Targeting of hexokinase 1 to liver and hepatoma mitochondria. The proportion of hexokinase (HK; EC 2.7.1.1) isozyme 1 (HK1) that is bound to the outer mitochondrial membrane is tissue specific and developmentally regulated. HK activity is known to be markedly elevated in many cancer cells and a significant fraction is mitochondrial bound. This study examined the role of the 15-amino acid N-terminal domain of HK1 in binding to liver and hepatoma mitochondria. A chimeric reporter construct, pCMVHKCAT, encoding this HK1 domain coupled to the chloramphenicol acetyltransferase (CAT) gene was electroporated into mouse Hepa 1-6 hepatoma cells. After digitonin treatment, cell fractions were assayed for HK, lactate dehydrogenase, and CAT activities. Digitonin (75 micrograms/mg of protein) caused cytosolic leak but 70% of HK remained with the pellet. HKCAT, like HK, remained predominantly with the pellet; CAT form the control, pCMVCAT, remained mostly unbound. Binding of membrane-free cell extracts to rat liver mitochondria in vitro showed 91% of the HKCAT bound, whereas only 12% of CAT bound. Specificity of HKCAT binding to mitochondria was demonstrated by competition of HK1 for HKCAT binding sites on rat liver mitochondria as well as by blockage of HKCAT binding by N,N'-dicyclohexylcarbodiimide, which covalently binds to porin and blocks HK1 binding. Deletional mutant constructs of HKCAT showed reduced binding with increasing deletion size. In summary, these studies demonstrate that the 15-amino acid N-terminal domain of HK1 is necessary and sufficient to confer mitochondrial binding properties to CAT and that there is specificity for this binding to the mitochondria.


Computational model of the on-alpha ganglion cell receptive field based on bipolar cell circuitry. The on-alpha ganglion cell in the area centralis of the cat retina receives approximately 450 synapses from type b1 cone bipolar cells. This bipolar type forms a closely spaced array (9 microns), which contributes from 1 to 7 synapses per b1 cell throughout the on-alpha dendritic field. Here we use a compartmental model of an on-alpha cell, based on a reconstruction from electron micrographs of serial sections, to compute the contribution of the b1 array to the on-alpha receptive field. The computation shows that, for a physiologic range of specific membrane resistance (9500-68,000 omega.cm2) and a linear synapse, inputs are equally effective at all points on the on-alpha dendritic tree. This implies that the electrotonic properties of the dendritic tree contribute very little to the domed shapes of the receptive field center and surround. Rather, these shapes arise from the domed distribution of synapses across the on-alpha dendritic field. Various sources of "jitter" in the anatomical circuit, such as variation in bipolar cell spacing and fluctuations in the number of synapses per bipolar cell, are smoothed by the overall circuit design. However, the computed center retains some minor asymmetries and lumps, due to anatomical jitter, as found in actual alpha-cell receptive fields.


Patchy accumulation of apical Na+ transporters allows cross talk between extracellular space and cell nucleus. Intracellular Na+ activities and local current densities were measured in fused Madin-Darby canine kidney cells using Na+ and voltage-sensing microelectrodes. Na+ that enters the cell across the apical plasma membrane accumulates initially in the nucleoplasm, several seconds ahead of its appearance in the cell cytoplasm. The spatial distribution of Na+ currents, produced by a local superfusion of the cell surface, indicates a nonuniform, patchy accumulation of apical Na+ transporters in the vicinity of the nucleus. Such pathways for direct Na+ flux between extracellular space and cell nucleus could be potentially important for gene activation.


Determination of ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single gene. Expression cDNA cloning and structural analysis of the human keratinocyte growth factor receptor (KGFR) revealed identity with one of the fibroblast growth factor (FGF) receptors encoded by the bek gene (FGFR-2), except for a divergent stretch of 49 amino acids in their extracellular domains. Binding assays demonstrated that the KGFR was a high-affinity receptor for both KGF and acidic FGF, while FGFR-2 showed high affinity for basic and acidic FGF but no detectable binding by KGF. Genomic analysis of the bek gene revealed two alternative exons responsible for the region of divergence between the two receptors. The KGFR transcript was specific to epithelial cells, and it appeared to be differentially regulated with respect to the alternative FGFR-2 transcript. Thus, two growth factor receptors with different ligand-binding specificities and expression patterns are encoded by alternative transcripts of the same gene.


A nuclear protein essential for binding of rat 1,25-dihydroxyvitamin D3 receptor to its response elements. Recombinant 1,25-dihydroxyvitamin D3 receptor from a baculovirus expression system requires a mammalian-derived nuclear accessory protein for binding to a vitamin D response element (DRE). This was established by electrophoretic mobility shift analyses using radiolabeled DNA probes consisting of DREs from two vitamin D-responsive genes. Mammalian nuclear extract was also required for the binding of wild-type porcine vitamin D receptor to a DRE. Surprisingly, the accessory factor-dependent formation of receptor-DRE complex was independent of exogenous 1,25-dihydroxyvitamin D3. A 59- to 64-kDa accessory protein from porcine intestinal nuclear extract was identified by size-exclusion chromatography. Nuclear extracts from rat liver and kidney contained accessory factor, whereas smaller amounts were detected in heart muscle. Spleen and skeletal muscle contained no detectable accessory factor.


