Tertiary Proteins Research Articles

An Improved Strategy for Task Scheduling in the Parallel Computational Alignment of Multiple Sequences.

Task scheduling in parallel multiple sequence alignment (MSA) through improved dynamic programming optimization speeds up alignment processing. The increased importance of multiple matching sequences also needs the utilization of parallel processor systems. This dynamic algorithm proposes improved task scheduling in case of parallel MSA. Specifically, the alignment of several tertiary structured proteins is computationally complex than simple word-based MSA. Parallel task processing is computationally more efficient for protein-structured based superposition. The basic condition for the application of dynamic programming is also fulfilled, because the task scheduling problem has multiple possible solutions or options. Search space reduction for speedy processing of this algorithm is carried out through greedy strategy. Performance in terms of better results is ensured through computationally expensive recursive and iterative greedy approaches. Any optimal scheduling schemes show better performance in heterogeneous resources using CPU or GPU.

How to build complexity

Summary The ribosome is the most complex molecular machine shared by all cellular life forms. Details of its evolution from an RNA world precursor are still encrypted in its molecular sequences, which are gradually yielding information beyond the reach of phylogenetic trees. Meanwhile, researchers can now study the assembly of the structure in real time, yielding further insights into the origins of complexity. Michael Gross reports.

Local Protein Structures to Bridge Sequence-Structure Knowledge

Protein sequences can be classified based on their structure similarity and/or common evolutionary origin called structural class. Information on structural class is readily available, easing the protein structure and protein function probing. SCOP and CATH are two prominent classification schemes used to assign the structural class of proteins. Both schemes determine the structural class manually base on known protein tertiary structures. However, the quantity of known protein sequences is growing exponentially with respect to the quantity of known tertiary proteins structures. Although SCOP and CATH are examples of well-established databases that contain more reliable information of structural class, yet the lack of known structural class of protein due to the laborious wet-lab experimental routine limits the high-throughput structural class assignment. The fact that this is a tedious and time-consuming manually-determined method has further limited the structural class assignment. As a consequence, the assignment of structural class by computational method suffers from the arbitrated statistical infer-ence. Thus, this study aims to provide a structural class prediction method that can acquire the knowledge of local protein structures, derived from known excessive primary sequences, in order to produce high-throughput sequence-structure class assignment instead of the laborious experimental based method. This structural class prediction method is termed as SVM-LpsSCPred.

Self-organizing mapping based swarm intelligence for secondary and tertiary proteins classification

Proteins have a significant role in animals and human health. Interactions among proteins are complex and large. Proteins separations are challenging process in molecular biology. Computational tools help to simulate the analysis in order to reduce the training data into small testing data. Large proteins have been mapped using self-organizing maps (SOMs). Neural network based SOMs has a significant role in reducing the irregular shapes of proteins interactions. Iterative checking enables the organizations of all proteins. In next stage, particle swarm intelligence is applied to classify the proteins’ families. In the current work, secondary (Two dimensional) and tertiary proteins (Three dimensional) proteins have been grouped. Two dimensional proteins contain fewer hydro-carbons than three dimensional proteins. For faster analysis, the angles of the proteins are taken into account. The SOMs is compared with Bounding Box approach. In final, the experimental evolutions show that swarm intelligence achieved faster processing through enabling less memory consumptions and time. Since PSO combines proteins datasets in fuzzy values, the compactness or integration of similar proteins are strong. On the other hand, Bounding Box uses the Crisp value. Therefore, it needs more space to organize the whole data. Without SOMs, swarm intelligence also results are poor due to the excessive time consuming and required storage area. Moreover, for almost all classification and clustering tools, it is observed that the overall classification task becomes slow, time consuming, space consuming and also less sensitive because of noises, irrelevant data in input datasets. Thus, the proposed SOM based PSO approach achieved less time consuming with efficient classification into secondary and tertiary proteins.

HPTLC and IR Spectral studies of the ethanolic extract of Phallusia nigra

Ascidians, commonly called as Sea Squirts are sedentary tunicates. Phallusia nigra is a simple ascidian belonging to the family Ascidiidae found in plenty throughout the year. The ethanolic extract of Phallusia nigra was subjected to HPTLC and IR spectral analysis to determine the possible bioactive components. In HPTLC studies, gallic acid, ferulic acid, caffeic acid and flavonoids such as rutin, isoquercitrin and quercetin were found to be present. The interpretation of the spectrum showed the presence of aliphatic bromo compounds, phenol or tertiary alcohols, carbonyl compound, carboxylic acids, lipids, proteins, alkanes and aromatic compound.

