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The alpha receptor of the ligand-binding ciliary neurotrophic factor (CNTF) in human recombinant cells function as a diffusible factor to regulate the cell-surface specificity, CNTF potency and ligand affinity. TF-1 cell responses indicate the requirement of sCNTF-alpha receptor for proliferation of the cells.

Universally present but overlooked cavities or pockets in long-lived biopharmaceuticals, such as monoclonal antibodies (mAbs), are capable of binding small drugs. Such direct interactions can alter the pharmacokinetics of drugs and potentially affect clinical outcome. The extreme differences in the pharmacokinetic properties of these 2 classes of drugs largely account for such effects. This overlooked mechanism of biologic-chemical drug interaction should be considered before approval of new long-lived entities.

Ciliary neurotrophic factor (CNTF) inhibits the production of tumor necrosis factor (TNF) in lipopolysaccharide (LPS)-treated mice and protects against LPS lethality when coadministered with its soluble receptor (sCNTFR alpha). Both of these activities are abolished in adrenalectomized (ADX) mice. LPS-induced pulmonary polymorphonuclear neutrophil (PMN) infiltration and nitric oxide (NO) production were also inhibited by CNTF + sCNTFR alpha but not by CNTF alone. sCNTFR alpha did not alter the clearance or tissue distribution of CNTF. Furthermore, CNTF variants coadministered with sCNTFR alpha protected against LPS toxicity in a manner related to their affinity for the beta components of CNTFR. Thus, inhibition of TNF production and protection against LPS lethality by CNTF/sCNTFR alpha require an intact hypothalamus-pituitary-adrenal axis (HPAA) and may be mediated by endogenous glucocorticoids. This protective effect is, at least in part, due to the inhibition of PMN infiltration and NO production, and appears to be mediated by cells displaying only beta-receptor subtypes.

Background: The receptor of ciliary neurotrophic factor (CNTF) contains the signal transduction protein gp130, which is also a component of the receptors of cytokines such as interleukin (IL)-6, leukemia-inhibitory factor (LIF), IL-11, and oncostatin M. This suggests that these cytokines might share common signaling pathways. We previously reported that CNTF augments the levels of corticosterone (CS) and of IL-6 induced by IL-1 and induces the production of the acute-phase protein serum amyloid A (SAA). Since the elevation of serum CS is an important feedback mechanism to limit the synthesis of proinflammatory cytokines, particularly tumor necrosis factor (TNF), we have investigated the effect of CNTF on both TNF production and lipopolysaccharide (LPS) toxicity., Materials and Methods: To induce serum TNF levels, LPS was administered to mice at 30 mg/kg i.p. and CNTF was administered as a single dose of 10 micrograms/mouse i.v., either alone or in combination with its soluble receptor sCNTFR alpha at 20 micrograms/mouse. Serum TNF levels were the measured by cytotoxicity on L929 cells. In order to measure the effects of CNTF on LPS-induced TNF production in the brain, mice were injected intracerebroventricularly (i.c.v.) with 2.5 micrograms/kg LPS. Mouse spleen cells cultured for 4 hr with 1 microgram LPS/ml, with or without 10 micrograms CNTF/ml, were also analyzed for TNF production., Results: CNTF, administered either alone or in combination with its soluble receptor, inhibited the induction of serum TNF levels by LPS. This inhibition was also observed in the brain when CNTF and LPS were administered centrally. In vitro, CNTF only marginally affected TNF production by LPS-stimulated mouse splenocytes, but it acted synergistically with dexamethasone (DEX) in inhibiting TNF production. Most importantly, CNTF administered together with sCNTFR alpha protected mice against LPS-induced mortality., Conclusions: These data suggest that CNTF might act as a protective cytokine against TNF-mediated pathologies both in the brain and in the periphery.

In human peripheral blood mononuclear cells, ciliary neurotrophic factor (CNTF) weakly suppressed endotoxin-induced interleukin (IL)-1 and prostaglandin E2(PGE2). Suppression of PGE2 and IL-8 synthesis was significantly greater (up to 42.6%, P < 0.05) by adding a 10-fold molar excess of soluble CNTF receptor (sCNTFR alpha). In cultured human fibroblasts, CNTF at 12 micrograms/ml did not suppress IL-1 alpha-induced IL-8. However, in the presence of a 10-fold excess of sCNTFR alpha, 300 ng/ml of CNTF suppressed IL-1 alpha-induced IL-8 by 44%. Therefore, sCNTFR alpha can confer to CNTF anti-inflammatory properties in vitro. IL-6 which, like CNTF, utilizes the gp130 signal transducer, possesses similar inhibitory effects. That CNTF and IL-6 share gp130 as a receptor component suggests that gp130 mediates these anti-inflammatory responses.