High-resolution tandem mass spectrometry of large biomolecules. Unit-resolution mass spectra have been obtained for peptides as large as 17 kDa, providing information on impurities and adduct ions, as well as accurate molecular weight values. Electrospray ionization produces many multiply-charged species of the same mass; isotopic peak resolution provides direct charge state assignment from the unit mass spacing of the isotopes. This is of special value when the spectrum also has many masses, such as from precursor ion dissociation or impurities. Mass measuring errors not only are concomitantly lower (less than 0.1 Da) than when the isotopic peaks are unresolved but also are independent of variations in 13C/12C natural isotopic abundances. Also, larger errors are avoided that occur when the measured peak envelope includes impurity or adduct ions. This also benefits tandem mass spectrometry; dissociation of peptide ions as large as 8.5 kDa yields fragment masses consistent (less than 0.1 Da) with their amino acid sequences.


Ongoing enterovirus-induced myocarditis is associated with persistent heart muscle infection: quantitative analysis of virus replication, tissue damage, and inflammation. Coxsackievirus B3-induced myocarditis in different immunocompetent mouse strains was used as a model to investigate interrelationships between virus replication and development of chronic enteroviral heart disease. Using in situ hybridization to detect enteroviral RNA, we show that heart muscle infection is not only detected in acute myocarditis but is also detected during the chronic phase of the disease. Coxsackievirus B3 could evade immunological surveillance in a host-dependent fashion, thus inducing a persistent infection of the myocardium in association with ongoing inflammation. Patterns of acute and persistent myocardial infection were quantitatively assessed in one representative mouse strain (A.CA/SnJ, H-2f) by applying computer-assisted digital image processing; these patterns were then related to the extent of myocardial tissue damage as well as to inflammation. We observed a strong correlation, both spatial and temporal, between viral replication and development of myocardial lesions, indicating that acute and chronic myocardial injuries are a consequence of multifocal organ infection. Analysis of strand-specific in situ hybridization revealed that viral replication in persistent infection is restricted at the level of RNA synthesis. The described procedure for quantitating organ infection provides a powerful tool for evaluating virus-host interactions and will be of particular interest to those studying human enterovirus-induced cardiomyopathies.


Hepatocyte growth factor inhibits growth of hepatocellular carcinoma cells. Hepatocyte growth factor (HGF) is a potent mitogen for primary hepatocytes. Therefore, we examined HGF as a possible autocrine growth factor in hepatocellular carcinoma (HCC). We introduced an albumin-HGF expression vector into Fao HCC cells and transgenic mice. Expression of the albumin-HGF vector in Fao HCC cells inhibited their growth in vitro. In vivo, FaoHGF cells produced tumors that averaged 10% of the sizes of G418-resistant controls when transplanted into nude mice. In contrast, hepatocytes from transgenic mice expressing HGF grew more rapidly than did those from normal siblings. Further, growth of eight additional HCC cell lines was inhibited by the addition of recombinant HGF. Finally, of 35 tumor cell lines surveyed, only 6 cell lines expressed HGF mRNA, and no HCC cell line expressed HGF. Although HGF stimulates normal hepatocytes, it is a negative growth regulator for HCC cells.


Viral proteins associated with the Epstein-Barr virus transactivator, ZEBRA. The BamHI Z Epstein-Barr replication activator (ZEBRA) mediates disruption of latency and induction of Epstein-Barr virus (EBV) early gene expression in latently infected lymphocytes. Polyclonal rabbit sera raised against ZEBRA were used to immunoprecipitate ZEBRA-associated proteins (ZAPs). ZAPs of 19, 21, 23, and 42 kDa were coimmunoprecipitated with ZEBRA from extracts of EBV-producing lymphoid cell lines. ZAPs were not recognized directly by the rabbit sera, but they were antigenic for EBV+ human sera. Immunoprecipitation of ZAPs by ZEBRA-specific antisera required the presence of ZEBRA. ZAPs were not coprecipitated with ZEBRA from mouse cells expressing only ZEBRA, from Raji (a cell line in which EBV is unable to complete lytic replication), or from cells treated with inhibitors of viral DNA synthesis. Thus, ZAPs are late EBV-encoded proteins. ZEBRA and ZAPs colocalized to a salt-insoluble nuclear fraction, and both were found extracellularly in crude preparations of virions. ZAPs might function to affect the cellular localization of ZEBRA, to alter its capacity to transactivate, or to influence its target gene specificity.


Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system. An isothermal in vitro DNA amplification method was developed based upon the following sequence of reaction events. Restriction enzyme cleavage and subsequent heat denaturation of a DNA sample generates two single-stranded target DNA fragments (T1 and T2). Present in excess are two DNA amplification primers (P1 and P2). The 3' end of P1 binds to the 3' end of T1, forming a duplex with 5' overhangs. Likewise, P2 binds to T2. The 5' overhangs of P1 and P2 contain a recognition sequence (5'-GTTGAC-3') for the restriction enzyme HincII. An exonuclease-deficient form of the large fragment of Escherichia coli DNA polymerase I (exo- Klenow polymerase) [Derbyshire, V., Freemont, P. S., Sanderson, M. R., Beese, L., Friedman, J. M., Joyce, C. M. & Steitz, T. A. (1988) Science 240, 199-201] extends the 3' ends of the duplexes using dGTP, dCTP, TTP, and deoxyadenosine 5'-[alpha-thio]triphosphate, which produces hemiphosphorothioate recognition sites on P1.T1 and P2.T2. HincII nicks the unprotected primer strands of the hemiphosphorothioate recognition sites, leaving intact the modified complementary strands. The exo- Klenow polymerase extends the 3' end at the nick on P1.T1 and displaces the downstream strand that is functionally equivalent to T2. Likewise, extension at the nick on P2.T2 results in displacement of a downstream strand functionally equivalent to T1. Nicking and polymerization/displacement steps cycle continuously on P1.T1 and P2.T2 because extension at a nick regenerates a nickable HincII recognition site. Target amplification is exponential because strands displaced from P1.T1 serve as targets for P2 and strands displaced from P2.T2 serve as targets for P1. A 10(6)-fold amplification of a genomic sequence from Mycobacterium tuberculosis or Mycobacterium bovis was achieved in 4 h at 37 degrees C.


