Impact of procedural multimedia instructions for pH BRAVO testing on patient comprehension: a prospective randomized study.

The positive impact on patient comprehension and improved procedural outcomes when multimedia is utilized to convey instructions preprocedurally has been previously shown for gastrointestinal procedures such as colonoscopy. However, in gastroesophageal reflux testing (GERD), we continue to utilize verbal and written instructions to establish this diagnosis when we use BRAVO pH testing. This is arguably a more complex procedure involving stopping medications, placement of a device, and maintaining an accurate diary for the duration of the testing. We hypothesize that by utilizing multimedia to relay complex textual information, patients will have improved comprehension of periprocedural instructions thereby improving data entry and satisfaction of expectations during the procedure. Prospective randomized study of 120 patients undergoing endoscopic placement of the BRAVO pH monitoring capsule for evaluation of GERD receive either written preoperative instructions (control) or written plus video instructions (video group). A composite comprehension score was calculated using procedure-specific parameters of data entry over the 48-hour monitoring period. Patient satisfaction was evaluated on the basis of a five-point Likert scale. Extent of patient satisfaction was defined by the fulfillment of patient expectations. Exclusion criteria included patients who did not have access to the video or did not complete follow-up. Seventy-eight patients completed all follow-up evaluations. The video group (n = 44) had a significantly higher mean comprehension score when compared to the control group (n = 34) (9.6 ± 1.4 vs. 7.4 ± 2.0, P = 0.01). Overall satisfaction with instructions was significantly higher in the intervention group (91% vs. 47%, p 0.01). We detected no significant difference in comprehension or satisfaction scores in subgroup analyses of the video group comparing patients <65 and ≥65 years of age and by education level. Compared to standard written instructions, video instructions improved patient comprehension based on data evaluation, and satisfaction. Therefore, clinicians should consider incorporation of multimedia instructions to enhance patient periprocedural expectations and understanding of reflux pH testing using the BRAVO procedure.

Intercellular adhesion molecule-1 (ICAM-1) is upregulated in aggressive papillary thyroid carcinoma.

BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) is implicated in carcinogenesis. In this study we examined the expression of ICAM-1 in papillary thyroid cancer (PTC). We hypothesized that ICAM-1 correlates with indicators of tumor aggressiveness in PTC. METHODS: Thirty-five primary and metastatic PTCs, five follicular adenomas, five Hashimoto thyroiditis, five nodular hyperplasia, and eight normal thyroid tissue samples were analyzed for ICAM-1 gene expression using quantitative reverse-transcription polymerase chain reaction (RT-PCR). ICAM-1 gene expression was analyzed at protein level by immunohistochemistry (IHC) using a semiquantitative score. Gene expression and intensity levels were correlated with markers of tumor aggressiveness including BRAF V600E mutation, tumor size, extrathyroidal extension (ETE), angiolymphatic invasion, and lymph node metastasis. RESULTS: ICAM-1 gene expression was higher in PTC (p = 0.01) and lymph node metastases (p = 0.03) when compared with benign tumors and Hashimoto's. Furthermore, PTCs exhibiting BRAF V600E mutation (p = 0.01), ETE (p < 0.01), and lymph node metastasis (p = 0.02) were associated with higher ICAM-1 levels. Gene expression correlated with protein levels on IHC. Additionally, poorly differentiated thyroid carcinoma had a higher ICAM-1 intensity score compared with well-differentiated carcinoma (p = 0.03). CONCLUSIONS: ICAM-1 expression is upregulated in papillary thyroid carcinoma. Furthermore, ICAM-1 upregulation correlated with aggressive tumor features such as BRAF V600E mutation, ETE, and lymph node metastasis, suggesting that ICAM-1 plays a role in thyroid cancer progression.

Bone metastases from differentiated thyroid carcinoma.