The alpha component of the receptor for ciliary neurotrophic factor (CNTF) differs from other known growth factor receptors in that it is anchored to cell membranes by a glycosylphosphatidylinositol linkage. One possible function of this type of linkage is to allow for the regulated release of this receptor component. Cell lines not normally responsive to CNTF responded to treatment with a combination of CNTF and a soluble form of the CNTF alpha receptor component. These findings not only demonstrate that the CNTF receptor alpha chain is a required component of the functional CNTF receptor complex but also reveal that it can function in soluble form as part of a heterodimeric ligand. Potential physiological roles for the soluble CNTF receptor are suggested by its presence in cerebrospinal fluid and by its release from skeletal muscle in response to peripheral nerve injury.

The human ciliary neurotrophic factor (CNTF) gene was identified and cloned, based on homology with the recently cloned rat cDNA. The gene encodes a protein of 200 amino acids, which shares about 80% sequence identity with rat and rabbit CNTF and, like these homologues, lacks an apparent secretion signal sequence. The human CNTF gene, like the rat gene, appears to contain a single intron separating two protein coding exons. An intronless human CNTF gene was constructed by the use of polymerase chain reactions and introduced into vectors designed for expression of foreign proteins in E. coli. The rat CNTF gene was also introduced into similar vectors. Both the human and rat proteins were expressed at exceptionally high levels, at 20-40% and 60-70% of total protein, respectively. Extraction of the recombinant proteins from inclusion bodies by guanidinium chloride, followed by two column chromatography steps, produced high yields of pure CNTF that supported survival and neurite outgrowth from embryonic chick ciliary neurons in culture. The biological activity of both recombinant proteins was comparable to that of native rat CNTF.

We recently described the purification and cloning of extracellular signal-regulated kinase 1 (ERK1), which appears to play a pivotal role in converting tyrosine phosphorylation into the serine/threonine phosphorylations that regulate downstream events. We now describe cloning and characterization of two ERK1-related kinases, ERK2 and ERK3, and provide evidence suggesting that there are additional ERK family members. At least two of the ERKs are activated in response to growth factors; their activations correlate with tyrosine phophorylation, but also depend on additional modifications. Transcripts corresponding to the three cloned ERKs are distinctly regulated both in vivo and in a differentiating cell line. Thus, this family of kinases may serve as intermediates that depend on tyrosine phosphorylation to activate serine/threonine phosphorylation cascades. Individual family members may mediate responses in different developmental stages, in different cell types, or following exposure to different extracellular signals.

The availability of relatively large amounts of nerve growth factor (NGF) has allowed extensive in vitro and in vivo characterization of the neuronal specificity of this neurotrophic factor. The restricted neuronal specificity of NGF (sympathetic neurons, neural crest-derived sensory neurons, basal forebrain cholinergic neurons) has long predicted the existence of other neurotrophic factors possessing different neuronal specificities. Whereas there have been many reports of "activities" distinct from NGF, full characterization of such molecules has been hampered by their extremely low abundance. The recent molecular cloning of brain-derived neurotrophic factor (BDNF) revealed that this protein is closely related to NGF and suggested that these two factors might be members of an even larger gene family. A PCR cloning strategy based on homologies between NGF and BDNF has allowed us to identify and clone a third member of the NGF family which we have termed neurotrophin-3 (NT-3). The establishment of suitable expression systems has now made available sufficient quantities of these proteins to allow us to begin to establish the neuronal specificity of each member of the neurotrophin family, and the role of each in development, maintenance and repair of the PNS and CNS. Using primary cultures of various PNS and CNS regions of the developing chick and rat, and Northern blot analysis, we describe novel neuronal specificities of BDNF, NT-3 and an unrelated neurotrophic factor-ciliary neurotrophic factor (CNTF).

The lack of reagents or molecular probes specific for the ciliary neurotrophic factor (CNTF) receptor has hindered characterization of the molecular mechanism(s) by which CNTF influences the proliferation, survival, and differentiation of cells of the vertebrate nervous system. We have developed methods for the detection and separation of cells expressing CNTF receptors by using a variety of binding assays based on a genetically engineered CNTF molecule containing an "epitope tag" at its C-terminus. These assays have allowed us to identify several neuronal cell lines, as well as embryonic and adult neurons in primary cultures, that bind CNTF and functionally respond to CNTF by rapidly activating the transcription of immediate early primary response genes.