Calcium channel blockers inhibit retinal degeneration in the retinal-degeneration-B mutant of Drosophila. Light accelerates degeneration of photoreceptor cells of the retinal degeneration B (rdgB) mutant of Drosophila. During early stages of degeneration, light stimuli evoke spikes from photoreceptors of the mutant fly; no spikes can be recorded from photoreceptors of the wild-type fly. Production of spike potentials from mutant photoreceptors was blocked by diltiazem, verapamil hydrochloride, and cadmium. Little, if any, effect of the (-)-cis isomer or (+)-cis isomer of diltiazem on the light response was seen. Further, the (+)-cis isomer was approximately 50 times more effective than the (-)-cis isomer in blocking the Ca2+ spikes, indicating that diltiazem action on the rdgB eye is mediated by means of blocking voltage-sensitive Ca2+ channels, rather than by blocking the light-sensitive channels. Application of the Ca(2+)-channel blockers (+)-cis-diltiazem and verapamil hydrochloride to the eyes of rdgB flies over a 7-day period largely inhibited light-dependent degeneration of the photoreceptor cells. Pulse labeling with [32P]phosphate showed much greater incorporation into eye proteins of [32P]phosphate in rdgB flies than in wild-type flies. Retarding the light-induced photoreceptor degeneration in the mutant by Ca(2+)-channel blockers, thus, suggests that toxic increase in intracellular Ca2+ by means of voltage-gated Ca2+ channels, possibly secondary to excessive phosphorylation, leads to photoreceptor degeneration in the rdgB mutant.


Components of the protein-excretion apparatus of Pseudomonas aeruginosa are processed by the type IV prepilin peptidase. In the Gram-negative pathogen Pseudomonas aeruginosa, mutants in the gene for the prepilin peptidase (pilD) are pleiotropic, as they not only fail to process pilin but also accumulate in the periplasm, in their mature form, several toxins and hydrolytic enzymes that are normally exported to the external medium (excreted). We have suggested that this excretion defect is due to the lack of PilD-dependent processing of proteins that share sequences in common with the prepilin subunit and that are components of a protein-excretion machinery. In this paper we report the isolation and characterization of transposon-induced excretion mutants with phenotypes similar to that of a pilD gene mutant. Using oligonucleotide probes designed to recognize sequences encoding the cleavage site of the type IV prepilins, we have isolated four linked genes with the predicted putative PilD-dependent cleavage site. Site-specific mutations within these genes have shown that they are required for protein excretion, and PilD-dependent processing of at least one of the four encoded proteins was demonstrated. Evidence suggests that similar components play a role in protein excretion in a wide variety of Gram-negative bacteria.


Immunoblots with rhodopsin antisera suggest that a purified mu opioid binding protein has structural characteristics of a G-protein-coupled receptor. A mu opioid binding protein (OBP), previously purified to homogeneity from bovine striatal membranes, was examined by immunoblotting with six antisera against bovine rhodopsin. An antibody against the carboxyl-terminal tail of rhodopsin and one against membrane-associated rhodopsin gave strong signals at the appropriate molecular mass (65 kDa). An antibody directed against the first cytoplasmic loop of rhodopsin was weakly reactive. Three other antibodies did not recognize OBP. This pattern of crossreactivity was identical to that previously seen with beta-adrenergic receptors. The existence of domains in the OBP, which are antigenically similar to those in two other guanine nucleotide regulatory protein-coupled receptors, supports the hypothesis that mu opioid receptors have the structure characteristic of this receptor family.


The cardiac conduction system in the rat expresses the alpha 2 and alpha 3 isoforms of the Na+,K(+)-ATPase. The sodium pump is crucial for the function of the heart and of the cardiac conduction system, which initiates the heartbeat. The alpha (catalytic) subunit of this pump has three isoforms; the alpha 1 isoform is ubiquitous, but the alpha 2 and alpha 3 isoforms are localized to excitable tissue. Because rodent alpha 2 and alpha 3 isoforms are relatively sensitive to ouabain, which also slows cardiac conduction, we studied heart-cell-specific expression of pump isoform genes. Multiple conduction-system structures, including sinoatrial node, bundle branches, and Purkinje strands, had prominent, specific hybridization signal for alpha 2 and alpha 3 isoforms compared with adjacent working myocytes. This gene-expression approach may be useful for labeling conduction tissue and also for localizing specific membrane channels and receptors in this system.