The Circulating Proteinase Inhibitor α-1 Antitrypsin Regulates Neutrophil Degranulation and Autoimmunity

Pathological inflammation and autoimmune disease frequently involve elevated neutrophil activity in the absence of infectious agents. Tumor necrosis factor-α (TNF-α) contributes to many of the problems associated with autoimmune diseases. We investigated the ability of serum α-1 antitrypsin (AAT) to control TNF-α biosynthesis and signaling in neutrophils and assessed whether AAT deficiency (AATD) is a TNF-α-related disease. In vitro studies demonstrate that serum AAT coordinates TNF-α intracellular signaling and neutrophil degranulation of tertiary and secondary granules via modulation of ligand-receptor interactions. AATD patients homozygous for the Z allele were characterized by increased activation of the TNF-α system, as demonstrated by increased membrane TNF-α levels and increased plasma concentrations of TNF receptor 1 and neutrophil-released secondary and tertiary granule proteins. The incidence of autoantibodies directed against degranulated lactoferrin and surface protein accessible to these antibodies was increased in ZZ-AATD, leading to an enhanced rate of neutrophil reactive oxygen species production. Treatment of ZZ-AATD individuals with AAT augmentation therapy resulted in decreased membrane TNF-α expression and plasma levels of granule antigenic proteins and immunoglobulin G class autoantibodies. These results provide a mechanism by which AAT augmentation therapy affects TNF-α signaling in the circulating neutrophil, indicating promising potential of this therapy for other TNF-α-related diseases.

In Vivo X-Ray Footprinting of Pre-30S Ribosomes Reveals Chaperone-Dependent Remodeling of Late Assembly Intermediates

Assembly of 30S ribosomal subunits from their protein and RNA components requires extensive refolding of the 16S rRNA and is assisted by 10-20 assembly factors in bacteria. We probed the structures of 30S assembly intermediates in E. coli cells, using a synchrotron X-ray beam to generate hydroxyl radical in the cytoplasm. Widespread differences between mature and pre-30S complexes in the absence of assembly factors RbfA and RimM revealed global reorganization of RNA-protein interactions prior to maturation of the 16S rRNA and showed how RimM reduces misfolding of the 16S 3' domain during transcription in vivo. Quantitative (14)N/(15)N mass spectrometry of affinity-purified pre-30S complexes confirmed the absence of tertiary assembly proteins and showed that N-terminal acetylation of proteins S18 and S5 correlates with correct folding of the platform and central pseudoknot. Our results indicate that cellular factors delay specific RNA folding steps to ensure the quality of assembly.

Escherichia coli rimM and yjeQ null strains accumulate immature 30S subunits of similar structure and protein complement

Assembly of the Escherichia coli 30S ribosomal subunits proceeds through multiple parallel pathways. The protein factors RimM, YjeQ, RbfA, and Era work in conjunction to assist at the late stages of the maturation process of the small subunit. However, it is unclear how the functional interplay between these factors occurs in the context of multiple parallel pathways. To understand how these factors work together, we have characterized the immature 30S subunits that accumulate in ΔrimM cells and compared them with immature 30S subunits from a ΔyjeQ strain. The cryo-EM maps obtained from these particles showed that the densities representing helices 44 and 45 in the rRNA were partially missing, suggesting mobility of these motifs. These 30S subunits were also partially depleted in all tertiary ribosomal proteins, particularly those binding in the head domain. Using image classification, we identified four subpopulations of ΔrimM immature 30S subunits differing in the amount of missing density for helices 44 and 45, as well as the amount of density existing in these maps for the underrepresented proteins. The structural defects found in these immature subunits resembled those of the 30S subunits that accumulate in the ΔyjeQ strain. These findings are consistent with an "early convergency model" in which multiple parallel assembly pathways of the 30S subunit converge into a late assembly intermediate, as opposed to the mature state. Functionally related factors will bind to this intermediate to catalyze the last steps of maturation leading to the mature 30S subunit.

