Human Keratinocyte growth factor (hKGF), a member of the FGF family of growth factors, contains five cysteines at amino acid positions 1, 15, 40, 102, and 106. We expressed five cysteine mutants of hKGF in which the cysteines were cumulatively replaced with alanine or serine, starting with cysteine-1. Recombinant hKGF has an inherently higher mitogenic activity and stability to heat and acid than reported for glycosylated hKGF. Mitogenic activity is increased an additional 2.6 fold by substitution of cysteine-1 with alanine. Mutants with the conserved cysteine substituted at position 40 were more susceptible to heat inactivation than rhKGF, but showed no significant difference in acid inactivation. Cysteine-free rhKGF is mitogenic, demonstrating that neither cysteines nor disulfide bonds are required for mitogenic activity. However, cysteine-free rhKGF does not bind Heparin-Sepharose and is unstable to heat and acid compared to rhKGF, suggesting that the cysteines have a role in maintaining KGF's structure. This information will useful in the development of a more stable and more potent wound healing agent from hKGF.

Syrian hamster DDT‐1 cells are derived from smooth muscle of the ductus deferens. DDT‐1 cell growth is increased by the addition of testosterone (T). Acidic fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF) also known as heparin binding growth factor I and II (HBGF‐I and HBGF‐II) can replace T in the stimulation of growth in these cells. This phenomenon is correlated with testosterone's ability to elevate aFGF/HBGF‐I mRNA. The increase steady‐state levels of aFGF/HBGF‐I mRNA were documented by northern blots and by in situ hybridization. Using a 520 bp human aFGF/HBGF‐I cDNA probe, a genomic clone with a 38 kb DNA insert was isolated from a cosmid library. By restriction enzyme analysis and southern hybridization, it was determined that there are three coding exons. DNA sequence analysis showed all of the coding region and 3′ noncoding sequences were on this clone. A 5′ noncoding exon not in the 38 kb insert is indicated, based on the cDNA sequences and genomic sequences of aFGF/HBGF‐I's from hamster DDT‐1 cells and several other species. The cDNA for hamster aFGF/HBGF‐I was isolated from a DDT‐1 lambda gt11 library and sequenced. Comparison of the coding region of aFGF/HBGF‐I from four species shows a >90% conservation of amino acid sequence.

Heparin-binding growth factor type 1 (HBGF-1; sometimes termed acidic fibroblast growth factor) is potentially an important factor in liver regeneration. HBGF-1 alone (half-maximal effect at 60 pM) stimulated hepatocyte DNA synthesis and bound to a high-affinity receptor (Kd = 62 pM; 5000 per cell). Epidermal growth factor (EGF) neutralized or masked the mitogenic effect of HBGF-1 concurrent with appearance of low-affinity HBGF-1 binding sites. HBGF-1 reduced the inhibitory effect of transforming growth factor type beta (TGF-beta) on the EGF stimulus. Nanomolar levels of HBGF-1 decreased the EGF stimulus. An increase in hepatic HBGF-1 gene expression after partial hepatectomy precedes increases in expression of the EGF homolog, TGF-alpha, and nonparenchymal-cell-derived TGF-beta in the regenerating liver. Expression of HBGF-1 mRNA occurs in both hepatocytes and nonparenchymal cells and persists for 7 days in liver tissue after partial hepatectomy. HBGF-1 acting through a high-affinity receptor is a candidate for the early autocrine stimulus that drives hepatocyte DNA synthesis prior to or concurrent with the EGF/TGF-alpha stimulus. It may allow hepatocyte proliferation to proceed in the presence of low levels of TGF-beta. An EGF/TGF-alpha-dependent change in HBGF-1 receptor phenotype and increasing levels of nonparenchymal-cell-derived HBGF-1 and TGF-beta may serve to limit hepatocyte proliferation.