Mammographic features predicting an extensive intraductal component in early-stage infiltrating ductal carcinoma. Several studies have shown that the presence of an extensive intraductal component in patients with infiltrating ductal carcinoma is a major factor for predicting local recurrence after breast-conserving surgery and radiotherapy. A prospective study of 101 consecutive mammograms in patients with stage I or II infiltrating ductal carcinoma was performed to determine the predictive values of mammographic features in determining the presence or absence of an extensive intraductal component. Thirty-five (35%) of the lesions contained a pathologically verified extensive intraductal component. Sixty-five percent (22/34) of lesions showing mammographic evidence of calcifications with or without a mass were associated with an extensive intraductal component (p less than .001). Lesions with calcifications greater than 3 cm in extent were significantly (p less than .05) more likely to have an extensive intraductal component (9/10; 90%) than those with calcifications less than 3 cm in extent (13/24; 54%). Only 17% (8/46) of patients in whom mammograms showed only a mass or architectural distortion and 24% (5/21) of patients who had a mass palpable clinically or who had normal findings on mammograms had lesions with an extensive intraductal component. We conclude that infiltrating ductal carcinomas associated with calcifications on mammography, especially if the calcifications are extensive, are likely to be associated with an extensive intraductal component. Carcinomas without calcifications that show masses or architectural distortion on mammography, or carcinomas with palpable masses and normal findings on mammography, are unlikely to have an extensive intraductal component.


Inotropic and lusitropic dysfunction in myocardium from patients with dilated cardiomyopathy. Isometric force of contraction (DT), peak rate of tension increase (+T), peak rate of tension decrease (-T), time to peak tension (TPT), and time to half-relaxation (T 1/2 T) were measured in electrically driven human papillary muscle strips (New York Heart Association [NYHA] class IV heart transplants, dilated cardiomyopathy; nonfailing (NF) donor hearts, brain dead) (1 Hz, 37 degrees C) under basal conditions (1.8 mmol/L Ca2+) and after stimulation with isoprenaline, ouabain, and Ca2+. There was no difference in the isometric contraction (+T, -T, TPT, and T 1/2 T) between NYHA IV hearts and NF hearts under basal conditions. Inotropic stimulation above 300% of basal DT increased -T significantly more in NF hearts (p less than 0.05) compared with NYHA IV hearts. The effectiveness of ouabain and Ca2+ to increase DT was not significantly changed in NYHA IV hearts compared with NF hearts. The isoprenaline-mediated increase in DT was reduced (p less than 0.05) in NYHA IV hearts to a similar extent (70%) as beta-adrenoceptors were downregulated. When the rate of stimulation was increased to 3 Hz (force-frequency relationship), force of contraction increased only in NF preparations, whereas it decreased in NYHA IV myocardium (p less than 0.05). It was concluded that the contractile apparatus in terminally failing human myocardium is sufficient to maximally increase DT. During inotropic stimulation, abnormalities in diastolic rather than systolic contraction become evident. This may indicate abnormal intracellular Ca2+ handling.


Increased oxygen free radical activity in patients on cardiopulmonary bypass undergoing aortocoronary bypass surgery. Cardiac dysfunction after cardiopulmonary bypass (CPB) has been reported by various investigators. Oxygen free radicals have been shown to depress cardiac function and contractility. To evaluate the possible role of oxygen free radicals (OFR) in post-pump cardiac dysfunction, measurements of cardiac function, OFR producing activity of polymorphonuclear (PMN) leukocytes (PMN chemiluminescence) and malondialdehyde (MDA), a lipid peroxidation product, in blood were made at induction of anesthesia (T1), before cross clamping of the aorta (T2), after closure of the chest (T3), and 24 hours postoperatively (T4) in 21 patients undergoing aortocoronary bypass surgery. The total OFR-derived chemiluminescence at T1, T2, T3, and T4 was 1590 +/- 156, 3169 +/- 338, 1972 +/- 214, and 2614 +/- 366 mv.min.10(6) PMN-1, respectively. Superoxide dismutase (SOD)-inhibitable chemiluminescence at T1, T2, T3, and T4 was 1214 +/- 129, 2674 +/- 328, 1752 +/- 215, and 2139 +/- 292 mv.min.10(6) PMN-1, respectively. Superoxide anion at T1, T2, T3, and T4 was 0.99 +/- 0.14, 1.30 +/- 0.17, 1.07 +/- 0.14, and 1.19 +/- 0.12 nmol.10(6) PMN-1.30 min-1, respectively. Blood MDA at T1, T2, T3, and T4 was 0.17 +/- 0.02, 0.25 +/- 0.03, 0.20 +/- 0.03, and 0.23 +/- 0.02 nmol/ml, respectively. OFR-derived and SOD inhibitable chemiluminescence, superoxide anion, and blood MDA increased significantly during CPB and postoperatively. There were decreases in the blood pressure and stroke volume, and increases in the central venous pressure, capillary wedge pressure, and heart rate during CPB and postoperatively. Cardiac output remained unchanged during this procedure. There was leukopenia during CPB.(ABSTRACT TRUNCATED AT 250 WORDS)