The presence of distant metastases from differentiated thyroid carcinoma decreases the 10-year survival of patients by 50%. Bone metastases represent a frequent complication especially of follicular thyroid cancer and severely reduce the quality of life causing pain, fractures, and spinal cord compression. Diagnosis is established by correlating clinical suspicion with imaging. Imaging is essential to detect, localize, and assess the extension of the lesions and should be used in conjunction with clinical evidence. Bone metastases are typically associated with elevated markers of bone turnover, but these markers have not been evaluated in differentiated thyroid cancer. Skeletal and whole-body magnetic resonance imaging and fusion 2-deoxy-2-[18F]fluoro-D-glucose whole-body positron emission tomography/computed tomography (PET/CT) are the best anatomic and functional imaging techniques available in specialized centers. For well-differentiated lesions, iodine-PET scan combined (124)I-PET/CT is the newest imaging development and (131)I is the first line of treatment. Bisphosphonates reduce the complications rate and pain, alone or in combination with radioiodine, radionuclides, or external beam radiotherapy and should be employed. Surgery and novel minimally invasive consolidation techniques demand an appropriate patient selection for best results on a multimodal approach. Basic research on interactions between tumor cells and bone microenvironment are identifying potential novel targets for future more effective therapeutic interventions for less differentiated tumors.

What is the quality of information about bariatric surgery on the internet?

BACKGROUND: The purpose of this study was to evaluate the role of Internet on patients scheduled for bariatric procedures and the quality of information available on different websites. METHODS: Between July 2003 to July 2005, patients undergoing bariatric surgical procedures completed a survey. Data were collected prospectively. One hundred valid surveys were returned. Independently, two bariatric surgeons evaluated available French and English websites using major search engines. RESULTS: Forty-two of 100 patients (42%) sought information about bariatric surgery on the Internet. Seventy-four percent of these patients (n = 31/42) used search engines with 81% visiting less than ten websites. According to the patient's evaluation, 58% of the websites visited did not provide technical details of any surgical bariatric procedures, and only 61% provided information regarding postoperative weight loss. Furthermore, 58% of websites did not provide information about the laparoscopic approach, and 54% did not give any information on potential postoperative complications. Bariatric surgeon's evaluation was similar except for two differences: laparoscopic approach and postoperative weight loss information were discussed in 90% (p < 0.001) and 43% (p < 0.1) of visited websites, respectively. CONCLUSION: When the Internet was used to search for information about bariatric surgery, search engines were preferentially used but search duration was short. Available Internet websites can be considered as moderately reliable; however, 25% of visited websites contain misleading information. Comparison between patients and surgeons views showed that patients were effective in detecting misleading information.

A novel transcriptional regulatory region within the core promoter of the hepatocyte growth factor gene is responsible for its inducibility by cytokines via the C/EBP family of transcription factors.

Hepatocyte growth factor (HGF) is an inducible cytokine that is essential for the normal growth and development of various tissues, such as the liver. To decipher the molecular mechanisms that regulate HGF gene induction at the transcriptional level, we carried out in vitro and in vivo studies on the mouse HGF gene promoter. We have identified a novel regulatory element, located between -6 and +7 bp (from the transcription start site) in the HGF basal promoter region, which binds to inducible transcription factors and dictates responsiveness to extracellular stimuli that activate this gene. The core binding sequence for the inducible cis-acting factors was determined to be TTTGCAA (-4 to +3 bp) within the HGF promoter. Competition and gel mobility supershift assays showed that these binding complexes are composed of C/EBPbeta (CCAAT/enhancer-binding protein beta) and C/EBPdelta. DNA binding analysis also revealed that the binding site for the C/EBP family of transcription factors in the HGF promoter region overlaps that of another binding protein (complex C1), which binds specifically to a novel sequence with a core binding site of ACCGGT located adjacent to the C/EBP site (-9 to -4 bp). C1 binds to this region of the promoter and represses the inducible upregulation by C/EBP through direct competition for their individual binding sites. Partial hepatectomy, which is known to activate HGF gene expression in the liver, increased C/EBP (especially C/EBPbeta) binding activity to this region of the HGF promoter. Thus, our present results provide a mechanistic explanation for the transcriptional induction of the HGF gene by extracellular signals (i.e., cytokines) that induce tissue growth and regeneration.

Identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse hepatocyte growth factor gene.

Hepatocyte growth factor (HGF), a cytokine with multiple functions, exhibits cell-type-specific as well as cytokine- and steroid hormone-regulated expression. The HGF gene is known to be expressed predominately in mesenchymal but not in epithelial cells. In this study, we report the identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse HGF gene, which is evidently responsible for the suppression of HGF expression in epithelial cells. Gel mobility shift assays and DNase I footprinting studies revealed that a 27-bp element (-16 to +11) around the transcription initiation site is responsible for the binding of a nuclear protein which is present in epithelial but not in mesenchymally derived cells. Further analysis of the binding activity of the DNA region with nuclear protein revealed that an approximately 19-bp sequence containing a unique palindromic structure (5'-AACCGACCGGTT-3') overlapped by a CAP box is essential for binding. Substitution of a single base (the contact site) within this region by site-directed mutagenesis resulted in total abrogation of the binding of the nuclear protein and a concomitant increase in the transcriptional activity of various lengths of HGF-chloramphenicol acetyltransferase fused genes when transfected into the epithelial cell line RL95-2 but not the mesenchymal cell line NIH 3T3. Southwestern (DNA-protein) analyses revealed that the nuclear protein which binds to this repressor element is a single polypeptide of approximately 70 kDa. Analysis of the nuclear extract prepared from regenerating mouse liver at various times after two-thirds partial hepatectomy by gel mobility shift assay revealed a substantial reduction (more than 75% within 3 h) in the binding of the repressor to its cognate binding site. Our results suggest that a cis-acting transcriptional repressor in the promoter region of the mouse HGF gene is involved in cell-type-specific regulation through binding to its cognate trans-acting protein which exists in epithelial cells but is absent in fibroblast cells.

Purification and biological characterization of human hepatopoietin A, a polypeptide growth factor for hepatocytes.

We have previously reported the presence of a high molecular weight polypeptide growth factor in the plasma of normal human or rat serum which stimulates DNA synthesis in primary cultures of normal rat hepatocytes. We referred to this activity as hepatopoietin A (HPTA) (Michalopoulos, G., Houck, K. A., Dolan, M. L., and Luetteke, N. C. Control of hepatocytes replication by two serum factors. Cancer Res., 44: 4414-4419, 1984; Thaler, J., and Michalopoulos, G. Hepatopoietin A. Partial characterization and trypsin activation of a hepatocyte growth factor. Cancer Res., 45: 2545-2549, 1985). At that time, however, complete purification of this growth factor had not been achieved. In the present report we describe the steps required for complete purification of HPTA from human plasma or rabbit serum. The purification involved sequential ammonium sulfate precipitation, heparin-affinity chromatography, anion-exchange high-performance liquid chromatography (HPLC), and reversed phase HPLC. The final purified product is a heterodimer consisting of a heavy and a light polypeptide chain with molecular weights of 70,000 and 35,000, respectively, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Under nonreducing conditions, however, the purified HPTA migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 69,000. The mitogenic activity of HPTA was associated with this band when it was eluted from unstained sodium dodecyl sulfate-polyacrylamide gels. Gel filtration HPLC under neutral isotonic conditions indicated that HPTA tends to form aggregates with molecular weights of greater than 300,000. Chromatofocusing indicated that HPTA is an acidic protein with an isoelectric point value of about 5.5. The mitogenic activity of HPTA was sensitive to heat, trypsin, and 2-mercaptoethanol, but relatively resistant to exposure to 1 N acetic acid, 2 M guanidine-HCl, and 0.1% sodium dodecyl sulfate. The stimulation of DNA synthesis induced by HPTA was totally abrogated by transforming growth factor-beta and markedly reduced in the presence of heparin. We present biochemical as well as biological evidence that HPTA is a hepatocyte growth factor distinct from other known polypeptide mitogens such as epidermal growth factor, transforming growth factor-alpha, platelet-derived growth factor, fibroblast growth factor, and thrombin.

