A novel putative tyrosine kinase receptor encoded by the eph gene.

Growth factors and their receptors are involved in the regulation of cell proliferation and also play a key role in oncogenesis. In this study, a novel putative kinase receptor gene, termed eph, has been identified and characterized by molecular cloning. Its primary structure is similar to that of tyrosine kinase receptors thus far cloned and includes a cysteine-rich region in the extracellular domain. However, other features of the sequence distinguish the eph gene product from known receptors with tyrosine kinase activity. Thus the eph protein may define a new class of these molecules. The eph gene is overexpressed in several human carcinomas, suggesting that this gene may be involved in the neoplastic process of some tumors.

Evidence that proteases are involved in superoxide production by human polymorphonuclear leukocytes and monocytes.

The possible participation of proteases in superoxide (O2-) production by human polymorphonuclear leukocytes (PMN) and monocytes was explores using various protease inhibitors and substrates. Protease inhibitors of serine proteases and synthetic inhibitors that modify the active site of serine proteases. Substrates used were synthetic substrates of the chymotrypsin type as well as trypsin type of protease. All these inhibitors and substrates inhibited O2- oroduction by human PMN and monocytes induced by cytochalasin E and concanavalin A, though PMN were more sensitive to these inhibitors and substrates than monocytes. Inhibition appeared rapidly even when the inhibitors were added at the same time as the stimulants, during the "induction time of O2-production" or at the time of maximum O2- production, whereas much greater inhibition was observed when the cells were preincubated with the inhibitors. These observations suggest that enzymatically active serine proteases are essential for these phagocytic cells to initiate and maintain the O2- production in response to the stimuli. The inhibitory effect of the inhibitor and substrate for chymotrypsin type protease was greater than that of those substances for trypsin-type protease. Macromolecular inhibitors also inhibited the O2- production. These findings suggest that the serine proteases involved in the O2- production by human PMN and monocytes are similar to chymotrypsin rather than trypsin, and are possibly located at the cell surface membrane.

Atrial natriuretic factor in human blood.

To determine whether atrial natriuretic factor (ANF) is a circulating hormone in men, a radioimmunoassay suitable for the estimation of ANF in human plasma was developed and the nature of plasma ANF was characterized. Plasma ANF was extracted before radioimmunoassay by affinity chromatography on a column of ANF antibody-coupled agarose. When plasma ANF extract was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with the radioimmunoassay of the eluted gel slices for ANF, almost all of the ANF activities ran in the 3,000-mol-wt area, while three peaks of ANF were observed in human atrial tissue extract, molecular weights of which corresponded to 14,000, 6,000, and 3,000, respectively. Reversed-phase high performance liquid chromatography of atrial tissue extract resolved multiple forms of ANF. In contrast, one major peak was observed in human plasma extract, and its retention time coincided with that of synthetic human alpha-atrial natriuretic polypeptide. When 500 ml of 0.9% saline was infused into six healthy subjects over 45 min, plasma levels of ANF were unequivocally elevated. The mean plasma ANF concentrations rose from the baseline (23.0 +/- 2.5 pg/ml, mean +/- SEM, n = 6) to the peak (41.8 +/- 4.9 pg/ml, mean +/- SEM) at 75 min postinfusion. No significant change in plasma ANF, on the other hand, was found in the control group. These results suggest that ANF is a circulating hormone in men and is secreted in response to isotonic volume expansion.

Measurement of delta-aminolevulinic acid synthetase activity in human erythroblasts.

A new, specific, and simple method for the determination of delta-aminolevulinic acid (ALA) synthetase activity in human bone marrow cells has been developed. ALA synthetase of erythroblasts was partially purified so as to permit the use of [(14)C]succinyl-CoA as a substrate for this enzyme. In this enzyme preparation there were negligible activities of succinyl-CoA hydrolase, alpha-ketoglutarate dehydrogenase, and succinyl-CoA synthetase and there was no activity of ALA dehydrase. The ALA formed from [(14)C]succinyl-CoA has been isolated by column chromatography. Radioactivity in the eluate from the column has been proved by paper chromatography to be exclusively that of [(14)C]ALA. The entire assay can be completed within 4 h, and [(14)C]succinyl-CoA was incorporated into [(14)C]ALA on the order of several percent. Moderate to marked decreases of ALA synthetase activity have been demonstrated in the erythroblasts of all cases of sideroblastic anemia. In the cases of iron deficiency anemia, on the other hand, normal or slightly elevated activity has been obtained.

