NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses.
The transcription factor NF-kappa B, more than a decade after its discovery, remains an exciting and active area of study. The involvement of NF-kappa B in the expression of numerous cytokines and adhesion molecules has supported its role as an evolutionarily conserved coordinating element in the organism's response to situations of infection, stress, and injury. Recently, significant advances have been made in elucidating the details of the pathways through which signals are transmitted to the NF-kappa B:I kappa B complex in the cytosol. The field now awaits the discovery and characterization of the kinase responsible for the inducible phosphorylation of I kappa B proteins. Another exciting development has been the demonstration that in certain situations NF-kappa B acts as an anti-apoptotic protein; therefore, elucidation of the mechanism by which NF-kappa B protects against cell death is an important goal. Finally, the generation of knockouts of members of the NF-kappa B/I kappa B family has allowed the study of the roles of these proteins in normal development and physiology. In this review, we discuss some of these recent findings and their implications for the study of NF-kappa B.

title: Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B.
abstract: 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.

title: Missing Pieces in the NF-κB Puzzle
abstract: The regulation of the transcription factor NF-kappaB activity occurs at several levels including controlled cytoplasmic-nuclear shuttling and modulation of its transcriptional activity. A critical component in NF-kappaB regulation is the IkappaB kinase (IKK) complex. This review is focused on recent progress as well as unanswered questions regarding the regulation and function of NF-kappaB and IKK.

title: Cloning of the p50 DNA binding subunit of NF-kappa B: homology to rel and dorsal.
abstract: The DNA binding subunit of the transcription factor NF-kappa B, p50, has been cloned. p50 appears to be synthesized as a larger protein that is then processed to its functional size. Sequence analysis reveals remarkable homology for over 300 amino acids at the amino-terminal end to the oncogene v-rel, its cellular homolog c-rel, and the Drosophila maternal effect gene dorsal. This establishes NF-kappa B as a member of the rel family of proteins, all of which display nuclear-cytosolic translocation. Protein sequence from the p65 polypeptide has established that it is not encoded in the same mRNA as p50. However, p65 appears homologous to c-rel, suggesting that c-rel may form heterodimers with p50 and rel may include a homodimerization motif.

title: New regulators of NF-κB in inflammation
abstract: Research on the biological function of nuclear factor-κB (NF-κB), a key mediator of inducible transcription in the immune system, has traditionally focused on its role in the initiation of innate and adaptive immune responses. These studies have largely concentrated on the mechanisms of signalling that lead to NF-κB activation and on the positive role of NF-κB in both physiological immunity and pathological inflammation. More recently, there has been growing interest in the mechanisms that directly regulate the NF-κB transcriptional programmes. As a result, several new NF-κB regulatory components have been identified and some of the known components have been assigned new roles. In this Review, we discuss these new insights into the regulation of NF-κB.

title: Regulation of inducible gene expression by the transcription factor NF-kappaB.
abstract: The transcription factor NF-kappaB plays a critical role in regulating inducible gene expression in immune responses. The activation of NF-kappaB is regulated at multiple levels, probably reflecting a need to maintain a tight control of its activity. We have recently discovered that direct phosphorylation of NF-kappaB itself is essential for its transcriptional activity and that phosphorylation acts as a switch to determine association of nuclear NF-kappaB with histone acetylases vs deacetylases.

title: Essential role of tuberous sclerosis genes TSC1 and TSC2 in NF-kappaB activation and cell survival.
abstract: The TSC1-TSC2 complex has recently been implicated in cell survival responses. We observed that NF-kappaB signaling is attenuated in TSC1- and TSC2-deficient MEFs concomitant with reduced survival following DNA damage or TNFalpha stimulation. Reconstitution of TSC2 expression in TSC2(-/-) MEFs rescued survival in an NF-kappaB activity-dependent manner. Furthermore, in TSC2(-/-) MEFs, the rapamycin-mediated inhibition of deregulated mTOR activity restored NF-kappaB activation and survival. This rapamycin-mediated effect was reversed by inhibition of NF-kappaB transcriptional activation or by inhibition of ERK1/2 MAP kinase or PI-3K pathways, which lie on signaling cascades that lead to NF-kappaB activation. These results provide evidence for a crosstalk between the TSC/Rheb/mTOR pathway and the NF-kappaB induction pathways and indicate that NF-kappaB functions as an important survival factor that regulates TSC2-dependent cell survival.