Mucolipidoses II and III variants with normal N-acetylglucosamine 1-phosphotransferase activity toward alpha-methylmannoside are due to nonallelic mutations. Normal N-acetylglucosamine 1-phosphotransferase activity toward mono- and oligosaccharide acceptor substrates was detected in cultured skin fibroblasts from mucolipidoses II and III patients who were designated as variants (one of four mucolipidosis II and three out of six mucolipidosis III patients examined). The activity toward natural lysosomal protein acceptors was absent or deficient in cell preparations from all patients with classical as well as variant forms of mucolipidoses II and III. Complementation analysis, using fused and cocultivated mutant fibroblast combinations, revealed that, while cell lines with variant mucolipidosis III constituted a complementation group distinct from that of classical forms of mucolipidoses II and III, the variant mucolipidosis II cell line belonged to the same complementation group as did the classical forms. In contrast to the mutant enzyme from variant mucolipidosis III patients that failed to recognize lysosomal proteins as the specific acceptor substrates, the activity toward alpha-methylmannoside in the variant mucolipidosis II patient could be inhibited by exogenous lysosomal enzyme preparations (bovine beta-glucuronidase and human hexosaminidase A). These findings suggest that N-acetylglucosamine 1-phosphotransferase is composed of at least two distinct polypeptides: (1) a recognition subunit that is defective in the mucolipidosis III variants and (2) a catalytic subunit that is deficient or altered in the classical forms of mucolipidoses II and III as well as in the mucolipidosis II variant.


[Etoposide and cisplatin combination chemotherapy in a patient with small cell lung carcinoma under artificial hemodialysis]. A 70-year-old man with small cell carcinoma of the lung who was under artificial hemodialysis, was treated by combined administration of etoposide and cisplatin. Fifty mg. of etoposide in combination with 35 mg. of cisplatin were infused before dialysis. Before one hour and during dialysis, plasma etoposide and platinum levels decreased rapidly. No severe side effect was observed after the administration of etoposide and cisplatin. In conclusion, it was considered that cisplatin and etoposide could be given to the patient with malignant tumor under artificial hemodialysis.


[A case of gastric cancer with multiple liver metastasis treated with transarterial and transportal chemoembolization of tegafur/lipiodol and epirubicin]. A 56-year-old woman was admitted for advanced gastric cancer (S3H3N3P2 Stage IV). She underwent subtotal gastrectomy, left ovariectomy, and catheterization of the hepatic artery. Pre- and postoperative adjuvant chemotherapies consisting of tegafur, epirubicin, mitomycin C and cisplatin were performed. Two months after surgery, combination of transhepatic arterial and transportal chemoembolization with tegafur 400 mg/lipiodol 3 ml and epirubicin 20 mg was especially effective for this patient. The metastatic lesions of the liver regressed by 85% on computed tomography and the CEA level in the plasma decreased from 51.3 to 5.1 ng/ml. The response was judged partial response (PR), and the patient is now in good general condition.


[Evaluation of chemotherapy of unresectable non-small cell lung cancer]. For this evaluation, a comparison was made of survival of 34 patients with non-small cell lung cancer who had undergone chemotherapy (Group B) with that of 38 patients without this chemotherapy (Group A). Group B had received neither radiotherapy for primary lesions nor bronchial artery infusion of anticancer drugs, and the chemotherapeutic regimen was CDDP + VDS or CDDP + VDS + MMC chemotherapy. MST of this group was significantly longer than that of Group A (4.5 months vs 7.3 months. P less than 0.01), but the PS features of the two groups differed significantly. A comparison was thus made of survival for PS 1-2 cases in both groups (Group A: 17 cases, Group B: 27 cases). MST of group A and B were 6.8 and 9.8 months, respectively (P less than 0.05). The efficacy of the treatment for prolonging survival in terms of age, sex, PS, histology, T factor, N factor and M factor was evaluated by multivariate analysis using the proportional hazard model of Cox. The results obtained indicated the chemotherapy significantly prolonged survival.


[Randomized trial of cisplatin plus ifosfamide versus cisplatin plus vindesine for non-small cell lung cancer (NSCLC)]. Between 2/87 and 10/89, 83 patients (pts) with previously untreated NSCLC were assigned at random to receive either the PI regimen (cisplatin 80 mg/m2 dl and ifosfamide 2 g/m2 d 1, 2, 3, q 3-4 wks) or the PV regimen (cisplatin 80 mg/m2 d 1 and vindesine 3 mg/m2 d 1, 8, 15, q 3-4 wks). Three pts (2 PI regimen, 1 PV regimen) were ineligible for the study and 13 pts (7 PI regimen, 6 PV regimen) did not complete it. Thirty-three PI regimen pts and 34 PV regimen pts completed 2 or more cycles of the treatment and were evaluated. Patient characteristics were almost identical in both groups in terms of sex, age, pathological types, stage, performance status and number of chemotherapy cycles. For the PI regimen, there were 1 C.R. and 6 P.R., and the response rate was 21.2% (7/33). In the PV regimen, there were 11 P.R., and the response rate was 32.4% (11/34). The median survival time (MST) for PI regimen was 29 weeks (range 12-156 wks) and for PV regimen 40 weeks (range 8-138 wks). The difference in the survival curves for the 2 regimens was not statistically significant. Toxicities for the 2 regimens were similar except for greater leukopenia and one treatment-related death due to renal toxicity on PI regimen. These results suggest that PI regimen is not superior to PV regimen in the treatment of NSCLC.


