Caspase-1 processes IFN-gamma-inducing factor and regulates LPS-induced IFN-gamma production.

Interferon-gamma-inducing factor (IGIF, interleukin-18) is a recently described cytokine that shares structural features with the interleukin-1 (IL-1) family of proteins and functional properties with IL-12. Like IL-12, IGIF is a potent inducer of interferon (IFN)-gamma from T cells and natural killer cells. IGIF is synthesized as a biologically inactive precursor molecule (proIGIF). The cellular production of IL-1beta, a cytokine implicated in a variety of inflammatory diseases, requires cleavage of its precursor (proIL-1beta) at an Asp-X site by interleukin-1beta-converting enzyme (ICE, recently termed caspase-1). The Asp-X sequence at the putative processing site in proIGIF suggests that a protease such as caspase-1 might be involved in the maturation of IGIF. Here we demonstrate that caspase-1 processes proIGIF and proIL-1beta with equivalent efficiencies in vitro. A selective caspase-1 inhibitor blocks both lipopolysaccharide-induced IL-1beta and IFN-gamma production from human mononuclear cells. Furthermore, caspase-1-deficient mice are defective in lipopolysaccharide-induced IFN-gamma production. Our results thus implicate caspase-1 in the physiological production of IGIF and demonstrate that it plays a critical role in the regulation of multiple proinflammatory cytokines. Specific caspase-1 inhibitors would provide a new class of anti-inflammatory drugs with multipotent action.

Proteolytic activation of protein kinase C delta by an ICE/CED 3-like protease induces characteristics of apoptosis.

Recent studies have shown that protein kinase C (PKC) delta is proteolytically activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. However, the relationship of PKC delta cleavage to induction of apoptosis is unknown. The present studies demonstrate that full-length PKC delta is cleaved at DMQD330N to a catalytically active fragment by the cysteine protease CPP32. The results also demonstrate that overexpression of the catalytic kinase fragment in cells is associated with chromatin condensation, nuclear fragmentation, induction of sub-G1 phase DNA and lethality. By contrast, overexpression of full-length PKC delta or a kinase inactive PKC delta fragment had no detectable effect. The findings suggest that proteolytic activation of PKC delta by a CPP32-like protease contributes to phenotypic changes associated with apoptosis.

Proteolytic activation of protein kinase C delta by an ICE-like protease in apoptotic cells.

These studies demonstrate that treatment of human U-937 cells with ionizing radiation (IR) is associated with activation of a cytoplasmic myelin basic protein (MBP) kinase. Characterization of the kinase by gel filtration and in-gel kinase assays support activation of a 40 kDa protein. Substrate and inhibitor studies further support the induction of protein kinase C (PKC)-like activity. The results of N-terminal amino acid sequencing of the purified protein demonstrate identity of the kinase with an internal region of PKC delta. Immunoblot analysis was used to confirm proteolytic cleavage of intact 78 kDa PKC delta in control cells to the 40 kDa C-terminal fragment after IR exposure. The finding that both IR-induced proteolytic activation of PKC delta and endonucleolytic DNA fragmentation are blocked by Bcl-2 and Bcl-xL supports an association with physiological cell death (PCD). Moreover, cleavage of PKC delta occurs adjacent to aspartic acid at a site (QDN) similar to that involved in proteolytic activation of interleukin-1 beta converting enzyme (ICE). The specific tetrapeptide ICE inhibitor (YVAD) blocked both proteolytic activation of PKC delta and internucleosomal DNA fragmentation in IR-treated cells. These findings demonstrate that PCD is associated with proteolytic activation of PKC delta by an ICE-like protease.

Effects of long-term tracheostomy on spectral characteristics of vowel production.

This study investigated the effects of long-term tracheostomy on the development of speech. Eight children who underwent tracheotomy during the prelingual period were compared to matched controls on selected spectral parameters of the speech acoustic signal and standard measures of oral-motor, phonologic, and articulatory proficiency. Analysis of formant frequency values revealed significant between-group differences. Children with histories of long-term tracheostomy showed reduced acoustic vowel space, as defined by group formant frequency values. This suggests that these children were limited in their ability to produce extreme vocal tract configurations for vowels (a,i,u) postdecannulation. Oral motor patterns were less mature, and sound substitutions were not only more variable for this group, but also reflected a persistent overlay of maladaptive compensations developed during cannulation.