Studies of the function of natural killer-interferon system in patients with Sjögren syndrome.

The natural killer (NK)-interferon (IFN) system is shown to be significantly involved in the resistance of host to viral infections and to tumours in numbers of animal models (1-4). The patients with Sjögren syndrome (SS) as well as those with collagen diseases were systematically investigated for the functions of NK-IFN system, including endogenous and augmented NK activity, IFN production, and responsiveness of NK cells to IFN stimulation, using virus persistently infected cells (heLa-measles cells) as target and stimulator cells. Although endogenous NK activity was not reduced, augmented NK activity by HeLa-measles cells in vitro was significantly depressed in patients with SS compared with that in age-matched normal controls. The patients with SS had also impaired capacity to produce IFN, which is shown to be a major factor regulating NK activity (5,6) in response to HeLa-measles cells in vitro. In three patients with SS who showed severely depressed NK activity, the effect of exogenous IFN was examined, and virtually no augmentation of NK activity was observed in all cases. Under the same condition, the normal controls demonstrated a dramatic increase in NK activity. The reduced IFN production was observed in all examined patients with SS, whereas impaired augmentation of NK activity by the stimulation with HeLa-measles cells as well as IFN seemed to be more striking in patients with the systemic manifestations of the disease, such as hypergammaglobulinemia and lymphoid hyperplasia. The possible involvement of dysfunction of NK-IFN system in the systemic manifestations of SS is discussed.

Molecular cloning and characterization of a Ca2+ + Mg2+-dependent adenosine triphosphatase from rat cardiac sarcoplasmic reticulum. Regulation of its expression by pressure overload and developmental stage.

To investigate the regulation of expression of cardiac Ca2+ + Mg2+-dependent ATPase (Ca2+-ATPase) in sarcoplasmic reticulum (SR), we isolated cDNA (pHA6) encoding a Ca2+-ATPase of rat cardiac SR. The clone consisted of 2,311 mRNA-derived nucleotides, which covered half the coding region and the entire 3'-untranslated regions. The nucleotides and deduced amino acid sequences of pHA6 showed striking homology, 89 and 98%, respectively, to those of rabbit Ca2+-ATPase of the slow-twitch form. Northern blot analyses revealed that the mRNA levels of Ca2+-ATPase were decreased by pressure overload and became 32% of sham in 1 mo. During the developmental stage the mRNA levels were very low in the fetal period and steeply increased around birth. These changes in mRNA levels were correlated with the corresponding protein levels. These results suggest that the expression of cardiac Ca2+-ATPase in SR is regulated by pressure overload and the developmental stage, at least in part, at the pretranslational level.

Distribution of cardiac myosin isozymes in human conduction system. Immunohistochemical study using monoclonal antibodies.

To determine the presence and distribution of cardiac myosin isozymes in the human conduction system, we performed an immunohistochemical study using monoclonal antibodies CMA19 and HMC14, which are specific for myosin heavy chains of human atrial type (alpha-type) and ventricular type (beta-type), respectively. Serial frozen sections of human hearts were obtained from autopsy samples and examined by indirect immunofluorescence. Alpha-type was found in all myofibers of sinus node and atrio-ventricular node, and in 55.2 +/- 10.2% (mean +/- SD, n = 5) of the myofibers of ventricular conduction tissue, which consists of the bundle of His, bundle branches, and the Purkinje network. In contrast, beta-type was found in all myofibers of the atrio-ventricular node and ventricular conduction tissue, whereas almost all myofibers of the sinus node were unlabeled by HMC14. Although the number of ventricular myofibers labeled by CMA19 was small, the labeled myofibers were more numerous in the subepicardial region than in the subendocardial region. These findings show that the gene coding for alpha-type is expressed predominantly in specialized myocardium compared with the adjacent ordinary working myocardium.

Molecular cloning and characterization of human cardiac alpha- and beta-form myosin heavy chain complementary DNA clones. Regulation of expression during development and pressure overload in human atrium.

We have constructed and characterized two types of myosin heavy chain (MHC) cDNA clones (pHMHC2, pHMHC5) from a fetal human heart cDNA library. Comparison of the nucleotide and deduced amino acid sequences between pHMHC2 and pHMHC5 shows 95.1 and 96.2% homology, respectively. The carboxyl-terminal peptide and 3'-untranslated (3'-UT) regions are highly divergent and specific for these cDNA clones. By using the synthetic oligonucleotide probes that are complementary to the unique 3'-UT regions of these cDNA clones, we demonstrate that pHMHC2 is exclusively transcribed in the atrium, whereas the mRNA for pHMHC5 is predominantly expressed in the ventricle. This result indicates that pHMHC2 and pHMHC5 code for alpha- and beta-form MHCs, respectively. Furthermore, we show that beta-form MHC mRNA is expressed in adult atrium at a low level but scarcely expressed in fetal atrium. Finally, we demonstrate that MHC isozymic transition in pressure-overloaded atrium is, at least in part, regulated at a pretranslational level.

