Sensory neuron regulation of gastrointestinal inflammation and bacterial host defence.

Sensory neurons in the gastrointestinal tract have multifaceted roles in maintaining homeostasis, detecting danger and initiating protective responses. The gastrointestinal tract is innervated by three types of sensory neurons: dorsal root ganglia, nodose/jugular ganglia and intrinsic primary afferent neurons. Here, we examine how these distinct sensory neurons and their signal transducers participate in regulating gastrointestinal inflammation and host defence. Sensory neurons are equipped with molecular sensors that enable neuronal detection of diverse environmental signals including thermal and mechanical stimuli, inflammatory mediators and tissue damage. Emerging evidence shows that sensory neurons participate in host-microbe interactions. Sensory neurons are able to detect pathogenic and commensal bacteria through specific metabolites, cell-wall components, and toxins. Here, we review recent work on the mechanisms of bacterial detection by distinct subtypes of gut-innervating sensory neurons. Upon activation, sensory neurons communicate to the immune system to modulate tissue inflammation through antidromic signalling and efferent neural circuits. We discuss how this neuro-immune regulation is orchestrated through transient receptor potential ion channels and sensory neuropeptides including substance P, calcitonin gene-related peptide, vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide. Recent studies also highlight a role for sensory neurons in regulating host defence against enteric bacterial pathogens including Salmonella typhimurium, Citrobacter rodentium and enterotoxigenic Escherichia coli. Understanding how sensory neurons respond to gastrointestinal flora and communicate with immune cells to regulate host defence enhances our knowledge of host physiology and may form the basis for new approaches to treat gastrointestinal diseases.

Major pol gene progenitors in the evolution of oncoviruses.

The genetic relationships among molecularly cloned prototype viruses representing all of the major oncovirus genera were investigated by molecular hybridization and nucleotide sequence analysis. One of the major progenitors of the pol genes of such viruses gives rise to mammalian type C viruses and another gives rise to type A, B, D, and avian type C oncoviruses. Evidence of unusual patterns of homology among the env genes of mammalian type C and D oncoviruses illustrates that genetic interactions between their progenitors contributed to the evolution of oncoviruses.

A new class of endogenous human retroviral genomes.

Human DNA contains multiple copies of a novel class of endogenous retroviral genomes. Analysis of a human recombinant DNA clone (HLM-2) containing one such proviral genome revealed that it is a mosaic of retroviral-related sequences with the organization and length of known endogenous retroviral genomes. The HLM-2 long terminal repeat hybridized with the long terminal repeat of the squirrel monkey virus, a type D retrovirus. The HLM-2 gag and pol genes share extensive nucleotide sequence homology with those of the M432 retrovirus (a type A-related retrovirus), mouse mammary tumor virus (a type B retrovirus), and the avian Rous sarcoma virus (a type C retrovirus). Nucleotide sequence analysis revealed regions in the HLM-2 pol gene that were as much as 70 percent identical to the mouse mammary tumor virus pol gene. A portion of the putative HLM-2 env gene hybridized with the corresponding region of the M432 viral genome.

Human endothelial cell growth factor: cloning, nucleotide sequence, and chromosome localization.

Several of the endothelial cell polypeptide mitogens that have been described probably play a role in blood vessel homeostasis. Two overlapping complementary DNA clones encoding human endothelial cell growth factor (ECGF) were isolated from a human brain stem complementary DNA library. Southern blot analysis suggested that there is a single copy of the ECGF gene and that it maps to human chromosome 5 at bands 5q31.3 to 33.2 A 4.8-kilobase messenger RNA was present in human brain stem messenger RNA. The complete amino acid sequence of human ECGF was deduced from the nucleic acid sequence of these clones; it encompasses all the well-characterized acidic endothelial cell polypeptide mitogens described by several laboratories. The ECGF-encoding open reading frame is flanked by translation stop codons and provides no signal peptide or internal hydrophobic domain for the secretion of ECGF. This property is shared by human interleukin-1, which is approximately 30 percent homologous to ECGF.

Cloning of the gene coding for human class 1 heparin-binding growth factor and its expression in fetal tissues.