The ductus deferens smooth muscle tumor cell line (DDT1-MF-2) is very sensitive to steroids. Treatment with 10 nM testosterone accelerates the growth of DDT1 cells in the absence of serum. Glucocorticoids in the presence or absence of androgens inhibits growth. Stimulation of growth of DDT1 cells by testosterone can be replaced by the addition of heparin-binding growth factor I and II (HBGF). Addition of testosterone plus HBGF growth factors results in a further increase in cell number. By in situ hybridization, accumulation of HBGF-I mRNA is significantly increased by testosterone treatment of low density cultures. Testosterone treatment of high density cultures results in no stimulation of HBGF-I mRNA accumulation. Glucocorticoids alone, which block growth of DDT1 cells, have no effect of HBGF-I mRNA accumulation. However, the simultaneous addition of glucocorticoid and androgens to DDT1 cells results in a rapid accumulation of HBGF-I mRNA by 12 h, although growth is inhibited by the presence of both steroid analogs.

Heparin-binding polypeptide growth factors (HBGF) are essential mitogens for isolated prostate cells. HBGF type one (HBGF-1) mRNA was expressed specifically in the epithelial cells of prostates from normal 6- to 8-week-old rats. Expression declined significantly at 14 weeks and was undetectable in 35-week-old animals. Slow-growing, androgen-responsive, nonmetastatic Dunning R3327PAP tumors, which are composed of a well-defined epithelium and stroma, expressed HBGF-1 mRNA constitutively in specifically the mesenchymal cells. A rapid-growing, androgen-independent, metastatic variant (Dunning R3327AT-3), which was composed of a single clonogenic cell type, expressed both HBGF-1 and HBGF type two (HBGF-2) mRNA. HBGF activity in the extracts of normal and tumor tissues correlated with mRNA levels. Epithelial cells from the R3327PAP tumor and the single cell type that composed the R3327AT-3 tumor exhibited alterations in HBGF receptor characteristics that correlated with increased sensitivity to mitogenic effects of HBGF. The results suggest that alterations in HBGF gene expression in both prostate epithelial and mesenchymal cells and in properties of the receptor in specifically epithelial cells may contribute to differential growth rates and malignancy of different prostatic tumors.

The differentiated human hepatoblastoma-derived cell line, HepG2, displayed two classes of specific membrane receptors for heparin-binding growth factor type 1 (HBGF-1). Specific membrane receptors were distinguished from nonreceptor heparin-like binding sites. Receptors with an apparent Kd of 9.2 +/- 0.9 pM and present at 15,000 +/- 900/cell correlated with HBGF-1 stimulation of HepG2 growth. Receptors with an apparent Kd of 2 +/- 0.4 nM and present at 180,000 +/- 18,000/cell correlated with inhibition of growth and changes in secretory products. Other hepatoma cell lines exhibited a simple positive mitogenic response to HBGF-1 and a single class of high affinity binding sites. HBGF-1 covalently cross-linked to hepatoma cell surface polypeptides of apparent mean molecular mass of 130 kilodaltons. At 37 degrees C, receptor-bound HBGF-1 was internalized (t 1/2 = 45 min) but not degraded for up to 6 h. The display of receptors decreased with increased cell density and expression of HBGF-1 mRNA and HBGF-1-like activity in the culture medium. Proliferating normal human hepatocytes also exhibited two classes of binding sites with affinities for HBGF-1 and apparent molecular weight similar to HepG2 cells. These results implicate HBGF-1 or homologues in human hepatoma cell growth and normal liver cell regeneration.

The 11S poly(A +)RNA from rat seminal vesicle was cloned. By hybrid selection of clones reacting to the low molecular weight region of the US peak, a clone containing a new seminal vesicle cDNA sequence called SVS VI was isolated. The DNA sequence of two overlapping cDNAs (pSV24 and pSV33) is presented. The sequence of SVS VI was compared to the previously isolated SVS IV and SVS V cDNAS. Dot hybridization showed that SVS VI is androgen responsive after giving testosterone to castrated rats. The hydrophilicity was analyzed using standard Bionet procedures. All three proteins are extremely variable, rich in α-helix and very water soluble. The computer predicted hydrophilicity is compared for SVS VI, V and IV. A small region in the 3'-non-coding area of SVS VI has high similarity to a region in SVS IV mRNA.