[Sequential MTX and 5-FU therapy of gastric cancer with systemic bone metastasis and disseminated intravascular coagulation]. Sequential therapy consisting of methotrexate (MTX) and 5-FU was performed together with the administration of heparin and FOY in 10 cases of gastric cancer with disseminated intravascular coagulation (DIC) causing systemic bone metastasis. The ages of the subjects ranged from 29 to 65 years (median: 49 years) with systemic bone metastasis and bone marrow carcinosis observed in all cases. Histological types consisted of 6 cases of poorly differentiated adenocarcinoma, 2 cases of signet-ring cell carcinoma, and one case each of mucocellular and tubular adenocarcinoma. Therapy consisted of intravenous injection of 30 mg-100 mg/m2 (one case, 20 mg) of MTX followed three hours later by intravenous injection of 600 mg/m2 of 5-FU weekly. Determination of DIC was made in accordance with the DIC diagnostic standards of the Ministry of Health and Welfare, and determination of tumor effectiveness was based on gastric cancer handling codes. PR was observed in 3 cases. Diffuse metastasis observed in the entire lung field disappeared in one case, while remarkable improvement was observed in systemic bone metastasis in scintigram findings for the other 2 cases. All 3 cases were able to be discharged. Reduction of DIC score and absence of pain were observed in 8 cases. Based on the above, aggressive implementation of this treatment method is suggested.


[Effect of dibutyryl cyclic AMP in the treatment of hepatocellular carcinoma--intraarterial infusion therapy combined with anticancer agent for hepatocellular carcinoma with portal vein thrombosis]. Dibutyryl cyclic AMP was administered to 7 cases with hepatocellular carcinoma and its tumor thrombosis in portal vein, combined with intraarterial infusion of Mitomycin C or Adriamycin with implanted reservoir. Among these cases, tumor regressed in 5 cases, and therapeutic effect on tumor thrombosis was observed in 4 cases. The median survival time after initial treatment was about 5 months in 5 cases of Vp3, and more than 18 months in 2 cases of Vp2. Reduction of liver dysfunction by cholinesterase and hepaplastin test was found in most cases, and no severe side effects were observed. It is suggested that dibutyryl cyclic AMP has an antitumor effect on hepatocellular carcinoma, especially on its tumor thrombosis in portal vein, and also may assist in recovery from liver dysfunction.


Cell fusion caused by herpes simplex virus-1 (HSV-1) strains tsB5 and MP is inhibited at pH 6.7 and pH 7.0. We investigated the effect of different pH conditions on Vero cell cultures infected with herpes simplex virus-1 (HSV-1) wild-type strain KOS, and syncytial mutants HSV-1 HFEM (tsB5) and HSV-1 mp (MP). Cell fusion was inhibited when infected cells were continuously incubated with culture media adjusted to pH 6.7 or pH 7.0. Inhibition of cell fusion was rapidly reversible when infected cell cultures were returned to pH 7.5. The rate of synthesis and cell-surface expression of virus-specified glycoproteins gB, gC, gD, and gH were not affected during continuous incubation at pH 7.0, but they were reduced at pH 6.7 in comparison to pH 7.5. At later hours p.i. however, these glycoproteins continued to accumulate at all tested pH levels. Accumulation of infectious virions was substantially reduced for MP, KOS, and tsB5 at pH 6.7. At pH 7.0, KOS and tsB5 titers were greatly reduced but MP titers were not affected.


Immunoperoxidase tracing of Junin virus neural route after footpad inoculation. To determine the pathway adopted by peripherally inoculated Junin virus (JV) to reach the CNS, rat tissues were serially harvested to trace the sequence of viral progression from right hind footpad to brain. Immunoperoxidase (PAP) labeling of viral antigen, concomitantly with infectivity assays and histological examination of each selected sample, were carried out. As from the 2nd week post-infection (pi), neurological disease inducing 100% mortality at 1 month was evident. At day 5 pi, viral antigen was first detected at footpad level in epidermic and dermic cells, as well as in neighbouring myocytes; labeled macrophages infiltrating small nerve branches were also disclosed. As from 10-15 days pi, viral antigen became apparent along ipsilateral sciatic nerve structures and within lumbar spinal ganglion neurons, followed by a fast viral spread throughout CNS neurons that involved spinal cord and brain. Concurrent histopathology featured minimal inflammatory reaction together with generalized astrocytic activation. Hematogenous viral transport was negligible, since JV was isolated much earlier and in higher infectivity titers in neural tissues than in blood. It may be concluded that after viral replication in footpad, JV neural route was demonstrated by its PAP labeling from peripheral nerves to cerebral cortex.


Maternally-derived passive immunity to enterotropic mouse hepatitis virus. Maternally-derived antibody to enterotropic mouse hepatitis virus (MHV) strain Y was transferred to pups by both intrauterine (IgG) and lactogenic (IgA and IgG) routes. Antibody present in the gastric whey of pups suckling immune dams dropped to undetectable levels by weaning age (21 days post partum). MHV-specific IgG was found in the serum of passively immune pups up to 10 weeks of age. Immune dams transferred equal levels of antibody to 3 consecutive litters of pups, without evidence of decline. Immunoblots showed that IgA and IgG in whey and serum were directed against nucleoprotein N and glycoprotein S. MHV-specific IgM was not detected in any sample.