The concerted regulatory functions of the transcription factors nuclear factor-1 and upstream stimulatory factor on a composite element in the promoter of the hepatocyte growth factor gene.

Hepatocyte growth factor (HGF) is an important multifunctional cytokine whose gene expression is regulated mainly at the transcriptional level. Previous studies using transgenic mice as well as in vitro analyses showed that a potential regulatory element(s) exists between -260 to -230 bp in the upstream region of the HGF gene promoter. In the present study, we have discovered that this region is a composite site through which members of the nuclear factor 1 (NF1) and upstream stimulatory factor (USF) families bind to and regulate HGF gene transcription. Gel mobility shift and supershift assays revealed that USF and NF1 have high binding affinity for this region and that the binding sites of the two different transcription factor families overlap. Functional studies showed that NF1 suppresses HGF gene promoter activity and that USF has an activating function. We found that the NF1/X and NF1/Red1 isoforms strongly suppressed HGF promoter activity while the NF1/L variant had no obvious effects. USF1, but not USF2, of the USF family stimulated HGF gene promoter activity. More interestingly, during liver regeneration after partial hepatectomy, a process which activates the HGF gene, we noted that the binding activity of USF to the HGF promoter element increased while that of NF1 decreased. These data provide insight into the molecular mechanisms that govern HGF gene transcription. Oncogene (2000).

Transcriptional activation of the hepatocyte growth factor receptor (c-met) gene by its ligand (hepatocyte growth factor) is mediated through AP-1.

Hepatocyte Growth Factor (HGF) exerts its biological effects via binding and activating a transmembrane protein tyrosine kinase receptor known as c-Met. Previous studies from our laboratory demonstrated that c-met gene expression is inducible by its own ligand (HGF). However, the molecular mechanism(s) involved in this process are unknown. The present study was carried out to address this question. Transfection of various c-met-CAT promoter constructs into the mouse hepatocellular carcinoma cell line Hepa 1-6 in combination with electrophoretic mobility shift assays (EMSA) identified the responsive element as an activated protein-1 (AP-1) binding site (TGAGTCA) within the c-met core promoter region at position -158 to -152. The c-met AP-1 element binds specifically to AP-1 protein as verified by supershift assays. EMSA studies and mutational analyses of the promoter region also revealed that the members of the Sp family of transcription factors (Sp-1 and Sp-3) bind to the c-met Sp-1 element (located at position -124) which is adjacent to the AP-1 site. We show that Sp binding dampens binding of AP-1 to its cognate site in the c-met promoter region. Stimulation of Hepa 1-6 cells with HGF resulted in a rapid and dramatic enhancement of the AP-1 binding activity as well as an overall increase in the level of AP-1 protein. Cotransfection of AP-1 expression vectors (c-Fos plus c-Jun) with c-met promoter constructs resulted in stimulation of c-met promoter activity. We found that transactivation of the c-met promoter by AP-1 can be blocked by Curcumin, an inhibitor of AP-1. Moreover, we found that the induction of the endogenous c-met gene by HGF is inhibited by the addition of Curcumin. The results demonstrate that the HGF-induced transcription of the c-met gene by HGF is, at least in part, due to activation of the AP-1 pathway.

Transcriptional regulation of the hepatocyte growth factor (HGF) gene by the Sp family of transcription factors.

