Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

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Differentiation and survival of neuronal cell types requires the action of neurotrophic polypeptides such as nerve growth factor (NGF). In the central and peripheral nervous system and the phaeochromocytoma cell model PC12, NGF exerts its effects through the activation of the signalling capacity of Trk, a receptor tyrosine kinase (RTK) which upon interaction with NGF becomes phosphorylated on tyrosines and thereby acquires the potential to interact with signal‐transducing proteins such as phospholipase C‐gamma (PLC gamma), phosphatidylinositol‐3′‐kinase (PI3′‐K) and SHC. Mutagenesis of the specific binding sites for these src homology 2 (SH2) domain‐containing substrates within the Trk cytoplasmic domain suggests a non‐essential function of PI3′‐K and reveals a major role for the signal controlled by the SHC binding site at tyrosine 490 and a co‐operative function of the PLC gamma‐mediated pathway for neuronal differentiation of PC12 cells.

The upstream regulatory region of the human papilloma virus‐16 (HPV‐16) genomic DNA contains a sequence element with a large degree of homology to the partially palindromic sequence GGTACANNNTGTTCT, which is the consensus sequence of the glucocorticoid responsive elements of known genes regulated by this steroid hormone. DNase I and dimethylsulfate protection experiments reveal the binding of this sequence by rat glucocorticoid receptor protein. A 400‐bp DNA segment centrally containing this sequence confers strong inducibility by dexamethasone to the promoter p97 of HPV‐16 and to the Herpes simplex virus thymidine kinase promoter, as judged by chloramphenicol acetyltransferase activity and RNase protection assays. The same DNA segment, that does not contain the consensus sequences of all papilloma viruses relevant for E2 protein‐mediated transcription enhancement, functions in an enhancer‐like fashion in addition to its glucocorticoid responsive action. This hormone‐independent transcription enhancement is absent in human MCF7 cells, but is strong in human HeLa cells where the combined activity of the constitutive and the steroid hormone‐dependent enhancer elements stimulate transcription by a factor of 500. This cell type specificity of the HPV‐16 enhancer may be responsible for the tissue tropism of the virus. These observations and the presence of numerous homologies to known enhancers of cellular and viral genes suggest a complex pattern of activation of the human papilloma virus‐16 promoters.

We have sequenced 1730 bp of human papilloma virus type 18 (HPV 18) DNA containing the open reading frames (ORF) E6, E7, the N‐terminal part of E1 and, additionally, 120 bp of the N‐terminal part of L1. Based on these sequencing data, together with the human papilloma virus type 16 (HPV 16) DNA sequence published recently, we identified and cloned the ORF E6, E7, E1 and L1 of HPV 18 and the ORF E6, E7, E1, E4, E5, L2 and L1 of HPV 16 into prokaryotic expression vectors. The expression system used provides fusions to the N‐terminal part of the MS2 polymerase gene controlled by the heat‐inducible lambda PL promoter. Using the purified fusion proteins as immunogens we raised antisera against the proteins encoded by the ORF E6, E7 and E1 of HPV 18 as well as those encoded by the ORF E6, E7, E4 and L1 of HPV 16. By Western blot analysis we could show that the E7 gene product is the most abundant protein in cell lines containing HPV 16 or HPV 18 DNA. It is a cytoplasmic protein of 15 kd in the SiHa and the CaSki cell lines which contain HPV 16 DNA, and 12 kd in the HeLa, the C4‐1 and the SW756 cell lines which contain HPV 18 DNA. These results were confirmed by in vitro translation of hybrid‐selected HPV 16 and HPV 18 specific poly(A)+ RNA from SiHa, CaSki and HeLa cells. Additionally, these experiments led to the identification of an 11‐kd E6 and a 10‐kd E4 protein in the CaSki cell line as well as a 70‐kd E1 protein in HeLa cells.

Interleukin-1␤ (IL-1␤) is cytotoxic to rat pancreatic ␤-cells by inhibiting glucose oxidation, causing DNA damage and inducing apoptosis. Nitric oxide (NO) is a necessary but not sufficient mediator of these effects. IL-1␤ induced kinase activity toward Elk-1, activation transcription factor 2, c-Jun, and heat shock protein 25 in rat islets. By Western blotting with phosphospecific antibodies and by immunocomplex kinase assay, IL-1␤ was shown to activate extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (p38) in islets and rat insulinoma cells. Specific ERK1/2 and p38 inhibitors individually reduced but in combination blocked IL-1␤-mediated islet NO synthesis, and reverse transcription-polymerase chain reaction of inducible NO synthase mRNA showed that ERK1/2 and p38 controlled IL-1␤-induced islet inducible NO synthase expression at the transcriptional level. Hyperosmolarity caused phosphorylation of Elk-1, activation transcription factor 2, and heat shock protein 25 and activation of ERK1/2 and p38 in islets comparable to that induced by IL-1␤ but did not lead to NO synthesis. Inhibition of p38 but not of ERK1/2 attenuated IL-1␤-mediated inhibition of glucose-stimulated insulin release. We conclude that ERK1/2 and p38 activation is necessary but not sufficient for IL-1␤-mediated ␤-cell NO synthesis and that p38 is involved in signaling of NO-independent effects of IL-1␤ in ␤-cells.

By Western blot technique, 519 samples of human sera were tested for the presence of antibodies to the human papillomavirus (HPV) type 16 proteins E4 and E7 that had been expressed in Escherichia coli as fusion proteins. Sera were obtained from patients attending the University hospitals for reasons unrelated to HPV infections (controls), from patients with HPV-associated lesions, as well as from patients suffering from cervical cancer. Within the control population, 18.1% of them had antibodies that reacted with the E4 protein, and 3.9% of them had antibodies that reacted with the E7 protein. No sex-specific difference in the antibody prevalence was observed. The highest proportion of anti-E4 antibody-positive individuals (40.7%) was observed in the age group between 11 and 20 years. The frequency of anti-E4-positive sera was threefold higher in patients with HPV-associated genital lesions than that in age-matched controls. Antibodies against the HPV16 E7 protein were found 14 times more frequently in patients with cervical cancer, compared with age- and sex-matched controls (P less than .00001). From these data, we concluded that anti-E4 antibodies may be correlated with virus replication and that anti-E7 antibodies may represent a marker for cervical cancer development.