Posttranslational cleavage of proinsulin is blocked by a point mutation in familial hyperproinsulinemia.

Familial hyperproinsulinemia is characterized by the accumulation of proinsulin-like material (PLM) in the plasma of affected patients. This disorder is inherited in an autosomal dominant fashion. The accumulation of PLM is thought to be due to the impaired conversion of proinsulin to insulin. Although PLM has been suggested to have an amino acid substitution, it has been impossible to locate and identify a substituted amino acid, due to the difficulty in isolating sufficient amounts of PLM from plasma samples. Therefore, we analyzed leukocyte DNA from one member of a proinsulinemic family, and we found a point mutation that changed guanine to adenine in the insulin gene. This transition implies that a substitution of histidine for arginine has occurred at amino acid position 65. Furthermore, it indicates that arginine at 65 is essential for the conversion of proinsulin to insulin. Our results suggest a novel mechanism by which disease can be incurred: a heritable disorder can result from a posttranslational processing abnormality caused by a point mutation.

Heterogeneity of beta-type myosin isozymes in the human heart and regulational mechanisms in their expression. Immunohistochemical study using monoclonal antibodies.

To investigate the existence of heterogeneity of beta-type myosin isozymes (HC beta) in human hearts, immunohistochemical studies using monoclonal antibodies (MoAbs) raised against human ventricular myosin heavy chains were performed. Two types of MoAbs recognized some muscle fibers in the atrium, whereas both reacted with all ventricular muscle fibers. Since atrial muscle fibers reactive with each MoAb were found to be clearly different, the existence of two immunologically distinct HC beta (beta 1, and beta 2) was suggested in the atrium. By using affinity chromatography, two molecular variants of HC beta were isolated from the bovine atrium, and differences in the primary structure of beta 1 and beta 2 were confirmed by analysis of peptides produced by chymotryptic digestion. In pressure-overloaded human atria, myofibers containing beta 1 and/or beta 2 increased in accordance with decrement of myofibers containing alpha-type myosin isozyme (P less than 0.01). But they differed in expression during the developmental stage, since beta 2 did not exist in the early embryonic bovine heart, but beta 1 did. Thus, there are two distinct HC beta whose expression is regulated by at least two factors: pressure overload and developmental stage.

Functional maturation of membrane potential changes and superoxide-producing capacity during differentiation of human granulocytes.

The alterations of stimulus-induced membrane potential changes, superoxide (O2-)-producing capacity and phagocytic activity during differentiation of human granulocytes were investigated in the human leukemia cell lines HL-60 and KG-1 differentiating in vitro and in human leukemic granulocytes obtained from chronic myelogenous leukemia patients. HL-60 cells incubated with dimethyl sulfoxide or with retinoic acid showed progressively increasing O2- production as well as membrane potential changes (depolarization) on contact with phorbol myristate acetate or the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, with a concomitant increase in the proportion of mature cells of the granulocytic type. Phagocytosis of latex particles, yeast, and oil droplets appeared 24 h after incubation with dimethyl sulfoxide and anteceded the increment of O2- production and membrane potential changes, both of which appeared concomitantly 3 d after incubation with dimethyl sulfoxide. Similar findings were observed when immature and mature granulocytes obtained from chronic myelogenous leukemia patients were stimulated by phorbol ester, the chemotactic peptide, or calcium ionophore A23187, and the amount of O2- production was parallel to the magnitude of membrane potential changes. HL-60 and KG-1 cells incubated for 1-6 d with phorbol myristate acetate showed neither O2- production nor membrane potential changes on contact with phorbol ester, chemotactic peptide, or A23187, although such cells resembled macrophages morphologically, and their phagocytic activity was significantly increased. O2- production and membrane potential changes in normal granulocytes induced by phorbol ester, chemotactic peptide and A23187 were inhibited by 2-deoxyglucose. These findings indicate that the O2--producing system and the system provoking membrane potential changes may develop concomitantly as human granulocytes mature and differentiate, and that the development of these systems and of phagocytic activity may be independently regulated.