In this study, we have localized an enhancer element in the 5'-flanking region of the HGF gene promoter and have identified its functional transcriptional factors. Through transient transfection of NIH3T3 fibroblast cells and gel mobility shift assays, the functional binding site was localized to a short region (-318 to -303 bp from the transcription start site) which has a CTCCC sequence. This motif is also conserved in the human HGF promoter and acts as a binding site for the Sp family of transcription factors. In the presence of NIH3T3 nuclear protein extracts, this enhancer region formed specific DNA-protein complexes which were totally abrogated by excess amounts of consensus Sp1 oligonucleotide. In gel mobility supershift assays, anti-Sp1 and anti-Sp3 antibodies specifically recognized these complexes as was evident by their slower migration. Site-specific mutation of this binding motif resulted in total loss of Sp1 and Sp3 binding and a concomitant loss of enhancer function within the context of the HGF promoter. Furthermore, in transient cotransfection assays, overexpression of Sp1 and/or Sp3 stimulated HGF promoter activity independently and additively through binding to the Sp1 binding site in the HGF gene promoter region. DNaseI hypersensitive analysis of freshly isolated nuclei from NIH3T3 cells revealed that five hypersensitive sites (HSS) are present within the 2 kb region immediately upstream of the HGF promoter. One of these sites was mapped to position -0.3 kb from the transcription start site. These data show that both Sp1 and Sp3 transcription factors upregulate HGF promoter activity by binding to the Sp1 binding site at position -318 to -303 bp in the HGF gene promoter.

The five amino acid-deleted isoform of hepatocyte growth factor promotes carcinogenesis in transgenic mice.

Hepatocyte growth factor (HGF) is a polypeptide with mitogenic, motogenic, and morphogenic effects on different cell types including hepatocytes. HGF is expressed as two biologically active isotypes resulting from alternative RNA splicing. The roles of each HGF isoform in development, liver regeneration and tumorigenesis have not yet been well characterized. We report the generation and analysis of transgenic mice overexpressing the five amino acid-deleted variant of HGF (dHGF) in the liver by virtue of an albumin expression vector. These ALB-dHGF transgenic mice develop normally, have an enhanced rate of liver regeneration after partial hepatectomy, and exhibit a threefold higher incidence of hepatocellular carcinoma (HCC) beyond 17 months of age. Moreover, overexpression of dHGF dramatically accelerates diethyl-nitrosamine induced HCC tumorigenesis. These tumors arise faster, are significantly larger, more numerous and more invasive than those appearing in non-transgenic littermates. Approximately 90% of female dHGF-transgenic mice had multiple macroscopic HCCs 40 weeks after injection of DEN; whereas the non-transgenic counterparts had only microscopic nodules. Liver tumors and cultured tumor cell lines from dHGF transgenics showed high levels of HGF and c-Met mRNA and protein. Together, these results reveal that in vivo dHGF plays an active role in liver regeneration and HCC tumorigenesis.

Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-MET.

The human proto-oncogene c-MET encodes a heterodimeric 190 kDa transmembrane protein (p190c-met) with structural features of a tyrosine kinase receptor. The ligand for this putative receptor has not yet been identified. By Northern blot hybridization we found that, among a restricted number of human tissues, c-MET is highly expressed in the liver. This prompted us to test the hypothesis of a functional interaction between the c-MET receptor and Hepatocyte Growth Factor (HGF), a heparin-binding polypeptide consisting of heavy and light chains of 65 and 35 kDa. Nanomolar concentrations of highly purified HGF added to GTL-16 cells, which overexpress the c-MET receptor, enhanced the phosphorylation on tyrosine of the p190c-met kinase. Addition of other known growth factors or serum was ineffective. The kinase activity of the c-MET receptor was also stimulated by HGF in an in vitro assay, after detergent solubilization and partial purification of p190c-met. Moreover, elution of immunoprecipitates obtained with anti-MET antibodies from GTL-16 cell lysates yielded an HGF-responsive kinase activity. These results suggest that HGF, or a growth factor structurally related to HGF, is a candidate ligand for the receptor encoded by c-MET.

Modulation of c-MET proto-oncogene (HGF receptor) mRNA abundance by cytokines and hormones: evidence for rapid decay of the 8 kb c-MET transcript.