We have identified four overlapping genomic DNA clones coding for human class 1 heparin-binding growth factor (HBGF-1), also known as acidic fibroblast growth factor, by screening genomic DNA libraries with an HBGF-1 cDNA probe. The exon-intron structure of the HBGF-1 gene was determined by Southern hybridization and nucleotide sequence analysis. The complete amino acid sequence of human HBGF-1 was deduced from the nucleotide sequence of these genomic DNA clones. The predicted amino acid sequence is identical to the published amino acid sequence determined by protein sequencing. Southern blot analysis of human DNA suggested that there is a single-copy gene coding for HBGF-1. A 4.5-kilobase mRNA and two minor species (3.4 and 2.0 kilobases) homologous to the HBGF-1 gene were detected in cellular RNA isolated from human adult brain and kidney. The HBGF-1 mRNAs from brain and kidney had slightly different sizes. The mechanism for the synthesis of different sizes of mRNA was not determined. We also detected HBGF-1 transcript from glioblastoma cells, fetal brain, and kidney but not from placenta or fetal liver. Since HBGF-1 is an angiogenic factor, these data suggest that it may play a role in embryonic angiogenesis during fetal development.

Multiple controlling mechanisms of FGF1 gene expression through multiple tissue-specific promoters.

We now know that fibroblast growth factor-1 (FGF1) transcription is controlled by at least four distinct promoters in a tissue-specific manner. Thus, promoter 1.A is active in the kidney, 1.B in the brain, and 1.C and 1.D in a variety of cultured cells including vascular smooth muscle cells. These promoters are separated from each other by up to 70 kbp. Multiple FGF1 transcripts arise from alternate promoter usage and alternative splicing of different 5'-untranslated exons. The 1.A and 1.B promoters are constitutively active in their respective cell types. In contrast, different biological response modifiers, including serum and transforming growth factor beta, can induce the 1.C and 1.D promoters. The 540-bp sequence upstream of the 1B transcription initiation site is sufficient to drive the expression of a heterologous luciferase reporter in cultured cells, and an 18-bp sequence within this region is important for the regulation of brain-specific gene expression. Furthermore, regulation occurs through the binding of the 18-bp sequence to a brain-specific 37-kDa protein and a ubiquitous basic helix-loop-helix protein, E2-2. We have produced transgenic mice bearing the brain-specific promoter of the human FGF1 gene joined to the SV40 immediate-early gene, which encodes the large T antigen. The resulting mice developed brain tumors that originated in the pontine gray, just rostral to the fourth ventricle. We have also identified a serum response element, comprising a CarG box and an Ets-binding site, in the 1.D promoter. Continued characterization of the mechanistic events that control the tissue-specific activation of FGF1 promoters will help us to understand the role of FGF1 in cancer, atherosclerosis, and neural development.

Alternative splicing generates two forms of mRNA coding for human heparin-binding growth factor 1.

Human class 1 heparin-binding growth factor (HBGF-1), also known as acidic fibroblast growth factor, is a mitogen for a variety of mesoderm- and neutroectoderm-derived cells in vitro as well as an angiogenic factor in vivo. Several oncogenes and growth factors have been shown to be homologous to HBGF-1. Four cDNA clones coding for HBGF-1 have been isolated from a human brain stem cDNA library. Nucleotide sequence analysis revealed that alternative splicing generated at least two different forms of HBGF-1 mRNA. Because the difference occurs in the 5'-untranslated regions, these transcripts may result from the usage of alternative promoters. One of the cDNA clones contains the polyadenylation signal, AATAAA, and a poly(A) tail, representing the 3'-end of an HBGF-1 mRNA. RNAase protection assays suggested this cDNA clone corresponds to a minor transcript, and the majority of the HBGF-1 mRNA terminates at 3.1 kbp downstream from the translation termination codon. The biological significance of this unusually long 3'-untranslated sequence is not known. To study the HBGF-1 gene structure, we have isolated 50 kbp of contiguous genomic DNA coding for the HBGF-1 protein. Both restriction enzyme mapping and nucleotide sequencing established that the distance between the first and second protein-coding exons is 13.6 kbp while that between the second and third is 5.3 kbp. By using the HBGF-1 cDNA as a probe, we showed that human fetal heart expresses high levels of HBGF-1 mRNA. Thus, HBGF-1 may be involved in mediating processes such as embryonic development and vascular growth in the heart.