Structures closed into cycles in globular proteins

Different types of structures closed into cycles are widespread at all the levels of structural organization of proteins. β-Hairpins, triple-stranded β-sheets, and βαβ-units represent simple structural motifs closed into cycles by systems of hydrogen bonds. Secondary closing of these simple motifs into larger cycles by means of different superhelices, split β-hairpins, or SS-bridges results in formation of complex structural motifs such as abcd-units, φ-motifs, five- and seven-segment α/β-motifs, etc. At the level of tertiary structure many proteins and domains fold into structures closed into cylinders. Apparently, closing the motifs and domains into cycles and cylinders results in formation of more cooperative and stable structures as compared with open ones, and this may be the reason for high frequencies of occurrence of the motifs in proteins.

Supramolecular Chirality in Crystalline Assemblies of Bile Acids and Their Derivatives; Three-Axial, Tilt, Helical, and Bundle Chirality

Steroidal bile acids and their derivatives exhibit characteristic inclusion behaviors in the crystalline state. Their crystals present varied assemblies due to asymmetric molecular structures, which relate to supramolecular properties through cooperative weak interactions. An overview indicates that the steroidal assemblies lie in an intermediate position among various molecules and have hierarchical structures such as primary, secondary, tertiary, and host-guest assemblies like proteins. Such an interpretation brought about the idea that the assemblies with dimensionality present supramolecular chirality such as three-axial, tilt, helical, bundle, and complementary chirality. This concept of the supramolecular chirality enables us to understand formation of chiral crystals starting from the molecular chirality of the steroidal molecules.

Low‐Resolution Molecular Dynamics Simulations of the 30S Ribosomal Subunit

Low-resolution molecular models can provide appropriate and efficient ways for studying large biomolecular systems such as the ribosome. We have developed computer codes that use the Yammp Under Python modeling package to assemble low-resolution force fields for RNA-protein complexes, and that connect these to the Amber molecular simulation package. This pipeline combines many of the complementary strengths of these two packages. Our target here is the 30S ribosomal subunit from Thermus thermophilus. One hundred nanosecond Langevin dynamics simulations were performed for the bound and the unbound 16S RNA, and conformational changes of the 16S RNA and its interaction with the 30S proteins were examined to establish the fidelity of our model. The S7 protein assembly pathway was also examined, and the effects of protein binding order on the 16S RNA were analyzed. The simulations suggest that ribosomal proteins play important roles in maintaining the native 16S RNA structure. Primary proteins (in terms of assembly) help more in stabilizing the conformation of the RNA than do secondary and tertiary proteins. Ribosomal proteins appear to bind to the RNA in an organized fashion wherein primary and secondary proteins help to prepare the binding sites for tertiary proteins. The methodology and tools described here should provide useful ways to explore other aspects of ribosomal conformational changes by means of molecular dynamics simulations.

C/EBPε Knockout (KO) EML and PBiIM Cell Lines Replicate the In Vivo Phenotype of the C/EBPε KO Mouse.

The appearance of the secondary granule proteins (SGP) lactoferrin (LF), transcobalamin I (TCI), neutrophil collagenase (NC) and neutrophil gelatinase (NG) marks the commitment to terminal neutrophil differentiation. C/EBPε plays a critical role in the coordinate upregulation of SGP genes. Disruption of the C/EBPε gene in mice leads to morphologic and functional defects in neutrophil maturation with a defective transition from the promyelocyte to the myelocyte stage. The neutrophils have bilobed nuclei, abnormal respiratory burst activity, and impaired chemotaxis and bactericidal activity. They lack specific granules and fail to express mRNAs encoding for secondary and tertiary granule content proteins. Phenotypic and functional defects of the C/EBPε −/− mice closely parallel those in patients with secondary granule deficiency. We have made 2 cell lines from the bone marrow of C/EBPε −/− mice and corresponding wildtype (WT) littermates. We first generated EML-like cell lines from WT and KO mice by transducing bone marrow with a retroviral vector expressing the dominant negative RARα and selecting for immortalized cells. We then induced the KO and WT EML cell lines with cytokines and ATRA to the neutrophil stage, and demonstrated that the KO cell morphology, functional responses (oxidative burst) and RNA expression patterns mimic the morphologic and functional abnormalities of the knockout mice. Transduction with a C/EBPε-expressing retrovirus corrects the phenotype of the KO cells, restoring normal morphology, oxidative burst, and secondary granule protein gene expression. Since C/EBPε is an RAR-responsive gene and because the dominant negative RARα also interferes with normal neutrophil maturation, we used an alternate approach to generate pBabePuro immortalized cell lines (PBiMs) by random insertional mutagenesis. We transduced WT and KO bone marrow cells with viral supernatent harboring the pBabepuro vector and selected for puromycin-resistant cells. Immortalized cells grew out 1–2 months after transduction and were maintained either in stem cell factor (SCF) alone or in SCF+IL-3 (interleukin 3). WT PBiMs are inducible with G-CSF to morphologically mature granulocytes or with GM-CSF to mature macrophages. KO PBiMs fail to mature to morphologically normal neutrophils, and form macrophage-like cells or die. By RNA analysis, both the EML and PBiM KO cells induced to differentiate toward neutrophils fail to express C/EBPε and lactoferrin, demonstrate absent oxidative burst, and fail to up-regulate Gr-1 upon induction as assayed by flow cytometry. Transduction of KO cells with a C/EBPε-containing retrovirus again restored respiratory burst activity. We have thus generated two independent C/EBPε −/− cell lines, each immortalized by a different mechanism, that should provide an important tool with which to study the transcriptional targets of C/EBPε in terminal myeloid differentiation. Since mutations in the C/EBPε gene have been reported in two patients with the rare clinical disorder specific granule deficiency, these lines will also serve as a convenient in vitro model for studying that disease.