The c-MET proto-oncogene product is a transmembrane tyrosine kinase receptor which was recently shown to transmit an array of important cellular responses induced by Hepatocyte Growth Factor (HGF). These biological effects include induction of mitogenesis, motogenesis, morphogenesis, metastogenesis and anti-tumor activity on a variety of epithelial cells. All of these processes are known to be associated with normal and abnormal tissue growth and development. The 190 kDa c-MET protein is encoded by a major transcript of 8 kilobases (kb), which is reported to be expressed predominantly in epithelial tissues. The expression pattern of c-MET mRNA and protein are drastically modified in many tumor tissues and cell lines. Currently, no information is available on the molecular mechanisms that regulate c-MET mRNA level. In the present communication, we report for the first time that the inflammatory cytokines such as IL-1 alpha, IL-6 and TNF-alpha, as well as TGF-beta 1, EGF, HGF and the steroidal hormones (estrogen, progesterone, tamoxifen and dexamethasone) markedly influence the steady-state levels of the 8 kb c-MET mRNA in human carcinoma cell lines derived from human tissues such as ovary, breast and endometrium. We demonstrate that c-MET receptor protein is present at high levels in primary tumors of human ovaries (clear cell carcinomas). We present evidence that the 8 kb c-MET mRNA undergoes rapid degradation with a half-life of less than 30 min and that this decay can be quickly inhibited by cycloheximide. Our results suggest that the expression of the c-met proto-oncogene resembles that of an immediate early response gene.

Structural and functional characterization of the mouse c-met proto-oncogene (hepatocyte growth factor receptor) promoter.

The c-met gene encoding Hepatocyte Growth Factor Receptor is predominantly expressed in epithelial cell types and overexpressed in a variety of human and mouse neoplastic tissues and cell lines. To understand the molecular mechanisms of the transcriptional regulation of this gene, we have cloned and functionally characterized the mouse c-met promoter region. Transient transfection analysis using a series of 5'-end deletion met-CAT chimeric constructs in epithelial (C-33A) and fibroblast (NIH3T3) cell lines demonstrated that the c-met promoter acts in a cell-type specific manner. These experiments also localized functionally important regulatory regions at -1390 to -279, relative to the transcription start site, which exert repressive activity, and at -278 to -77 which exhibit enhancing effects on c-met promoter activity. Further analysis by electrophoretic mobility shift assays using specific competitors and antibodies identified Sp1 protein binding to two cognate response elements at -221 and -124 within the enhancer region. Cotransfection experiments revealed that Sp1 stimulated promoter activity of the met-CAT constructs containing the two Sp1 binding sites. These results demonstrate that Sp1 is actively involved in the transcriptional regulation of the c-met promoter.

Molecular cloning and characterization of cDNA encoding mouse hepatocyte growth factor.

A cDNA encoding mouse hepatocyte growth factor (HGF) has been cloned and completely sequenced by use of reverse transcriptase-polymerase chain reaction (RT-PCR) and subsequent cloning. Sequence analysis reveals that mouse HGF, similar to its human and rat counterparts, consists of 728 amino acids, and both the alpha- and beta-chains are encoded in a single open reading frame. Strong homology exists in the primary structure of HGF among the three species of mouse, rat and human (more than 90%), especially in Kringle 1 of the alpha chain which is assumed to be an essential domain for binding of HGF to its receptor, c-MET, a proto-oncogene product. Our results suggest the existence of evolutionary pressure to conserve the distinct structure, and presumably the biological functions, of HGF.

Risk Factors for Postoperative Morbidity After Totally Robotic Gastric Bypass in 302 Consecutive Patients.