Anti-idiotypic antibodies to bovine herpesvirus-1 inhibit virus infection in cell cultures. A panel of murine monoclonal antibodies (MAbs) to bovine herpesvirus-1 (BHV-1) was prepared. Three of them were neutralizing MAbs and reacted against 130/75/50 kDa, 77 kDa, or 97 kDa glycoproteins (gp). A fourth non-neutralizing MAb recognized the 97 kDa gp. Competition radioimmunoassay demonstrated that each of the four MAbs reacted against a different virus epitope. Anti-idiotypic antibodies (anti-id) to the four MAbs were produced in rabbits and purified by sequential immunoaffinity chromatography. Each anti-id inhibited the binding of its respective MAb to BHV-1 in competitive ELISA and blocked BHV-1 neutralizing activity of the MAb. This inhibition suggested that the anti-ids were specific for the antigen binding site of the MAbs. Treatment of MDBK cells with anti-ids inhibited BHV-1 infection, which suggested that the anti-ids block a cellular component essential for virus infection. Absence of significant cross-reactivity among the anti-ids for heterologous MAbs indicated that they recognized unique determinants on the antigen binding site of the homologous MAb.


Adaptation of transmissible gastroenteritis virus to growth in non-permissive Vero cells. The CPK cells derived from swine kidney were infected with the attenuated TO-163 strain of transmissible gastroenteritis (TGE) virus, and fused with uninfected Vero cells in the presence of polyethylene glycol. Repeated cocultivation of the fused cells with uninfected Vero cells rendered the virus to grow in Vero cells. The Vero cell-adapted virus acquired the ability to infect and produce cytopathic effects in several other non-permissive cell lines of non-porcine origin. No major differences in viral polypeptides were shown between the Vero cell-adapted TO-163 strain and its parent strain by indirect immunofluorescence and Western blotting using monoclonal and polyclonal antibodies to TGE virus.


Latency and reactivation of a thymidine kinase-negative bovine herpesvirus 1 deletion mutant. A bovine herpesvirus 1 (BHV 1) mutant variant with a deletion in the thymidine kinase (TK) gene was assessed for its ability to establish latency and be reactivated in cattle. After treatment with dexamethasone, reactivated TK- BHV 1 was isolated from one of four cattle that received virus by intravenous inoculation only, and from four of four cattle that received virus by intranasal, intravaginal, and intravenous inoculation. Results prove that TK- BHV 1 will establish latency and can be reactivated in the natural host.


Follicular dendritic cell-B cell interactions in virus disease. Common localization but different cell damage caused by antibody immobilized virus? Follicular dendritic cells (FDC) are involved in the trapping and retention of antigen-antibody complexes in lymphoid follicles. This FDC immobilized antigen is thought to be involved in the generation of memory B-lymphocytes. Follicular trapping of both Aleutian disease virus and HIV particles has been demonstrated. However as far as known their affects on FDC and follicular B-cells are completely different. It is hypothesized that the trapping of (antibody-complexed) virus particles by the FDC-network may have an important role in several virus diseases.


Hybridization relatedness of Israeli and U.S. bluetongue (BLU) serotypes using cDNA probes from BLU virus strain 11-UC8. Partial cDNA clones representing 47%, 96%, and 98% of genome segments 7, 9, and 10, respectively, of a US bluetongue virus (BLU) 11 virulent strain were used to study, for the first time, the genetic relationships between Israeli BLU proto-serotypes and field isolates, and US BLU proto-serotypes. Their usefulness as group-specific identification probes was also determined. The viral nucleic acid was extracted from the infected cells and the purified dsRNA genome segments were fractionated by polyacrylamide gel electrophoresis, transferred to a nylon membrane and hybridized to the 32P labeled DNA probes. The three probes recognized all the samples tested. Genome segment 7, that code for the mayor inner capsid protein VP7, showed the most variation in the hybridization signal with the US proto-serotypes and all the Israeli samples studied. The genome segments 9 and 10 that code for the minor inner capsid protein VP6 and the nonstructural protein NS3, respectively, were highly conserved in all the samples tested despite their distant geographical regions of origin. The last two mentioned clones showed to be good group-specific probes for the identification of BLU samples from Israel and United States. The obtained cloned genetic probes were also tested against US epizootic haemorrhagic disease virus (EHDV) serotype 1 and 2 viral dsRNA, a distantly related orbivirus. None of them hybridized with the viral dsRNA of these two viruses.


Viremic dissemination of mouse hepatitis virus-JHM following intranasal inoculation of mice. Using a sensitive infant mouse bioassay to detect infectious virus, the pattern of mouse hepatitis virus (MHV) JHM dissemination in blood and other tissues was examined during the first 5 days following intranasal inoculation. MHV replicated in nasal turbinates of both susceptible BALB and resistant SJL mice from days 1 through 5, but BALB mice had higher titers on days 1 and 2. Viremia was detectable on days 1 through 5 in BALB mice, but only on days 3 and 5 in SJL mice. Transient virus replication occurred in the lungs of both mouse genotypes at 1 and 2 days, then ceased. This correlated with more consistently demonstrable virus in blood collected from the left atrium of the heart, compared to jugular vein, portal vein and right atrial blood. Virus was associated equally with the plasma and cellular fractions of blood on day 3, but was primarily in the buffy coat of the cellular fraction on day 5. Interferon-alpha/beta was detected in serum and spleen, but not liver or brain of BALB mice or in any tissue of SJL mice. BALB serum and spleen interferon was first detected at 36 h, peaked between 48 and 72 h, and was undetectable by 108 h. The distribution of virus in nose, cervical, axillary and mesenteric lymph nodes, spleen, Peyer's patch, thymus, bone marrow and liver was examined at 1, 2, and 3 days. The resulting pattern suggested lymphatic spread of virus to cervical lymph node and mesenteric lymph node as pathways of dissemination in addition to viremia.