The rat seminal vesicle secretion IV (SVS IV) gene was isolated from a lambda Charon 4A library. The SVS IV gene transcription unit was found to be on one 3.3-kilobase (kb) EcoRI fragment. Restriction mapping and DNA sequence analysis demonstrated that the entire length of the SVS IV transcription unit is 1,930 base pairs (bp) and contains two introns. The 3.3-kb EcoRI fragment contains 144 bp of 5'-flanking region. At -113 bp from the presumed transcription initiation site an interesting structure with perfect dyad symmetry is noted. In another lambda clone, a 3.5-kb EcoRI fragment was isolated that contains the SVS IV gene and was shown to be identical to the 3.3-kb EcoRI fragment except for 180 bp of DNA in the second intron. The extra DNA consists of several (8-10) 20-bp tandem repeats flanked on each side by seven or eight copies of this same 20-bp repeat. Fisher X Sprague-Dawley hybrid rats, which contain both the EcoRI 3.5-kb form and the 3.3-kb form of the SVS IV gene, were crossed with each other. Analysis of the F1 generation demonstrated that the presence or absence of the 180-bp intronic insertion in the SVS IV gene defines an allelic difference. This report also presents the DNA sequence of the transcription unit and flanking regions of the SVS IV gene.

The abundant class of poly(A+)RNA [poly(A+)RNA11S] from rat seminal vesicle was used to synthesize ds-cDNA11S. The ds-cDNA11S was inserted and cloned into the Pst I site of pBR-322 using E. coli RR1 as host. Colony filter hybridization and restriction mapping was used to demonstrate that a 620 NTP long insert in a plasmid clone (pSV2) represents the almost full length structural gene coding for a precursor to the seminal vesicle secretion protein IV (SVS IV). The entire insert was sequenced and the coding region was matched with the known amino acid sequence. Most of the signal peptide sequence was derived from the DNA sequence. The insert in pSV2 was labelled and used to study the effect of testosterone on the accumulation of mRNA SVS IV. Administration of testosterone to castrated rats resulted in the induction of mRNA SVS IV from a few molecules per cell to levels of over 100,000 after 96 h of hormone treatment.

Total poly (A)-containing messenger RNA [poly(A)-mRNAT] was isolated from seminal vesicles of adult male rats. By preparative 5%-20% linear sucrose gradient centrifugation in 1% SDS, a single peak was seen in the 11S region [poly(A)-mRNA11S]. Poly (A)-mRNAT and poly(A)-mRNA11S were translated in the wheat germ S-30 in vitro translation system. Laemmli-SDS-polyacrylamide slab gel electrophoresis of translation products of poly(A)-mRNAT revealed three major protein bands at approximately 18,500, 15,000, and 14,000 daltons. In vitro translation of poly(A)-mRNA11S across the 11S peak revealed partial purification of the mRNAs for the three predominant proteins. The lower molecular weight part of the 11S peak enriched for the mRNAs coding for the 15,000 and 14,000 dalton polypeptides, and the higher molecular weight side of the peak enriched for the 18,500 dalton mRNA. The highly abundant class of mRNA from rat seminal vesicle can be purified with one sucrose gradient centrifugation.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTComplexity of poly(A)-containing heterogeneous nuclear ribonucleic acid from mouse embryoid bodies (OTT6050)Per Erik Mansson and Stephen E. HarrisCite this: Biochemistry 1979, 18, 10, 2073–2078Publication Date (Print):May 1, 1979Publication History Published online1 May 2002Published inissue 1 May 1979https://doi.org/10.1021/bi00577a036RIGHTS & PERMISSIONSArticle Views16Altmetric-Citations8LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (708 KB) Get e-Alerts Get e-Alerts