Growth Factor Independence 1 (Gfi-1) Plays a Role in Mediating Specific Granule Deficiency (SGD) in a Patient Lacking an Abnormality in the C/EBPε Gene.

Neutrophil specific granule deficiency (SGD) is a rare congenital disorder marked by recurrent bacterial infections of the skin and respiratory system. Neutrophils from SGD patients lack secondary and tertiary granules and their content proteins and exhibit defects in chemotaxis and bactericidal activity. A mouse model deficient for the transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) manifests a similar phenotype as SGD patients and prompted examination of the human C/EBPε gene for mutation in this disease. Mutations in the C/EBPε gene have been identified by others in two patients with SGD, resulting in loss of gene expression. However, other patients with a similar disease phenotype have a normal C/EBPε coding sequence, suggesting that other genetic abnormalities in myelopoiesis can lead to SGD. Studies in our laboratory on one such patient lacking a C/EBPε mutation demonstrated an elevated level of the C/EBPε protein in the patient's peripheral blood neutrophils as compared to the level in normal control neutrophils. Microarray analysis of this patient's bone marrow compared with that of a normal control revealed, among other genes, elevated levels of the transcription factors C/EBPε and PU.1. As a consequence, several PU.1 target genes showed increased expression in the SGD patient bone marrow. This observation was confirmed by both real time PCR and western blot analysis. PU.1 is a hematopoietic-specific transcription factor belonging to the Ets family of DNA binding proteins and plays a critical role in B-cell, macrophage and late stage neutrophil development. Sequence analysis of the PU.1 gene from our SGD patient however, did not reveal any mutation in the coding region of the gene. Western blot analysis of nuclear extracts prepared from peripheral blood neutrophils from this patient did however reveal significantly decreased levels of the transcription factor Gfi-1 (Growth factor independent-1), although no mutation has been found thus far in the coding region of the Gfi-1 gene from the SGD patient. Gfi-1 belongs to a family of zinc finger containing transcriptional repressor oncoproteins. Mice lacking Gfi-1 were found not only to be neutropenic, but also demonstrated defects in neutrophil differentiation, including loss of neutrophil secondary and tertiary granule proteins, reminiscent of SGD. More recently, heterozygous germline mutations of Gfi-1 were shown to cause severe congenital neutropenia (SCN) in humans. It has been suggested that Gfi1 represses neutrophil elastase (Ela2), germline mutations within which are a major contributor to hereditary neutropenias. Our data suggest that decreased levels of Gfi1 in our SGD patient result in increased levels of PU.1 and C/EBPε; an effect consistent with observations first made in the neutrophils of Gfi-1-null mice. The increased PU.1 levels might then act to sequester C/EBPε protein via direct protein-protein interaction. This in turn could explain the loss of secondary granule protein gene expression in the SGD patient by inducing functional C/EBPε deficiency.