Isozymic changes in myosin of human atrial myocardium induced by overload. Immunohistochemical study using monoclonal antibodies.

An immunohistochemical study using monoclonal antibodies specific for the heavy chains of either human atrial (HC alpha) or ventricular (HC beta) myosin was performed to clarify the distribution of each isozyme in normal as well as pressure-overloaded human hearts. In normal human ventricles, all muscle fibers were stained by a monoclonal antibody (HMC14) specific for HC beta, whereas a small number of fibers reacted with a monoclonal antibody (CMA19) specific for HC alpha. In contrast, in normal human atria, almost all muscle fibers were stained by CMA19, and a relatively larger number of muscle fibers also reacted with HMC14. Furthermore, in pressure-overloaded atria, muscle fibers reactive with HMC14 were strikingly increased while those reactive with CMA19 showed a corresponding decrease. The extent of this isozymic redistribution was in good correlation with atrial pressure. These results not only confirmed the existence of isoforms of myosin heavy chain in human hearts, but also demonstrated that redistribution of iso-myosins could occur as an adaptation to pressure overload.

Increased angiotensin-converting enzyme in peripheral blood monocytes from patients with sarcoidosis.

Angiotensin-converting enzyme (ACE) activity was measured in isolated peripheral blood monocytes and culture medium from 28 patients with sarcoidosis and compared with values obtained from monocytes of 25 normal control subjects. ACE activity was determined by radioimmunoassay of angiotensin II produced from angiotensin I. While there was no measurable ACE activity in monocytes or culture medium from normal controls under the conditions of our study, monocytes from patients with sarcoidosis all showed activity both in cells and culture medium. The mean ACE activity of monocytes from patients with sarcoidosis was 2.0 pg angiotensin II formed/min per 10(5) cells, and that released into medium over a 24-h interval was 30.4 pg angiotensin II/min per 10(5) cells. The monocyte ACE from patients with sarcoidosis was activated by chloride ions and inhibited by EDTA, captopril, and rabbit antiserum to purified human plasma ACE, indicating that enzymatic activity was effected specifically by ACE. Thus, our studies show a significant elevation and release of ACE by peripheral blood monocytes of patients with sarcoidosis under conditions where monocytes of normal control subjects do not demonstrate ACE activity.

Heterogeneous mutations in the human lipoprotein lipase gene in patients with familial lipoprotein lipase deficiency.

The DNA sequences were determined for the lipoprotein lipase (LPL) gene from five unrelated Japanese patients with familial LPL deficiency. The results demonstrated that all five patients are homozygotes for distinct point mutations dispersed throughout the LPL gene. Patient 1 has a G-to-A transition at the first nucleotide of intron 2, which abolishes normal splicing. Patient 2 has a nonsense mutation in exon 3 (Tyr61----Stop) and patient 3 in exon 8 (Trp382----Stop). The latter mutation emphasizes the importance of the carboxy-terminal portion of the enzyme in the expression of LPL activity. Missense mutations were identified in patient 4 (Asp204----Glu) and patient 5 (Arg243----His) in the strictly conserved amino acids. Expression study of both mutant genes in COS-1 cells produced inactive enzymes, establishing the functional significance of the two mis-sense mutations. In these patients, postheparin plasma LPL mass was either virtually absent (patients 1 and 2) or significantly decreased (patients 3-5). To detect these mutations more easily, we developed a rapid diagnostic test for each mutation. We also determined the DNA haplotypes for patients and confirmed the occurrence of multiple mutations on the chromosomes with an identical haplotype. These results demonstrate that familial LPL deficiency is a heterogeneous genetic disease caused by a wide variety of gene mutations.

Rat c-raf oncogene activation by a rearrangement that produces a fused protein.

In a previous study, activated rat c-raf was detected by an NIH 3T3 cell transfection assay, and a rearrangement was demonstrated in the 5' half of the sequence of the gene. In the present study, the cDNAs of normal and activated rat c-raf were analyzed. Results showed that the activated c-raf gene is transcribed to produce a fused mRNA, in which the 5' half of the sequence is replaced by an unknown rat sequence. This mRNA codes a fused c-raf protein. The normal and activated c-raf cDNAs were each connected to the long terminal repeat of Rous sarcoma virus and transfected into NIH 3T3 cells. Only the activated form had transforming activity. We conclude that the rearrangement is responsible for the activation of c-raf.

Evolution, expression, and chromosomal location of a novel receptor tyrosine kinase gene, eph.