Tumorigenesis in transgenic mice in which the SV40 T antigen is driven by the brain-specific FGF1 promoter.

Gene expression can be manipulated by the introduction of a hybrid gene formed by linking a highly tissue-specific regulatory element to a gene whose expression might be expected to alter cellular function. Previously, we have shown that the human FGF1 gene contains four distinct tissue-specific promoters. In an effort to perturb the programming of proliferation and differentiation in a subset of neural cells, we have produced transgenic mice bearing the brain-specific promoter of the human FGF1 gene joined to the SV40 immediate early gene, which encodes the large T antigen. The resulting mice, and offspring from four individual lines, developed brain tumors that originated in the pontine gray, just rostral to the fourth ventricle. Tumors were moderately vascularized, as demonstrated by staining with both hematoxylin and eosin and antibodies to three different endothelial cell markers, but vessels were histologically normal. Scattered tumor foci were present as early as postnatal day 26; and affected animals died between 5 - 8 months of age. In mature animals, tumors lacked terminal differentiation markers for astrocytes (glial fibrillary acidic protein) or neurons (synaptophysin and neuron-specific enolase). However, they expressed high levels of proliferating cell nuclear antigen and vimentin, markers for proliferating cells. This immunophenotype is consistent with the tumor being at an early stage of differentiation. Therefore, these mice may provide a valuable tool for the study of tumorigenesis, replenishment and differentiation of neural stem cells.

Different fibroblast growth factor 1 (FGF-1) transcripts in neural tissues, glioblastomas and kidney carcinoma cell lines.

We have previously reported the tissue specific distribution of four different FGF-1 transcripts containing alternative 5' untranslated exons spliced to the first protein coding exon. The predominant transcript in brain is FGF-1.B and in kidney FGF-1.A. Others have shown, by in situ hybridization and immunohistochemical analysis, that expression of FGF-1 in the brain is exclusively in neural cells but not in glial cells. Here we have examined the distribution of FGF-1.B and FGF-1.A transcripts in glioblastoma and retinal tissues and in kidney carcinoma cell lines. Our results show that FGF-1.B is the predominant transcript in neural derived tissues including both the diabetic retina and normal retina tissues. Surprisingly, FGF-1.B transcript is highly expressed in glioblastoma tissues. In contrast, a normal brain glial cell line, CHII, expresses very low levels of FGF-1 mRNA. These results strongly implicate the role of FGF-1 in the etiology of glioblastoma. We also examined several kidney carcinoma derived cell lines for the expression of FGF-1 mRNA. Most of these kidney cell lines do not express any FGF-1 transcripts. An interpretation by deduction is that kidney adenocarcinomas are derived from cortex but medulla has been reported as the site of FGF-1 synthesis. Of the kidney derived cell lines which are positive for FGF-1 message, only one expressed FGF-1.A transcript. The data may suggest that the establishment of kidney cell lines results in a switch of promoter usage from the 1.A seen in kidney tissue. Similarly, culturing of glioma cell lines may result in a switch from FGF-1.B seen in glioma tissues to FGF-1.D seen in most glioma cell lines. Continued studies of the FGF-1 transcripts, their functional promoters and their tissues distribution will provide insight into the potential role of FGF-1 in cell growth, tissue differentiation and malignant transformation.

The human FGF-8 gene localizes on chromosome 10q24 and is subjected to induction by androgen in breast cancer cells.

