cDNAs encoding the large subunit of human replication factor C.

Replication factor C (RFC) is a multisubunit, DNA polymerase accessory protein required for the coordinated synthesis of both DNA strands during simian virus 40 DNA replication in vitro. Previous studies have shown that RFC is a DNA-dependent ATPase that binds in a structure-specific manner to the 3' end of a primer hybridized to a template DNA, an activity thought intrinsic to the 140-kDa component of this multisubunit complex. Here, the isolation and analysis of cDNAs encoding this subunit is described. Analysis of the full-length coding sequence revealed an open reading frame of 3.4 kb, encoding an 1148-amino acid protein with a predicted molecular mass of 130 kDa. A putative ATP-binding motif was observed that is similar to a motif in several of the smaller subunits of RFC and in functionally homologous replication factors of bacterial and viral origin. A "DEAD" box is also conserved among these proteins. The predicted protein shows significant identity with a DNA-binding protein of murine origin (B. Luckow, P. Lichter, and G. Schütz, personal communication). Regions of similarity were also seen between the amino acid sequences of the 140-kDa subunit of RFC, poly(ADP-ribose) polymerase, and bacterial DNA ligases--possibly representing a conserved structural feature of these proteins that bind similar DNA substrates.     

Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene.

Mutations in the APC (adenomatous polyposis coli) gene appear to be responsible for not only familial adenomatous polyposis but also many sporadic cases of gastrointestinal cancers. Using homologous recombination in mouse embryonic stem cells, we constructed mice that contained a mutant gene encoding a product truncated at a 716 (Apc delta 716). Mendelian transmission of the gene caused most homozygous mice to die in utero before day 8 of gestation. The heterozygotes developed multiple polyps throughout the intestinal tract, mostly in the small intestine. The earliest polyps arose multifocally during the third week after birth, and new polyps continued to appear thereafter. Surprisingly, every nascent polyp consisted of a microadenoma covered with a layer of the normal villous epithelium. These microadenomas originated from single crypts by forming abnormal outpockets into the inner (lacteal) side of the neighboring villi. We carefully dissected such microadenomas from nascent polyps by peeling off the normal epithelium and determined their genotype by PCR: all microadenomas had already lost the wild-type Apc allele, whereas the mutant allele remained unchanged. These results indicate that loss of heterozygosity followed by formation of intravillous microadenomas is responsible for polyposis in Apc delta 716 intestinal mucosa. It is therefore unlikely that the truncated product interacts directly with the wild-type protein and causes the microadenomas by a dominant negative mechanism.     

Isolation of endopeptidase-24.11 (EC 3.4.24.11, "enkephalinase") from the pig stomach. Hydrolysis of substance P, gastrin-releasing peptide 10, [Leu5] enkephalin, and [Met5] enkephalin

The purpose of this investigation was to isolate the cell-surface enzyme endopeptidase-24.11 from the stomach wall of the pig and to examine the hydrolysis of the gastric neuropeptides. Endopeptidase-24.11 was isolated from gastric membranes by immunoadsorbent chromatography using a monoclonal antibody to porcine kidney endopeptidase-24.11. The enzyme was purified with a yield of 1.2 micrograms/g wet wt of fundic muscle. A single polypeptide chain of apparent subunit molecular weight of 90,000 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gastric endopeptidase-24.11 hydrolyzed substance P, gastrin-releasing peptide 10, [Leu5] enkephalin, and [Met5] enkephalin by cleavage of peptide bonds on the N-terminal side of hydrophobic amino acids. The enzymatic activity was inhibited completely by phosphoramidon (10(-6) M) and strongly by 1,10-phenanthroline (10(-3) M), but was unaffected by captopril (10(-5) M).     