Partial sequence analysis of the genomic eph locus revealed that the splicing points of kinase domain-encoding exons were completely distinct from those of the other protein tyrosine kinase members reported, suggesting that this is the earliest evolutionary split within this family. In Northern (RNA) blot analysis, the eph gene was expressed in liver, lung, kidney, and testis of rat, and screening of 25 human cancers of various cell types showed preferential expression in cells of epithelial origin. Overexpression of eph mRNA was found in a hepatoma and a lung cancer without gene amplification. Comparison of cDNA sequences derived from a normal liver and a hepatoma that overproduces eph mRNA demonstrated that two of them were completely identical throughout the transmembrane to the carboxy-terminal portions. Southern blot analysis of DNAs from human-mouse hybrid clones with an eph probe showed that this gene was present on human chromosome 7.

Unregulated expression of the erythropoietin receptor gene caused by insertion of spleen focus-forming virus long terminal repeat in a murine erythroleukemia cell line.

A murine erythroleukemia (MEL) cell line, F5-5, expressed 10,000 binding sites for erythropoietin (EPO) per cell, 10-fold more than was expressed by other murine erythroleukemia cell lines and normal erythroid progenitors. Northern (RNA) and Southern blot analyses revealed overexpression of mRNA for the EPO receptor (EPOR) and rearrangement of one of the EPOR gene alleles in F5-5 cells, respectively. Molecular cloning of F5-5-derived cDNA encoding EPOR revealed that the 5' noncoding region of the EPOR cDNA corresponds to the 3' long terminal repeat sequence of the polycythemic strain of Friend spleen focus-forming virus (F-SFFVP). The aberrant EPOR transcripts containing the 3' long terminal repeat sequence were mainly expressed in F5-5 cells. The same integration upstream of the EPOR gene was also observed in other subclones and the parent cell line. It is possible that overexpression of EPOR by viral promoter insertion will confer growth advantage to an F-SFFVP-infected erythroid progenitor cell, leading to positive clonal selection through further leukemogenic steps.

Organization and chromosomal localization of the human platelet-derived endothelial cell growth factor gene.

Human platelet-derived endothelial cell growth factor (hPD-ECGF) is a novel angiogenic factor which stimulates endothelial cell growth in vitro and promotes angiogenesis in vivo. We report here the cloning and sequencing of the gene for hPD-ECGF and its flanking regions. This gene is composed of 10 exons dispersed over a 4.3-kb region. Its promoter lacks a TATA box and a CCAAT box, structures characteristic of eukaryotic promoters. Instead, six copies of potential Sp1-binding sites (GGGCGG or CCGCCC) were clustered just upstream of the transcription start sites. Southern blot analysis using genomic DNAs from several vertebrates suggested that the gene for PD-ECGF is conserved phylogenetically among vertebrates. The gene for hPD-ECGF was localized to chromosome 22 by analysis of a panel of human-rodent somatic cell hybrid lines.

Clinical trials and cancer risk in Japan.

This paper presents the results of a clinical trial with vitamin A that was performed in Japan for the treatment of various cancers. The results of epidemiologic studies on the relationship between food intake and development of lung cancer in Japan also are reported. Several Japanese groups are studying the combined effect of vitamin A and its derivatives and anticancer chemotherapeutic agents on the in vitro and in vivo growth of various cancer cells, however, few clinical trials of these combinations for cancer treatment have been conducted. The results of these few studies are reviewed.

Augmentation of mouse natural killer cell activity by a streptococcal preparation, OK-432.

Streptococcal immunopotentiator OK-432 (NSC-B116209) augmented the natural killer (NK) cell activity of peritoneal exudate cells (PEC) in inbred C57BL/6 mice given ip injections of 0.1 mg OK-432 per mouse. The cytotoxic activity of PEC increased as early as 1 day after inoculation, reached its peak on day 3, and gradually declined thereafter, YAC-1, K562, and MOLT-4 target cells were more sensitive to PEC than were EL 4 and P815 target cells. The elimination of adherent cells by a nylon wool column enriched the proportion of cytotoxic cells among PEC. Nylon wool column-passed PEC were resistant to treatment with anti-Thy 1.2 antibody plus complement and sensitive to anti-asialo GM1 serum plus complement. Because 1:40-diluted rabbit antiserum against glycosphingolipid asialo GM1 is capable of eliminating mouse NK cell activity and is not cytotoxic to killer T-cells, the above results strongly suggest that OK-432 augments the NK cell activity in mice.