Androgen-induced growth factor (AIGF or FGF-8) was originally isolated from the conditioned medium of an androgen-dependent Shionogi carcinoma, SC-3, cell line. It shares structural similarity with other members of the FGF family. The temporal and spatial expression patterns of the FGF-8 gene suggest its involvement in gastrulation, regionalization of the brain, and organogenesis of the limb and face as an embryonic epithelial factor. In the adult, expression of FGF-8 is restricted to gonads including testes and ovaries. Since FGF-8 is identified as a corroborating gene in MMTV-induced mammary tumors in Wnt-1 transgenic mice and because FGF-8 manifested its autocrine mitogenic activity in SC-3 cells, it is possible that aberrant expression of FGF-8 may be present in human cancers which are hormone dependent. However, very little is known about human FGF-8. To determine whether FGF-8 plays a role in human breast cancer, we have isolated the full-length cDNA from SK-BR-3 breast cancer cells. We have also isolated the corresponding genomic DNA in a P1 cloning vector. The FGF-8 gene has been mapped to chromosome 1Oq24 using both somatic cell hybrid genetic analysis and fluorescence in situ hybridization. Finally, we show that FGF-8 gene expression in a human breast cancer cell line, MDA-MB-231, is inducible by androgen. The findings presented here will facilitate our understanding of the molecular mechanism underlying hormone-responsive breast and prostate cancers.

Cloning and sequence analysis of the human acidic fibroblast growth factor gene and its preservation in leukemia patients.

Acidic fibroblast growth factor (aFGF), also known as heparin-binding growth factor 1, is a mitogen for a variety of mesoderm- and neuroectoderm-derived cells. Several different aFGF mRNA species resulting from alternative splicing have been reported. These results suggest that the gene structure and regulatory mechanism for gene expression of aFGF are complex. As a first step toward understanding aFGF gene structure, we have isolated nine overlapping genomic DNA clones spanning 54 kbp and determined the complete DNA sequences of all three coding exons. Comparison of the nucleotide sequences between the human and bovine DNA showed that the sequence similarity extended 2400 bp downstream from the coding region. Cloning of the aFGF gene allowed us to characterize this locus in acute nonlymphocytic leukemia (ANLL) patients. A fraction of ANLL patients (10-20%) have a deletion in the long arm of chromosome 5, whose distal breakpoint overlaps the aFGF locus. Therefore, a prospective cohort of eight ANLL patients was screened using three different repetitive sequence-free probes derived from the aFGF locus. Using beta-globin gene as a normalization probe for hybridizing band intensities, we conclude that there is no allelic loss or gross rearrangement within the 40 kbp stretch of the aFGF gene locus in ANLL patients with or without 5q- deletion. Consistent with this observation, the aFGF mRNA was not detected in the mononuclear cells derived from either an ANLL patient or a normal individual as judged by the reverse transcription and polymerase chain reaction. We also identified a DNA fragment, 10.7 kbp upstream from the first coding exon of human aFGF, whose sequence is conserved in both the primate and rodent genomes. Further characterization of this fragment is likely to provide insight into the significance of this high degree of conservation.

Gene structure and differential expression of acidic fibroblast growth factor mRNA: identification and distribution of four different transcripts.

We have isolated four cDNA clones coding for human acidic fibroblast growth factor (aFGF) containing alternative 5' untranslated exons. Using RNAase protection analyses, we demonstrated the presence of at least four upstream, untranslated exons that are alternatively spliced to the first protein-coding exon. We designate these four untranslated exons, -1A, -1B, -1C and -1D. Splicing of these exons to the first coding exon will generate mRNA 1.A, 1.B, 1.C and 1.D respectively. Expression of these transcripts is regulated in a tissue-specific manner, as the major aFGF transcript in human brain frontal cortex differs from that in kidney. Furthermore, the pattern of aFGF transcripts in several glioblastoma cell lines tested is different from that in normal brain tissue. We isolated nine overlapping genomic clones containing these four upstream, untranslated exons. These four exons were localized on these clones by Southern hybridization and nucleotide sequence analysis. The overlapping clones are shown to be contiguous with our previously isolated genomic clones that contain the three aFGF-coding exons. The sizes of the four introns are 82.9, 71.1, 29.3 and 6.9 kbp. The transcriptional start sites of the two most upstream exons (-1A and -1B) have been mapped using RNAase protection and primer extension analyses. The sequences upstream of the start sites for aFGF 1.B mRNA do not contain a consensus TATA box. In contrast, the canonical CCAAT and TATA sequences are located at the proper distances from the transcription start site of aFGF 1.A mRNA.