Effects of angiotensin converting enzyme inhibitor and calcium channel blocker on normoxic and hypoxic pulmonary vascular tone in unanesthetized sheep

We evaluated the effects of captopril and nifedipine on normoxic and hypoxic pulmonary vascular tone in unanesthetized sheep. Infusion of captopril (10 micrograms/kg/min) in normoxia revealed a tendency to increase the mean pulmonary arterial pressure (Ppa) and the pulmonary vascular resistance (PVR) following the systemic vasodilation. A statistically significant increase was reached by 20 minutes. Hypoxia of 10% oxygen in nitrogen produced a prominent pulmonary hypertensive response. Captopril significantly decreased the hypoxic values of Ppa and PVR from 20.3 +/- 1.3 to 17.1 +/- 1.1 mmHg (p less than 0.01) and from 4.31 +/- 0.45 to 3.49 +/- 0.45 mmHg/L/min (p less than 0.01), respectively. Infusion of nifedipine (10 micrograms/kg/min) in normoxia caused an increase in Ppa from 15.5 +/- 0.9 to 18.9 +/- 1.0 mmHg (p less than 0.01), but not in PVR. This elevation in Ppa was considered to be derived from the significant increase in the cardiac output. Nifedipine significantly decreased the hypoxic values of Ppa and PVR from 21.3 +/- 1.5 to 19.3 +/- 1.5 mmHg (p less than 0.05) and from 3.88 +/- 0.30 to 27.3 +/- 0.13 mmHg/L/min (p less than 0.01), respectively. Captopril and nifedipine produced systemic hypotensive responses during both normoxic and hypoxic ventilation. It is concluded that both captopril and nifedipine are potent pulmonary vasodilating drugs in animal subjects with a hypoxic condition and that they might be useful in the clinical vasodilator therapy of hypoxic pulmonary hypertension in man.     

A targeting model of boron neutron-capture therapy to hepatoma cellsin vivo with a boronated anti-(α-fetoprotein) monoclonal antibody

We described previously that¹⁰B atoms delivered by monoclonal antibody (mAb) exerted a cytotoxic effect on AH66 cells in a dose-dependent manner upon thermal neutron irradiationin vitro. In the present study, the delivering capacity of boronated anti-(-fetoprotein) (AFP) mAb to carry¹⁰B atoms to AFP-producing tumor xenografts in nude mice was determined. Boronated mAb was prepared by conjugating 50 mM ¹⁰B compound to an anti-AFP mAb (2 mg/ml) usingN-succinimidyl-3-) (2-pyridyldithio) propionate. The number of¹⁰B atoms conjugated directly to the mAb was estimated to be 459/antibody by prompt -ray spectrometry. Boron concentrations in tumor tissue obtained 12, 24, 72, and 120 h after injection of 3.0 mg¹⁰B-conjugated anti-AFP mAb were 11.10±3.12 (SD,n=6), 29.30±5.11, 33.02±11.8, and 12.91±5.62 ppm respectively. For control¹⁰B-conjugated anti-dinitrophenol (DNP) mAb, the values were 9.59±0.99, 10.37±2.86, 10.00±2.95, and 8.83±4.71 ppm respectively. The concentrations in blood were less than 0.40±0.10 ppm with anti-AFP mAb and less than 0.51±0.15 ppm with anti-DNP mAb at each sampling time (12, 24, 72, and 120 h). The number of¹⁰B atoms delivered to the tumor cells was calculated to be 0.62×10⁹, 1.63×10⁹, 1.84×10⁹ and 0.72×10⁹ at each sampling time after injection of¹⁰B-anti-AFP mAb. The amount of¹⁰B atoms necessary for effective boron neutron-capture therapy was estimated to be 10⁹/tumor cell. We were able to carry 1.84×10^{9 10}B atoms to AH66 tumor cells by using¹⁰B-anti-AFP mAb. The accumulation reached its peak 72 h after injection. These data indicated that the¹⁰B-conjugated antitumor mAb could deliver a sufficient amount of¹⁰B atoms to the tumor cells to induce cytotoxic effects 72 h after injection upon thermal neutron irradiation.Abbreviations BNCT boron neutron-capture therapy - AFP -fetoprotein - SPDP N-succinimidyl-3-(2-pyridyldithia) propionate - DNP dinitrophenol     

Studies on insulin-like growth factors (IGF-I, -II) and there binding proteins in normal human pregnancy