Restoration of C/EBPα Expression in a BCR-ABL+ Cell Line Induces Terminal Granulocytic Differentiation

The transcription factor C/EBPalpha plays a critical role in the process of granulocytic differentiation. Recently, mutations that abrogated transcriptional activation of C/EBPalpha were detected in acute myeloid leukemia patient samples. Moreover, the progression of chronic myelogenous leukemia (CML) to blast crisis in patients was correlated with down-modulation of C/EBPalpha. The KCL22 cell line, derived from BCR-ABL+ CML in blast crisis, expressed wild-type C/EBPepsilon protein but not a functional C/EBPalpha, -beta, and -gamma. Restoration of C/EBPalpha expression in KCL22 cells triggered a profound proliferative arrest, a block in the G2/M phase of the cell cycle and a gradual increase in apoptosis. Within 3 days of inducing expression of C/EBPalpha, a remarkable neutrophilic differentiation of the KCL22 blast cells occurred as shown by morphologic changes, induction of expression of CD11b, primary, secondary, and tertiary granule proteins, and granulocyte colony-stimulating factor receptor. Using high density oligonucleotide microarrays, the gene expression profile of KCL22 cells stably transfected with C/EBPalpha was compared with that of empty vector, and we identified genes not previously known to be regulated by C/EBPalpha. These included the up-regulation of those genes important for regulation of hematopoietic stem cell homing, granulocytic differentiation, and cell cycle, whereas down-regulation occurred for genes coding for signaling molecules and transcription factors that are implicated in regulation of proliferation and differentiation of hematopoietic cells. Our study showed that restoration of C/EBPalpha expression in BCR-ABL+ leukemic cells in blast crisis is sufficient for rapid neutrophil differentiation suggesting a potential therapeutic role for ectopic transfer of C/EBPalpha in acute phase of CML.

Neutrophil specific granule deficiency and mutations in the gene encoding transcription factor C/EBP(epsilon).

Neutrophil specific granule deficiency (SGD) is a rare congenital disorder. The neutrophils of these patients display atypical bilobed nuclei; lack expression of at least one primary and all secondary and tertiary granule proteins; and possess defects in chemotaxis, disaggregation, receptor upregulation, and bactericidal activity. SGD patients suffer frequent and severe bacterial infections. Although the first of five patients worldwide was reported in the early 1970s, the molecular basis for the defect was discovered only recently. This review presents data implicating the functional loss of the myeloid transcription factor CCAAT/enhancer binding protein (C/EBP(epsilon)) as a causative agent in the development of SGD. The murine model for SGD provides evidence for defects in eosinophil granule gene expression and indicates abnormalities in macrophage maturation and function. Deficiencies in multiple myeloid lineages, in addition to neutrophils, indicate the importance of C/EBP(epsilon) in regulating important innate immune and inflammatory responses critical for host defense.

Neutrophil-specific granule deficiency: homozygous recessive inheritance of a frameshift mutation in the gene encoding transcription factor CCAAT/enhancer binding protein–ε

AbstractNeutrophil-specific granule deficiency (SGD) is a rare congenital disorder. The neutrophils of individuals with SGD display atypical bi-lobed nuclei, lack expression of all secondary and tertiary granule proteins, and possess defects in chemotaxis, disaggregation, receptor up-regulation, and bactericidal activity, resulting in frequent and severe bacterial infections. Previously, a homozygous mutation in theCCAAT/enhancer binding protein–ε (C/EBPε) gene was reported for one case of SGD. To substantiate the role of C/EBPε in the development of SGD and elucidate its mechanism of inheritance, the mutational status of the gene was determined in a second individual. An A-nucleotide insertion in the coding region of the C/EBPε gene was detected. This mutation completely abolished the predicted translation of all C/EBPε isoforms. Microsatellite and nucleotide sequence analyses of the C/EBPε locus in the parents of the proband indicated that the disorder may have resulted from homozygous recessive inheritance of the mutant allele from an ancestor shared by both parents. The mutant C/EBPε32 protein localized in the cytoplasm rather than the nucleus and was unable to activate transcription. Consistent with this, a significant decrease in the levels of the messenger RNAs (mRNAs) encoding the secondary granule protein human 18-kd cationic antimicrobial protein (hCAP-18)/LL-37 and the primary granule protein bactericidal/permeability-increasing protein were observed in the patient. The hCAP-18 mRNA was induced by overexpression of C/EBPε32 in the human myeloid leukemia cell line, U937, supporting the hypothesis that C/EBPε is a key regulator of granule gene synthesis. This study strongly implicates mutation of theC/EBPε gene as the primary genetic defect involved in the development of neutrophil SGD and defines its mechanism of inheritance.

