls many virulence factors by using two quorum-sensing systems, las and rhl. The las system is composed of the LasR regulator protein and its cell-to-cell signal, N-(3-oxododecanoyl) homoserine lactone, and the rhl system is composed of RhlR and the signal N-butyryl homoserine lactone. A third intercellular signal, the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4-quinolone), also regulates numerous virulence factors. PQS synthesis requires the expression of multiple operons, one of which is pqsABCDE. Previous experiments showed that the transcription of this operon, and therefore PQS production, is negatively regulated by the rhl quorum-sensing system and positively regulated by the las quorum-sensing system and PqsR (also known as MvfR), a LysR-type transcriptional regulator protein. With the use of DNA mobility shift assays and beta-galactosidase reporter fusions, we have studied the regulation of pqsR and its relationship to pqsA, lasR, and rhlR. We show that PqsR binds the promoter of pqsA and that this binding increases dramatically in the presence of PQS, implying that PQS acts as a coinducer for PqsR. We have also mapped the transcriptional start site for pqsR and found that the transcription of pqsR is positively regulated by lasR and negatively regulated by rhlR. These results suggest that a regulatory chain occurs where pqsR is under the control of LasR and RhlR and where PqsR in turn controls pqsABCDE, which is required for the production of PQS.

OBJECTIVE

Insulin secretion involves complex events in which the mitochondria play a pivotal role in the generation of signals that couple glucose detection to insulin secretion. Studies on the mitochondrial generation of reactive oxygen species (ROS) generally focus on chronic nutrient exposure. Here, we investigate whether transient mitochondrial ROS production linked to glucose-induced increased respiration might act as a signal for monitoring insulin secretion.

METHODS

ROS production in response to glucose was investigated in freshly isolated rat islets. ROS effects were studied using a pharmacological approach and calcium imaging.

RESULTS

Transient glucose increase from 5.5 to 16.7 mmol/l stimulated ROS generation, which was reversed by antioxidants. Insulin secretion was dose dependently blunted by antioxidants and highly correlated with ROS levels. The incapacity of beta-cells to secrete insulin in response to glucose with antioxidants was associated with a decrease in ROS production and in contrast to the maintenance of high levels of ATP and NADH. Then, we investigated the mitochondrial origin of ROS (mROS) as the triggering signal. Insulin release was mimicked by the mitochondrial-complex blockers, antimycin and rotenone, that generate mROS. The adding of antioxidants to mitochondrial blockers or to glucose was used to lower mROS reversed insulin secretion. Finally, calcium imaging on perifused islets using glucose stimulation or mitochondrial blockers revealed that calcium mobilization was completely reversed using the antioxidant trolox and that it was of extracellular origin. No toxic effects were present using these pharmacological approaches.

CONCLUSIONS

Altogether, these complementary results demonstrate that mROS production is a necessary stimulus for glucose-induced insulin secretion.

The voltage-gated calcium channel α(2)δ and β subunits are traditionally considered to be auxiliary subunits that enhance channel trafficking, increase the expression of functional calcium channels at the plasma membrane and influence the channels' biophysical properties. Accumulating evidence indicates that these subunits may also have roles in the nervous system that are not directly linked to calcium channel function. For example, β subunits may act as transcriptional regulators, and certain α(2)δ subunits may function in synaptogenesis. The aim of this Review is to examine both the classic and novel roles for these auxiliary subunits in voltage-gated calcium channel function and beyond.

The recent development of molecular biology and mouse genetics and the analysis of the skeletal phenotype induced by genetic mutations in humans led to a better understanding of the role of transcription factors that govern bone formation. This review summarizes the role of transcription factors in osteoblastogenesis and provides an integrated perspective on how the activities of multiple classes of factors are coordinated for the complex process of developing the osteoblast phenotype. The roles of Runx2, the principal transcriptional regulator of osteoblast differentiation, Osterix, beta-Catenin and ATF which act downstream of Runx2, and other transcription factors that contribute to the control of osteoblastogenesis including the AP1, C/EBPs, PPARgamma and homeodomain, helix-loop-helix proteins are discussed. This review also updates the regulation of transcription factor expression by signaling factors and hormones that control osteoblastogenesis.

The trefoil peptides, a recently recognized family of protease-resistant peptides, expressed in a regional specific pattern throughout the normal gastrointestinal tract. Although these peptides have been hypothesized to act as growth factors, their functional properties are largely unknown. Addition of recombinant trefoil peptides human spasmolytic polypeptide (HSP), rat and human intestinal trefoil factor (RITF and HITF) to subconfluent nontransformed rat intestinal epithelial cell lines (IEC-6 and IEC-17), human colon cancer-derived cell lines (HT-29 and CaCO2) or nontransformed fibroblasts (NRK and BHK) had no significant effect on proliferation. However addition of the trefoil peptides to wounded monolayers of confluent IEC-6 cells in an in vitro model of epithelial restitution resulted in a 3-6-fold increase in the rate of epithelial migration into the wound. Stimulation of restitution by the trefoil peptide HSP was enhanced in a cooperative fashion by the addition of mucin glycoproteins purified from the colon or small intestine of either rat or man, achieving up to a 15-fold enhancement in restitution. No synergistic effect was observed by the addition of nonmucin glycoproteins. In contrast to cytokine stimulation of intestinal epithelial cell restitution which is mediated through enhanced TGF beta bioactivity, trefoil peptide, and trefoil peptide-mucin glycoprotein stimulation of restitution was not associated with alteration in concentrations of bioactive TGF-beta and was not affected by the presence of immunoneutralizing anti-TGF beta antiserum. Collectively, these findings suggest that the trefoil peptides which are secreted onto the lumenal surface of the gastrointestinal tract may act in conjunction with the mucin glycoprotein products of goblet cells to promote reestablishment of mucosal integrity after injury through mechanisms distinct from those which may act at the basolateral pole of the epithelium.