In order to elucidate the roles of Insulin-like Growth Factors (IGF-I, -II) in human pregnancy, the levels of IGF-I and IGF-II, the distribution of binding proteins for IGF-I or IGF-II and profiles of unsaturated somatomedin binding proteins (USBP) were estimated in maternal and cord plasma. IGF-I levels in maternal plasma gradually elevated in late gestation, reaching 304.4 +/- 130.1ng/ml at term, and were significantly higher than those in nonpregnant women (188 +/- 58). IGF-II levels, which were 414.9 +/- 75.4ng/ml in women in the third trimester of gestation, did not produce any remarkable changes in the levels in nonpregnant women (395.9 +/- 72.6). On the other hand, IGF-I in cord plasma also increased according to gestational age, reaching 37.3 +/- 14.6ng/ml at term, but it was significantly lower than that in maternal weight (r = 0.50, p less than 0.005) and relative birth weight (r = 0.41, p less than 0.005). IGF-II in cord plasma showed no significant changes during gestation, however, IGF-II levels in AFD (appropriate for date) newborns delivered at term (203.8 +/- 59.4) were significantly lower than those in maternal plasma. And they had no positive correlations with birth weight and relative birth weight. On Sephadex G150 gel-chromatography of cord plasma from AFD newborns at term, more than 70% of immunoreactive IGF-I in plasma was eluted at 150K region, and 150K USBP could be detected as observed in adult plasma. On the other hand, most of the immunoreactive IGF-II was eluted at 40K region, and 150K USBP was not detected in AFD newborns at term. In adult plasma, most of the immunoreactive IGF-II was eluted at 150K region, but 150K USBP could not be detected. From these results, it is supposed that IGF-I plays an essential role in fetal growth rather than IGF-II.     

Reproducibility of head-up tilt-table testing for eliciting susceptibility to neurally mediated syncope in patients without structural heart disease.

Head-up tilt testing has gained acceptance as a tool for assessing susceptibility to neurally mediated syncopal syndromes (e.g., vasovagal syncope), and is currently being evaluated as a means of testing therapeutic interventions in these conditions. To assess reproducibility of head-up tilt testing and thereby assess the potential of such testing for immediate evaluation of a planned treatment, findings during 2 sequential 80 degrees head-up tilt tests were compared in 23 patients (age range 6.5 to 74 years) undergoing evaluation of syncope of unknown origin. Upright tilt was performed initially in the absence of drugs, and repeated if necessary during pharmacologic provocation by means of isoproterenol infusions of 1 and 3 micrograms/min (tilt 1). End points were syncope, maximal tolerated isoproterenol dose, or a tilt duration of 10 minutes. The second tilt test (tilt 2) was conducted after approximately 30 minutes of supine rest using the maximal provocative conditions used in tilt 1. Fifteen of 23 patients (65%) developed syncope in either tilt 1 or 2, while 8 of 23 (35%) remained asymptomatic. Tilt testing results were concordant (i.e., positive in both tests, or negative in both tests) in 20 of 23 (87%) patients. Concordance was, however, less among tilt-positive patients (12 of 15, 80%) since 3 patients were tilt-positive in tilt 1 only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Src family kinases and nitric oxide production are required for hepatocyte growth factor-stimulated endothelial cell growth

Hepatocyte growth factor (HGF) is a potent mitogen for vascular endothelial cells (EC); however, signal transduction pathways for HGF-stimulated EC growth remain unclear. In the present study we investigated the role of Src family kinases and nitric oxide (NO) in HGF-stimulated EC growth. Human umbilical vein endothelial cells (HUVEC) were stimulated with HGF and NO was measured by an NOx analyzing HPLC system. Activation of ERK1/2 and p38 MAPK was assessed by Western blot. NO production in HUVEC increased 1.8-fold by HGF. A Src family kinases inhibitor PP1 inhibited HGF-stimulated NO production by 71%. HUVEC growth increased 1.9-fold in cell number by HGF. PP1 and Nitro-L-arginine methylester (L-NAME) inhibited HGF-stimulated HUVEC growth by 51 and by 71%. ERK1/2 and p38 MAPK were phosphorylated by HGF and a MEK inhibitor PD98059 and a p38 MAPK inhibitor SB203580 inhibited HGF-stimulated HUVEC growth by 66% and by 58%; however, HGF-induced phosphorylation of ERK1/2 and p38 MAPK was not inhibited by L-NAME, indicating that NO is not an upstream activator of ERK1/2 and p38 MAPK. These findings demonstrated that Src family kinases regulate HGF-stimulated NO production in HUVEC and that HGF stimulates HUVEC growth through NO-dependent and NO-independent pathways.