Impaired development of oral-motor functions required for normal oral feeding as a consequence of tube feeding during infancy.

The child who is tube fed during the first two years of life is at risk for impaired development of normal oral-motor patterns and coordination during feeding. In order to ensure as smooth a transition to oral feeding as possible, it is necessary to provide the tube-fed child with a framework of normal oral sensitization from which he can develop the level of trust in his own oral mechanism required to achieve the mature, coordinated oral movements essential to development of the normal bite-chew-suck-swallow sequence. While this can be a tedious and taxing process, an ongoing program of oral stimulation, training and development should be included in the overall management of every young child receiving tube feedings.

The myeloid blood cell differentiation-inducing protein MGI-2A is interleukin-6.

The mouse myeloid blood cell differentiation-inducing protein, macrophage and granulocyte inducer, type 2A (MGI-2A), was purified, and the amino acid sequence of a CNBr cleavage peptide (22 residues) was determined. This amino acid sequence is identical to the sequence found in positions 73 to 94 of mouse interleukin-6 (IL-6). Recombinant mouse IL-6 protein induces differentiation of mouse myeloid leukemic cells that are induced to differentiation by MGI-2, and monoclonal antimouse-MGI-2 antibody, which neutralizes MGI-2, also completely neutralizes this IL-6-induced differentiation. These results show that the major type of mouse myeloid differentiation-inducing protein (MGI-2A) and IL-6 are very similar and most likely identical proteins. Recombinant human IL-6 (also called interferon-beta 2 or B-cell differentiation factor), which shows only a 41% similarity to mouse IL-6, has 11 identical amino acid residues out of the 22 in the mouse MGI-2A peptide and also induces differentiation of the same myeloid leukemic cells.

The human hematopoietic colony-stimulating factors.

The complementary DNAs and genes encoding the four major human myeloid growth factors--granulocyte colony-stimulating factor, macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3--have all been molecularly cloned. These DNA clones have proved valuable for studying the molecular biology of these important regulatory molecules as well as for the large-scale production of the recombinant growth factor proteins. These advances have led to a much better understanding of the role of the myeloid growth factors in regulating hematopoiesis in vivo that should soon find practical application in clinical medicine.

Immortalization of rat embryo fibroblasts by mutant polyomavirus large T antigens deficient in DNA binding.

We have identified a putative DNA-binding domain in polyomavirus large T antigen. Mutations introduced into the gene between amino acids 290 and 310 resulted in proteins that no longer bound to the high-affinity binding sites on the polyomavirus genome, showed no detectable nonspecific DNA binding, and were not able to initiate DNA replication from the viral origin. These mutant T antigen genes were introduced into rat embryo fibroblasts together with the neomycin resistance gene to allow selection for growth in the presence of G418. All the mutations tested facilitated the establishment of these cells in long-term culture at an efficiency indistinguishable from that of the wild-type protein.

A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediates selective mRNA degradation.

The mRNAs of transiently expressed genes frequently contain an AU-rich sequence in the 3' untranslated region. We introduced a 51 nucleotide AT sequence from a human lymphokine gene, GM-CSF, into the 3' untranslated region of the rabbit beta-globin gene. Our experiments demonstrate that this caused the otherwise stable beta-globin mRNA to become highly unstable in vivo. The instability conferred by the AU sequence in the mRNA was partially alleviated by treatment of the cells with cycloheximide. We propose that the AU sequences are the recognition signal for an mRNA processing pathway which specifically degrades the mRNAs for certain lymphokines, cytokines, and proto-oncogenes.

Stimulation of haematopoiesis in primates by continuous infusion of recombinant human GM-CSF.