title: Laboratory rearing of Theileria annulata-free Hyalomma anatolicum anatolicum ticks
abstract: Hyalomma anatolicum anatolicum is a three-host tick which transmits Theileria annulata infection in Indian cattle. Laboratory rearing of ixodid ticks is an essential requirement of any laboratory engaged with research on ticks and tick borne diseases. The Entomology laboratory of Indian Veterinary Research Institute is fully equipped with all the facilities and skilled manpower to maintain a homogenous H. a. anatolicum population throughout the year. The continuous supply of eggs, larvae and adults of H. a. anatolicum is maintained to meet out the demand of different experiments viz., preparation of tick antigens for immunization of animals, experimental challenge, isolation of genomic DNA and RNA. Maintenance of a H. a. anatolicum colony free of T. annulata infection is imperative for the experimental challenge infestation on cross-bred (Bos indicus × B. taurus) calves, in order to prevent the transmission of T. annulata infection to the experimental animals. A system has been developed in the laboratory in which the larvae of H. a. anatolicum were fed on New Zealand white rabbits and the dropped fed nymphs molted to adults are fed on cross-bred calves free of T. annulata infection. This synthetic cycle prevents the transstadial transmission of T. annulata as the rabbits are unsusceptible to T. annulata infection and only the adults were fed on cross-bred animals. Moreover, absence of transovarial transmission of T. annulata prevents the chance of carry over infection to experimental animals in the next cycle.

title: The effects of subtherapeutic antibiotic use in farm animals on the proliferation and persistence of antibiotic resistance among soil bacteria
abstract: The use of antibiotics at subtherapeutic concentrations for agricultural applications is believed to be an important factor in the proliferation of antibiotic-resistant bacteria. The goal of this study was to determine if the application of manure onto agricultural land would result in the proliferation of antibiotic resistance among soil bacteria. Chlortetracycline-resistant bacteria were enumerated and characterized from soils exposed to the manure of animals fed subtherapeutic concentrations of antibiotics and compared to the chlortetracycline-resistant bacteria from soils at farms with restricted antibiotic use (dairy farms) and from non-agricultural soils. No significant differences were observed at nine different study sites with respect to the numbers and types of cultivated chlortetracycline-resistant bacteria. Genes encoding for tetracycline resistance were rarely detected in the resistant bacteria from these sites. In contrast, soils collected from a tenth farm, which allowed manure to indiscriminately accumulate outside the animal pen, had significantly higher chlortetracycline-resistance levels. These resistant bacteria frequently harbored one of 14 different genes encoding for tetracycline resistance, many of which (especially tet(A) and tet(L)) were detected in numerous different bacterial species. Subsequent bacterial enumerations at this site, following the cessation of farming activity, suggested that this farm remained a hotspot for antibiotic resistance. In conclusion, we speculate that excessive application of animal manure leads to the spread of resistance to soil bacteria (potentially by lateral gene transfer), which then serve as persistent reservoir of antibiotic resistance.

title: Endotoxin-induced organ injury.
abstract: To review the effects of endotoxemia on the major organ systems of the body and discuss potential mechanisms of tissue injury.



Appraisal of 60 articles representing a cross section of studies relating to in vivo and in vitro responses to endotoxin.



Cell cultures, isolated tissue preparations, animal and human studies.



Endotoxemia results in the activation of numerous cellular and hematogenous mediators. These mediators range from prostaglandins, thromboxanes, and leukotrienes, to complement components. Tumor necrosis factor may be responsible for initiating many of the observed responses to endotoxin. Species and tissue specificity are a prominent feature of the response to endotoxin.



No single agent can yet be implicated as the common mediator of endotoxin-induced organ injury. Endotoxin initiates the elaboration of a cascade of secondary mediators that amplify the response to the initial insult. The relative importance of individual agents as mediators of the response to endotoxin varies with the experimental model studied.

title: Both avian and mammalian embryonic myoblasts are intrinsically heterogeneous
abstract: Adult skeletal muscles are composed of different fibre types. What initiates the distinctive muscle fibre type-specific specialization in a developing embryo is still controversial. In vitro studies of avian muscles have shown the expression of one of the slow myosin heavy chains, SM2, in only some myotubes. In this report we demonstrate the expression of another slow myosin heavy chain, SM1, restricted to only some chicken myotubes (presumptive slow) in vitro. We also demonstrate that as is the case for avian species, distinct fast and slow myogenic cells are detectable in mammalian species, human and rat, during in vitro development in the absence of innervation. While antibodies to fast myosin heavy chains stained all myotubes dark in these muscle cell cultures, antibodies to slow myosin heavy chains stained only a proportion of the myotubes (presumptive slow). The other myotubes were either unstained or only weakly stained with slow myosin heavy c hain antibodies. The muscle cell cultures prepared from different developmental stages of rat skeletal muscles showed a reduction in the number of slow myosin heavy chain-positive myotubes with advancing foetal growth. It is concluded that embryonic myogenic cells that are likely to form distinct fast or slow muscle fibre types are intrinsically heterogeneous, not only in avian but also in mammalian species, although extrinsic factors reinforce and modify such commitment throughout subsequent development.