Because of the palindromic character of most 6-bp restriction sites, filling-in and ligation of the protruding ends create symmetric sequences which include new 6-bp restriction sites. The old site is, in most cases, lost. After cleavage at the new palindromic site and removal of the protruding ends, a new center of symmetry is created which is often part of yet another 6-bp restriction site. A compilation of potential and available sites as presented should prove useful in genetic engineering.

ColE1 derivative plasmids were constructed in which the natural promoter that primes replication or, in addition, the region coding for the RNA I control element had been deleted. In all of these molecules priming of the origin was effected by read-through transcription from constitutive or inducible (lacUV5) promoters inserted farther upstream. In the latter case, regulation of lac repressor activity with IPTG resulted in controlled plasmid levels in vivo. These results indicated that, at least in the absence of other control elements, regulation of the priming promoter was sufficient to control DNA replication and determine plasmid copy number.

Commonly used expression vectors direct the synthesis of antibiotic-resistance proteins at unnecessarily high levels that complicate purification of the desired recombinant product. To overcome this problem, the promoter of the kanamycin-resistance gene (kanR) was altered by site-specific mutagenesis. As a result, synthesis of KanR protein was greatly reduced to the minimum required for host selection. At the same time, recombinant protein production was increased up to two-fold. Since the mutations did not alter any coding sequences and had no effect on plasmid copy number, the results suggest that plasmid-coded protein production can be limited, at least in part, by other genes expressed from the same plasmid. Because of the dependence of protein synthesis on gene dosage, an important aspect of minimizing antibiotic resistance is continuous selection for cells harboring the maximum vector-copy number, thus ensuring maximal product synthesis.

We report that processing at a cloned bacteriophage T7 RNase III site results in strong stabilization of the mRNA relative to the full-length transcript. In contrast, processing by RNase III of the bacteriophage lambda int transcript leads to rapid degradation of the messenger. It is proposed that the mode of cleavage within the RNase III site determines mRNA stability. Single cleavage leaves part of the phage T7 RNase III site in a folded structure at the generated 3' end and stabilizes the upstream mRNA whereas double cleavage at the lambda int site removes the folded structure and accelerates degradation. In addition, the processed transcript is as active a messenger as the unprocessed one and can direct protein synthesis for longer times. This increased efficiency is accompanied by a proportional (3-4 fold) increase in protein levels. In contrast, processing at the lambda int site reduces Int synthesis. Thus, processing may either stabilize mRNA and stimulate gene expression or destabilize a messenger and prevent protein synthesis. The end result appears to be determined by the mode of cleavage within the RNase III site.

The phage T7 endonuclease gene was fused to the 3' end of the lac repressor gene. The hybrid protein exhibits repressor and nuclease functions in a manner dependent on the conformation of the DNA. With supercoiled DNA, nuclease activity is directed to the major cruciform, whereas with linear DNA, the enzyme cleaves preferentially restriction fragments carrying the operator. These properties render the hybrid protein a unique probe of DNA conformation in vitro and in vivo.

A genetically engineered lac repressor/T7 endonuclease hybrid protein shows repressor-like binding toward restriction fragments carrying the lac operator. In addition, fragments carrying the operator near a particular pBR322 sequence are cleaved into specific products. Cleavage occurs at precise positions within that sequence, is independent of the orientation of the operator, is inhibited by isopropyl-1-thio-beta-D-galactopyranoside, and is observed when the target is separated from the operator by at least as few as 150 and as many as 240 base pairs. This evidence indicates that the hybrid protein is a site-directed nuclease that requires the following two structural elements for activity: the lac operator and a target. Repressor-like binding directs the enzyme to the operator and nearby single-stranded DNA targets. The discovery of an unusual target in a well-studied DNA sequence is evidence of the power of this approach for probing unusual structures in non-supercoiled duplex DNA.

The mechanism of inhibition of RNA polymerase-catalyzed synthesis of RNA by actinomycin D and the phenan-thridinium derivatives ethidium bromide and 3,8-diamino-6-ethylphenanthridinium bromide (DEMB) is examined. A general kinetic equation describing the dependence of RNA synthesis on DNA template concentration is derived and distinct expressions corresponding to various possible mechanisms of inhibition are subsequently obtained by introducing into the equations assumptions as appropriate for the individual mechanisms. The fitting of the experimental results of inhibition into the resulting equations suggested that the ethidium bromide and DEMB inhibit RNA polymerase by forming an inhibitor-template complex which interferes with enzyme recognition of, and binding to, appropriate sites on the template (binding inhibition). The fitting of the dependence of the rate of RNA synthesis on the bound-inhibitor to DNA ratios to the derived kinetic expressions also allows a tentative distinction to be made as to whether ethidium bromide and DEMB interfere with RNA synthesis by a mechanism of 'partial' or 'complete' inhibition.