Certain proteins are known to play an important part in the proliferation, differentiation and functional activation of haematopoietic progenitor cells in vitro. These proteins include erythropoietin and various colony-stimulating factors (CSFs), one of which is granulocyte-macrophage colony-stimulating factor (GM-CSF). Recently, both murine and human GM-CSF have been purified to homogeneity and complementary DNAs encoding them have been cloned. Although the in vitro activity of recombinant human GM-CSF has been investigated intensively, little is known about the functional activity of this protein in vivo. There is strong evidence that colony-stimulating activities produced by various human and murine tumour tissues and cell lines can stimulate granulopoiesis in mice, as can human urinary extracts. A partially purified preparation of human urinary colony-stimulating factor, however, proved only marginally effective in stimulating granulopoiesis in humans. All these studies suffer from the lack of a homogeneous preparation of colony-stimulating factor. It has recently been shown that recombinant murine multi-CSF or interleukin-3 can stimulate haematopoiesis in mice in vivo. Large-scale production of recombinant human GM-CSF now permits us to examine its effects in vivo using a primate model. We find that the continuous infusion of GM-CSF in healthy monkeys rapidly elicits a dramatic leukocytosis and a substantial reticulocytosis. A similar effect has been observed in one pancytopenic, immunodeficient rhesus macaque. These results suggest that GM-CSF could prove useful in several clinical situations.

Guanine nucleotide contacts within viral DNA sequences bound by polyomavirus large T antigen.

Essential nucleotide contacts between the polyomavirus large T antigen and its multiple specific binding regions within the regulatory sequences of the polyomavirus genome were determined in vitro by methylation interference. Methylation of any of the guanine residues of the 5'-G(A/G)GGC-3' pentanucleotide repeats in large-T-antigen-binding regions A, B, C, and 3 (A. Cowie and R. Kamen, J. Virol. 52:750-760, 1984) interfered with T antigen binding. Within regions A, B, and C these pentanucleotides are spaced 5 or 6 base pairs apart. Therefore, the clusters of contacted nucleotides within each of these binding regions are localized along one face of the DNA helix. Methylation of guanines within the sequences between the pentanucleotide repeats did not interfere with binding. The ORI binding region contains four additional pentanucleotide sequences within a region of dyad symmetry. Methylation of only particular guanines of these pentanucleotides interfered with T antigen binding. The spatial arrangement of the pentanucleotides in the ORI is such that the clusters of contacted guanines are situated around the DNA helix, thereby forming a very different arrangement from that found in the other binding regions. A model is discussed in which cooperative interactions between T antigen protomers, recognizing individual pentanucleotides, determines the strength and the function of different T antigen-DNA interactions.

Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression.

Sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral DNA replication (Tyndall et al., Nucleic Acids Res. 9:6231-6250, 1981). We have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. One approach, recently described by de Villiers et al. (Nature [London], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptional enhancers suggested that an enhancer function is required for polyomavirus DNA replication. The other approach, described in this paper, was to analyze a series of deletion mutants that functionally dissect the enhancer region and enabled us to localize four sequence elements in this region that are involved in the activation of replication. These elements, which have little sequence homology, are functionally redundant. Element A (nucleotides 5108 through 5130) was synthesized as a 26-mer with XhoI sticky ends, and one or more copies were introduced into a plasmid containing the origin of replication, but lacking the enhancer region. Whereas one copy of the 26-mer activated replication only to 2 to 5% of the wild-type level, two copies inserted in either orientation completely restored replication. We found that multiple copies of the 26-mer were also active as a transcriptional enhancer by measuring the beta-globin mRNA levels expressed from a plasmid that contained either the polyomavirus enhancer or one or more copies of the 26-mer inserted in a site 3' to the beta-globin gene. We observed a correlation between the number of inserted 26-mers and the level of beta-globin RNA expression.

Multiple binding sites for polyomavirus large T antigen within regulatory sequences of polyomavirus DNA.

Polyomavirus large T antigen binds specifically to multiple sites within the regulatory region of the viral genome. Experiments done with crude extracts from wild-type virus-infected mouse cells and immunoprecipitation of protein-DNA complexes localized two high-affinity binding sites on the early region side of the DNA replication origin. Purification of the large T antigen by immunoaffinity chromatography made it possible to refine the analysis through application of DNase I footprinting. The high-affinity interactions were resolved into three closely spaced, but distinct, binding regions. These begin at a site only slightly overlapping the early boundary of the core replication origin, a location highly homologous to that of simian virus 40 large T antigen-binding site I, but then extend away from the origin toward the early coding sequence and thus span the early region transcriptional initiation sites. Each tight-binding region contains from two to four copies of the sequence 5'-(A = T)G(A greater than G)GGC-3' repeated at 9- to 11-base-pair spacing. At high protein concentrations and at low ionic strength, additional sites within the core replication origin and in the enhancer region were protected from DNase I digestion. These minor binding sites also included repeats of sequences related to the consensus, but at different spacings. Our results suggest that, unlike simian virus 40 DNA, the polyomavirus genome may have distinct regions of interaction with its large T antigen which separately are involved in initiation of DNA replication and the regulation of viral transcription.