Memory CD8(+) T cells in the lung airways provide protection from secondary respiratory virus challenge by limiting early viral replication. Here, we demonstrate that although airway-resident memory CD8(+) T cells were poorly cytolytic, memory CD8(+) T cells recruited to the airways early during a recall response showed markedly enhanced cytolytic ability. This enhanced lytic activity did not require cognate antigen stimulation, but rather was dependent on STAT1 transcription factor signaling through the interferon-alpha receptor (Ifnar1), resulting in the antigen-independent expression of granzyme B protein in both murine and human virus-specific T cells. Signaling through Ifnar1 was required for the enhanced lytic activity and control of early viral replication by memory CD8(+) T cells in the lung airways. These findings demonstrate that innate inflammatory signals act directly on memory T cells, enabling them to rapidly destroy infected host cells once they enter infected tissues.

Blimp1, a zinc-finger containing DNA-binding transcriptional repressor, functions as a master regulator of B cell terminal differentiation. Considerable evidence suggests that Blimp1 is required for the establishment of anteroposterior axis formation and the formation of head structures during early vertebrate development. In mouse embryos, Blimp1 is strongly expressed in axial mesendoderm, the tissue known to provide anterior patterning signals during gastrulation. Here, we describe for the first time the defects caused by loss of Blimp1 function in the mouse. Blimp1 deficient embryos die at mid-gestation, but surprisingly early axis formation, anterior patterning and neural crest formation proceed normally. Rather, loss of Blimp1 expression disrupts morphogenesis of the caudal branchial arches and leads to a failure to correctly elaborate the labyrinthine layer of the placenta. Blimp1 mutant embryos also show widespread blood leakage and tissue apoptosis, and, strikingly, Blimp1 homozygous mutants entirely lack PGCs. At the time of PGC allocation around 7.25 days post coitum, Blimp1 heterozygous embryos exhibit decreased numbers of PCGs. Thus Blimp1 probably acts to turn off the default pathway that allows epiblast cells to adopt a somatic cell fate, and shifts the transcriptional program so that they become exclusively allocated into the germ cell lineage.

Soluble oligomers of Abeta42 peptide are believed to play a major role in the pathogenesis of Alzheimer disease (AD). It was recently found that at least some of the neurotoxic effects of these oligomers may be mediated by specific binding to the prion protein, PrP(C), on the cell surface (Laurén, J., Gimbel, D. A., Nygaard, H. B., Gilbert, J. W., and Strittmatter, S. M. (2009) Nature 457, 1128-1132). Here we characterized the interaction between synthetic Abeta42 oligomers and the recombinant human prion protein (PrP) using two biophysical techniques: site-directed spin labeling and surface plasmon resonance. Our data indicate that this binding is highly specific for a particular conformation adopted by the peptide in soluble oligomeric species. The binding appears to be essentially identical for the Met(129) and Val(129) polymorphic forms of human PrP, suggesting that the role of PrP codon 129 polymorphism as a risk factor in AD is due to factors unrelated to the interaction with Abeta oligomers. It was also found that in addition to the previously identified approximately 95-110 segment, the second region of critical importance for the interaction with Abeta42 oligomers is a cluster of basic residues at the extreme N terminus of PrP (residues 23-27). The deletion of any of these segments results in a major loss of the binding function, indicating that these two regions likely act in concert to provide a high affinity binding site for Abeta42 oligomers. This insight may help explain the interplay between the postulated protective and pathogenic roles of PrP in AD and may contribute to the development of novel therapeutic strategies as well.

BACKGROUND

Immunoglobulin A (IgA) deficiency is the most common primary immunodeficiency defined as decreased serum level of IgA in the presence of normal levels of other immunoglobulin isotypes. Most individuals with IgA deficiency are asymptomatic and identified coincidentally. However, some patients may present with recurrent infections of the respiratory and gastrointestinal tracts, allergic disorders, and autoimmune manifestations. IGA AND ITS FUNCTIONS: Although IgA is the most abundant antibody isotype produced in the body, its functions are not clearly understood. Subclass IgA1 in monomeric form is mainly found in the blood circulation, whereas subclass IgA2 in dimeric form is the dominant immunoglobulin in mucosal secretions. Secretory IgA appears to have prime importance in immune exclusion of pathogenic microorganisms and maintenance of intestinal homeostasis. Despite this critical role, there may be some compensatory mechanisms that would prevent disease manifestations in some IgA-deficient individuals.

METHODS

In IgA deficiency, a maturation defect in B cells to produce IgA is commonly observed. Alterations in transmembrane activator and calcium modulator and cyclophilin ligand interactor gene appear to act as disease-modifying mutations in both IgA deficiency and common variable immunodeficiency, two diseases which probably lie in the same spectrum. Certain major histocompatibility complex haplotypes have been associated with susceptibility to IgA deficiency.

CONCLUSIONS

The genetic basis of IgA deficiency remains to be clarified. Better understanding of the production and function of IgA is essential in elucidating the disease mechanism in IgA deficiency.