Detection of the PTC/retTPC oncogene in human thyroid cancers.

We have investigated the PTC/retTPC oncogene, an activated form of ret proto-oncogene with a specific rearrangement, in thyroid malignancies. Southern analysis was used to screen 36 thyroid papillary carcinomas (PC), 22 normal thyroid tissues from glands with PC elsewhere, three follicular carcinomas, eight follicular adenomas and 30 other non-malignant thyroids. Rearrangements were detected in four PCs (11%) using probes derived from the ret proto-oncogene. Genomic breakpoints from a PC and a PC cell line (TPC-1) were cloned and sequenced. The rearrangement points of ret proto-oncogene were found in the intron between the exon for the transmembrane domain and the first exon for the tyrosine kinase domain. Furthermore, the PTC/retTPC chimeric transcripts were detected in two PCs with the rearrangement by reverse transcription polymerase chain reaction. Distant metastases were present in 50% (2/4) of PCs with the rearrangement, but in only two out of 32 PCs without a detectable rearrangement (P = 0.05, Fisher exact test). Our study suggests that the rearrangement of the ret proto-oncogene may be involved in the development of distant metastases in patients with papillary thyroid carcinomas. However, a larger clinical study will be required to verify this observation.

Nucleotide sequences of two newt (Notophthalmus viridescens) fibroblast growth factor receptor-2 variants.

The FGF receptor tyrosine kinase family consists of four members. We report the sequence of two newt (Notophthalmus viridescens) FGFR2 cDNAs which were isolated from a forelimb blastema cDNA library and represent the newt cognates of two different isoforms of FGFR2, one homologous to bek the other to the KGFR.

Uncoordinate synthesis of histone H1 in cells arrested in the G1 phase.

It has been known for several years that DNA replication and histone synthesis occur concomitantly in cultured mammalian cells. Normally all five classes of histones are synthesized coordinately. However, mouse myeloma cells, synchronized by starvation for isoleucine, synthesize increased amounts of histone H1 relative to the four nucleosomal core histones. This unscheduled synthesis of histone H1 is reduced within 1 h after refeeding isoleucine, and is not a normal component of G1. The synthesis of H1 increases coordinately again with other histones during the S phase. The DNA synthesis inhibitors, cytosine arabinoside and hydroxyurea, block all histone synthesis in S-phase cells. The levels of histone H1 mRNA, relative to the other histone mRNAs, is increased in isoleucine-starved cells and decreases rapidly after refeeding isoleucine. The increased incorporation of histone H1 is at least partially due to the low isoleucine content of histone H1. Starvation of cells for lysine resulted in a decrease in H1 synthesis relative to core histones. Again the ratio was altered on refeeding the amino acid. 3T3 cells starved for serum also incorporated only H1 histones into chromatin. The ratio of different H1 proteins also changed. The synthesis of the H1(0) protein was predominant in G0 cells, and reduced in S-phase cells. These data indicate the metabolism of H1 is independent of the other histones when cell growth is arrested.

Differential expression of two clusters of mouse histone genes.

The mouse histone mRNAs coded for by three different cloned DNA fragments have been characterized. Two of these cloned DNA fragments, MM221 and MM291, located on chromosome 13, code for H3, H2b and H2a histone mRNAs, which are expressed at low levels in cultured mouse cells and fetal mice. The other DNA fragment, MM614, located on chromosome 3, codes for an H3 and an H2a mRNA, which are expressed at high levels in these cells. The mRNAs for each histone protein share common coding region sequences, while the untranslated regions of all the genes have diverged significantly, as judged by S1 nuclease mapping. Amino acid substitutions in some H3, H2a and H2b proteins are detected as internal cleavages in the S1 nuclease maps. All of these genes code for replication variant histone mRNAs, which are regulated in parallel with DNA synthesis.

Cloning and characterization of the mouse Fgf-1 gene.