BACKGROUND: Totally robotic gastric bypass (robotic Roux-en-Y gastric bypass, R-RYGBP) has been adopted in some centers on the basis of large retrospective studies. In view of some data showing higher morbidity and higher costs, some authors have considered that robotic gastric bypass may no longer be justified with the existing system. Although low postoperative complication rates after R-RYGBP have been reported, risk factors for postoperative morbidity have never been evaluated. The goal of this study was to identify risk factors for postoperative morbidity after R-RYGBP. METHODS: A retrospective analysis of a prospectively maintained database was performed and included 302 consecutive patients after R-RYGBP performed between 2007 and 2013. This subset of patients represented 34 % of all gastric bypass procedures performed during this study period. Univariate and multivariate analyses were performed in order to identify risk factors for postoperative overall morbidity (Clavien scores 1-4 versus 0) and major morbidity (Clavien score ≥3 versus 0-1-2). RESULTS: Postoperative morbidity and mortality rates were 24.4 and 0.6 %, respectively. In multivariate analysis, independent risk factors for overall morbidity were American Society of Anesthesiologists (ASA) score ≥3 (odds ratio (OR) 2.0) and previous bariatric surgery (revisional gastric bypass) (OR 2.0). Independent risk factors for major morbidity (Clavien ≥3) were previous bariatric surgery (revisional gastric bypass) (OR 3.7), low preoperative hematocrit level (OR 0.9), and revisional gastric bypass procedure with concomitant gastric banding removal (OR 5.7). CONCLUSIONS: R-RYGBP is prone to increased complications in the setting of a high preoperative ASA score and revisional surgery. This should be taken into consideration by clinicians when evaluating R-RYGBP.

The mouse hepatocyte growth factor-encoding gene: structural organization and evolutionary conservation.

A mouse genomic phage library was screened by using a cDNA probe coding for mouse hepatocyte growth factor (HGF). Five overlapping genomic clones which contained the entire mouse HGF gene were isolated and characterized by restriction mapping, Southern hybridization and DNA sequencing. HGF spans about 65 kb and consists of 18 exons separated by 17 introns, similar to its human counterpart. The nucleotide (nt) sequences of the introns at the exon-intron junctions are GT-AG, analogous to those found in other eukaryotic genes. The exon-intron gene organization of HGF is highly homologous to that of several other genes encoding kringle-containing proteins, especially HGF-like protein and plasminogen. This result suggests that HGF probably evolved through gene duplication and/or exon shuffling events from an ancestral gene. Southern hybridization of genomic DNA from different species revealed that a high degree of homology exists among a variety of vertebrates, including chicken, when a mouse HGF cDNA was used as a probe. This evolutionary conservation of HGF strongly suggests that the protein may play an important role in normal cell physiology. Our current results on mouse HGF structure provide basic and detailed information to carry out further manipulation, such as gene targeting.

Long-term treatment with hepatic tumor promoters inhibits mitogenic responses of hepatocytes to acidic fibroblast growth factor and hepatocyte growth factor.

Studies with hepatocyte cultures have defined four hepatocyte mitogens which can transmit a complete mitogenic signal in cultures kept in completely defined conditions. These four mitogens are epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), hepatopoietin A/hepatocyte growth factor (HPTA/HGF) and hepatopoietin B (HPTB). In this study, we investigated the effect of aFGF, HGF and the mito-inhibitor transforming growth factor beta (TGF-beta) on cultured hepatocytes isolated from livers of rats treated with the xenobiotic hepatic tumor promoters phenobarbital (PB) and alpha-hexachlorocyclohexane (alpha-HCH). Male F344 rats were treated with each of these two xenobiotics to stimulate hepatic DNA synthesis and augmentative hepatomegaly. At different times on the regimens with tumor promoters, hepatocytes were isolated and placed in primary culture. DNA synthesis of hepatocytes in culture stimulated by these two growth factors and the suppression of DNA synthesis affected by TGF-beta were examined as a function of time of treatment in vivo with these two promoters. Following day 10, hepatocytes from both promoter regimens became unresponsive to these two growth factors for the rest of the duration of the treatment (day 90). TGF-beta suppressed DNA synthesis stimulated by growth factors but did not affect the high background DNA synthesis stimulated by xenobiotics themselves.