Polyoma virus DNA replication requires an enhancer.

Sequences which activate polyoma virus DNA replication are located within a region that also includes the transcriptional enhancer. We demonstrate a cis involvement of enhancers in DNA replication by showing that this region can be replaced by other enhancers, in a position- and orientation-independent manner, and that an immunoglobulin gene enhancer confers tissue-specific replicatory ability.

Construction and functional characterization of polyomavirus genomes that separately encode the three early proteins.

Modified polyomavirus genomes that individually encode the large and small T proteins were constructed by exchanging restriction endonuclease fragments between cDNA copies of the respective mRNAs and cloned genomic DNA. The efficacies of the new constructs, and that of the middle T protein gene described previously (R. Treisman , U. Novak, J. Favaloro , and R. Kamen , Nature [London] 292:595-600, 1981), were demonstrated with simian virus 40 (SV40)-polyomavirus recombinants in which part or all of the SV40 late region was replaced with the modified polyomavirus early genes. Each of the three recombinant viruses induced the synthesis of only the expected polyomavirus early protein in infected CV-1 cells. The rates of synthesis of large, middle, and small T proteins were ca. 1.5, 4.0, and 9.0 times the rate of synthesis of SV40 large T protein, respectively. The deletion of introns had no detrimental effect on mRNA biogenesis. Indeed, a further polyomavirus-SV40 recombinant, containing wild-type polyomavirus early region DNA, expressed an aberrant 58,000-dalton form of the middle T protein which we believe to result from utilization of a cryptic splice site. Immunofluorescence studied with monkey cells infected by the recombinant viruses allowed us to determine the cellular locations of the polyomavirus early proteins. Overproduction of the middle T protein did not result in a corresponding overproduction of the middle T protein-associated tyrosine phosphokinase activity.

DNA binding activity of polyoma virus large tumor antigen.

Polyoma virus large tumor antigen from productively infected mouse cells has been purified to greater than 50% homogeneity by a simple immunoaffinity procedure using monoclonal antibodies. A radioimmunoreaction was devised for assaying purity. The purified large tumor antigen retained its antigenicity and its ability to bind DNA specifically. The regions on the polyoma virus genome recognized by the protein were characterized. Three binding regions were localized within the portion of the genome between the viral origin of DNA replication and the protein coding sequence, overlapping the early promoter and the sites of initiation of mRNAs that specify the viral tumor antigens. The binding regions each contain direct repeats of the pentanucleotide sequence G-R-G-G-C.

Expression of the large T protein of polyoma virus promotes the establishment in culture of "normal" rodent fibroblast cell lines.

Transfer into mouse and rat embryo fibroblasts in primary culture of cloned polyoma virus genes encoding only the large T protein led to the establishment of flat colonies in sparse subcultures at a frequency equal to that of transformation by wild-type virus. Cell lines could be derived from such colonies and maintained in culture for large numbers of generations without entering crisis. They exhibited a normal phenotype, by the criteria of growth on plastic to a low saturation density and of anchorage dependency. However, they required a lower serum concentration for growth than spontaneously established 3T3 cells. Similar results were obtained after transfer of recombinant DNA molecules encoding only the amino-terminal 40% of the large T protein, suggesting that this "immortalization" function corresponds to the activity of an amino-terminal domain of the protein. Immunoprecipitation analysis of T antigens in cell lines established after transfer of the full-size and of the truncated large T genes demonstrated the expression of the full-size large T protein and of a Mr 40,000 antigen expressed from the amino-terminal part of the gene, respectively. After transfer of a "large T only" plasmid that carries a tsa mutation, cell lines were established at 33 degrees C with the same efficiency as with the wild-type large T gene, but their growth was arrested after a shift to 40 degrees C, with a progressive loss in cell viability. This result indicates a continuous requirement for a large T function in the maintenance of "immortality."