Fibroblast growth factor 1 (FGF-1 or aFGF), is a mitogen for a variety of mesoderm- and neuroectoderm-derived cells, as well as an angiogenic factor in vivo. It has been implicated in angiogenic diseases including atherosclerosis, cancer and inflammatory autoimmune diseases. As part of an effort to understand the role of FGF-1 in the pathobiology of inflammation, we have isolated and characterized the mouse Fgf-1 gene. Southern blot analysis of mouse genomic DNA using the mouse Fgf-1 cDNA as a probe revealed that mouse FGF-1 is encoded by a single copy gene. Comparison of the available mouse Fgf-1 cDNA sequence with newly obtained genomic sequence allowed us to establish the exon/intron boundaries. The mouse Fgf-1 coding region is comprised of three protein coding exons, which we determined to be separated by an 11.4-kb and a 4.9-kb intron. The elucidation of the mouse Fgf-1 coding region revealed great similarity between the mouse and human Fgf-1 gene structure.

Cloning and characterization of a novel upstream untranslated exon of the mouse Fgf-1 gene.

Fibroblast growth factor 1 (FGF-1 or aFGF), is the prototype member of the heparin-binding growth factors which are capable of angiogenesis in vivo. FGF-1 has been implicated in atherosclerosis, cancer, wound repair and inflammatory autoimmune diseases. As part of an effort to understand the role of FGF-1 in the etiopathogenesis of inflammation and cancer, we have undertaken steps to isolate and characterize the mouse Fgf-1 gene. Southern blotting and sequence analysis displayed considerable conservation within the coding and upstream untranslated regions of Fgf-1 in human, mouse, hamster, rat and bovine. By using primers derived from the 5'-untranslated exon of a rat prostate-specific Fgf-1 cDNA, a 220-bp product was amplified from mouse genomic DNA via PCR. Sequence analysis of this amplicon showed that there was 80% similarity with the corresponding region of the rat FGF-cDNA sequence. Primers designed from this amplicon and the Fgf-1 coding region were used to isolate multiple overlapping genomic clones spanning the entire mouse Fgf-1 gene. Sequencing analysis of the genomic sequence upstream from this novel 5'-untranslated exon did not reveal typical TATA, CCAAT sequences. It appears that the occurrence of multiple untranslated exons for FGF-1 is a highly conserved theme for this gene across species.

Novel receptor protein tyrosine phosphatase (RPTPrho) and acidic fibroblast growth factor (FGF-1) transcripts delineate a rostrocaudal boundary in the granule cell layer of the murine cerebellar cortex.

We have identified a novel receptor-like protein tyrosine phosphatase (RPTPrho) transcript whose expression in the cerebellar cortex is restricted to the granule cell layer of lobules 1-6. Acidic fibroblast growth factor (FGF-1) mRNA follows a similar cerebellar expression pattern. Together, the two markers define a sharp boundary in lobule 6, slightly caudal to the primary fissure. Anterior and posterior compartments became discernible only during postnatal weeks two and six, for RPTPrho and FGF-1, respectively. A rostrocaudal boundary in lobule 6 of the murine cerebellar cortex has also been identified morphologically by the effects of the meander tail mutation. The position of the RPTPrho and FGF-1 boundary on the rostrocaudal axis of the cerebellar cortex was close to, but not coincident with, the caudal extent of the disorganized anterior lobe of meander tail and the rostral extent of Otx-2 expression. The restricted pattern of FGF-1 and RPTPrho implies that these molecules may have specific signaling roles in the tyrosine phosphorylation/dephosphorylation pathway in the anterior compartment of the adult cerebellar cortex.

Genetic relatedness between intracisternal A particles and other major oncovirus genera.

Intracisternal A particles represent a major oncovirus genus. By reciprocal hybridization between molecularly cloned A particles and representatives of other oncovirus genera, we established pol gene homology with type B, type D and avian type C viruses. The most extensive homology was with mammalian type D viruses. The transcriptional orientation of the IAP genome was determined, as well as evidence indicating that its pol gene, which is apparently defective, contains coding regions for both reverse transcriptase and endonuclease proteins.