A hierarchy of RNA subdomains in assembly of the central domain of the 30 S ribosomal subunit.

Beginning with the framework that has been developed for the assembly of the 30 S ribosomal subunit, we have identified a series of RNAs that are minimal binding sites for proteins S15, S6, S18, and S11 in the central domain from Thermus thermophilus. The minimal binding RNA for proteins S15, S6, and S18 consists of helix 22 and three-way junctions at both ends composed of portions of helices 20, 21, and 23. Addition of the remaining portion of helix 23 to this construct results in the minimal site for S11. Surprisingly, almost half of the central domain (helices 24, 25, and 26) is dispensable for binding the central domain proteins. Thus, at least two classes of RNA elements can be identified in ribosomal RNA. A protein-binding core element (such as helices 20, 21, 22, and 23) is required for the association of ribosomal proteins, whereas secondary binding elements (such as helices 24, 25, and 26) associate only with the preformed core RNP complex. Apparently, there may be a hierarchy of ribosomal RNA elements similar to the hierarchy of primary, secondary, and tertiary binding ribosomal proteins.

Myeloid Transcription Factor C/EBPɛ Is Involved in the Positive Regulation of Lactoferrin Gene Expression in Neutrophils

AbstractTargeted mutation of the myeloid transcription factor C/EBPɛ in mice results in gram-negative septic death at 3 to 5 months of age. This study defines the underlying molecular defects in their terminal granulocytic differentiation. The mRNA for the precursor protein of the cathelin-related antimicrobial peptides was almost completely absent in the bone marrow cells of C/EBPɛ−/− mice. This finding may help explain their susceptibility to gram-negative sepsis, because both are bacteriocidal peptides with potent activity against gram-negative bacteria. Superoxide production was found to be reduced in both granulocytes and monocytes of C/EBPɛ−/− mice. While gp91 phox protein levels were normal, p47phox protein levels were considerably reduced in C/EBPɛ −/− granulocytes/monocytes, possibly limiting the assembly of the NADPH oxidase. In addition, expression of mRNA of the secondary and tertiary granule proteins, lactoferrin and gelatinase, were not detected, and levels of neutrophil collagenase mRNA were reduced in bone marrow cells of the knock-out mice. The murine lactoferrin promoter has a putative C/EBP site close to the transcription start site. C/EBPɛ bound to this site in electromobility shift assay studies and mutation of this site abrogated binding to it. A mutation in the C/EBP site reduced the activity of the promoter by 35%. Furthermore, overexpression of C/EBPɛ in U937 cells increased the activity of the wild-type lactoferrin promoter by 3-fold. In summary, our data implicate C/EBPɛ as a critical factor of host antimicrobial defense and suggests that it has a direct role as a positive regulator of expression of lactoferrin in vivo.

Myeloid Transcription Factor C/EBPɛ Is Involved in the Positive Regulation of Lactoferrin Gene Expression in Neutrophils

Targeted mutation of the myeloid transcription factor C/EBPɛ in mice results in gram-negative septic death at 3 to 5 months of age. This study defines the underlying molecular defects in their terminal granulocytic differentiation. The mRNA for the precursor protein of the cathelin-related antimicrobial peptides was almost completely absent in the bone marrow cells of C/EBPɛ−/− mice. This finding may help explain their susceptibility to gram-negative sepsis, because both are bacteriocidal peptides with potent activity against gram-negative bacteria. Superoxide production was found to be reduced in both granulocytes and monocytes of C/EBPɛ−/− mice. While gp91 phox protein levels were normal, p47phox protein levels were considerably reduced in C/EBPɛ −/− granulocytes/monocytes, possibly limiting the assembly of the NADPH oxidase. In addition, expression of mRNA of the secondary and tertiary granule proteins, lactoferrin and gelatinase, were not detected, and levels of neutrophil collagenase mRNA were reduced in bone marrow cells of the knock-out mice. The murine lactoferrin promoter has a putative C/EBP site close to the transcription start site. C/EBPɛ bound to this site in electromobility shift assay studies and mutation of this site abrogated binding to it. A mutation in the C/EBP site reduced the activity of the promoter by 35%. Furthermore, overexpression of C/EBPɛ in U937 cells increased the activity of the wild-type lactoferrin promoter by 3-fold. In summary, our data implicate C/EBPɛ as a critical factor of host antimicrobial defense and suggests that it has a direct role as a positive regulator of expression of lactoferrin in vivo.