Inhibition of herpes simplex virus production in vitro by cyclosporin A. Nontoxic concentrations of Cyclosporin A (CyA) dose-dependently inhibited herpes simplex virus (HSV) production in resting monkey kidney cells. The block was at the step of virus DNA synthesis as assessed by [3H]thymidine incorporation and by dot blot hybridization of infected cell DNA using a cloned 32P-labelled HSV DNA fragment (BamHI X) as probe. This was further supported by analysis of HSV protein synthesis in the presence of CyA as assessed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot. A relative accumulation of HSV alpha- (e.g., ICP 4) and beta 1-proteins (e.g., ICP 6 and 8) was found, whereas HSV gamma 1-proteins were slightly decreased and gamma 2-proteins were markedly decreased by CyA. The production of thymidine kinase and DNA polymerase was decreased when CyA was added to HSV infected cells. The sensitivity to CyA was not escaped by thymidine kinase nor DNA polymerase deficient mutants. Passage of HSV in presence of CyA did not result in induction of drug resistance.


The influence of arrestin (48K protein) and rhodopsin kinase on visual transduction. The shutoff of the phototransduction cascade in retinal rods requires the inactivation of light-activated rhodopsin. The underlying mechanisms were studied in functionally intact detached rod outer segments by testing the effect of either sangivamycin, an inhibitor of rhodopsin kinase, or phytic acid, an inhibitor of 48K protein binding to phosphorylated rhodopsin, on light responses recorded in whole-cell voltage clamp. The results suggest that isomerized rhodopsin is inactivated fully by multiple phosphorylation and that the binding of 48K protein accelerates recovery by quenching partially phosphorylated rhodopsin. Higher concentrations of sangivamycin cause changes in the light response that cannot be explained by selective inhibition of rhodopsin kinase and suggest that other protein kinases are needed for normal rod function.


Calcium influx through nicotinic receptor in rat central neurons: its relevance to cellular regulation. The Ca2+ permeability of a nicotinic acetylcholine receptor (nAChR) in the rat CNS was determined using both current and fluorescence measurements on medial habenula neurons. The elementary slope conductance of the nAChR channel was 11 pS in pure external Ca2+ (100 mM) and 42 pS in standard solution. Ca2+ influx through nAChRs resulted in the rise of cytosolic Ca2+ concentration ([Ca2+]i) to the micromolar range. This increase was maximal under voltage conditions (below -50 mV) in which Ca2+ influx through voltage-activated channels was minimal. Ca2+ influx through nAChRs directly activated a Ca(2+)-dependent Cl- conductance. In addition, it caused a decrease in the GABAA response that outlasted the rise in [Ca2+]i. These results underscore the physiological significance of Ca2+ influx through nAChR channel in the CNS.


Ciliary neurotrophic factor prevents neuronal degeneration and promotes low affinity NGF receptor expression in the adult rat CNS. Recombinant human ciliary neurotrophic factor (CNTF) was infused for 2 weeks into the lateral ventricle of fimbria-fornix transected adult rats, and its effects were compared with those of purified mouse nerve growth factor (NGF). We provide evidence that CNTF can prevent degeneration and atrophy of almost all injured medial septum neurons (whereas NGF protects only the cholinergic ones). CNTF is also involved in up-regulation of immunostainable low affinity NGF receptor (LNGFR) in cholinergic medial septum and neostriatal neurons and in a population of lateral septum neurons. In contrast to NGF, CNTF did not stimulate choline acetyltransferase in the lesioned septum and normal neostriatum (pointing to different mechanisms for the regulation of choline acetyltransferase and LNGFR), cause hypertrophy of septal or neostriatal cholinergic neurons, or cause sprouting of LNGFR-positive (cholinergic) septal fibers.


A family of metabotropic glutamate receptors. Three cDNA clones, mGluR2, mGluR3, and mGluR4, were isolated from a rat brain cDNA library by cross-hybridization with the cDNA for a metabotropic glutamate receptor (mGluR1). The cloned receptors show considerable sequence similarity with mGluR1 and possess a large extracellular domain preceding the seven putative membrane-spanning segments. mGluR2 is expressed in some particular neuronal cells different from those expressing mGluR1 and mediates an efficient inhibition of forskolin-stimulated cAMP formation in cDNA-transfected cells. The mGluRs thus form a novel family of G protein-coupled receptors that differ in their signal transduction and expression patterns.


Efficient expression of rat brain type IIA Na+ channel alpha subunits in a somatic cell line. Type IIA rat brain Na+ channel alpha subunits were expressed in CHO cells by nuclear microinjection or by transfection using a vector containing both metallothionein and bacteriophage SP6 promoters. Stable cell lines expressing Na+ channels were isolated, and whole-cell Na+ currents of 0.9-14 nA were recorded. The mean level of whole-cell Na+ current (4.5 nA) corresponds to a cell surface density of approximately 2 channels active at the peak of the Na+ current per microns 2, a density comparable to that observed in the cell bodies of central neurons. The expressed Na+ channels had the voltage dependence, rapid activation and inactivation, and rapid recovery from inactivation characteristic of Na+ channels in brain neurons, bound toxins at neurotoxin receptor sites 1 and 3 with normal properties, and were posttranslationally processed to a normal mature size of 260 kd. Expression of Na+ channel cDNA in CHO cells driven by the metallothionein promoter accurately and efficiently reproduces native Na+ channel properties and provides a method for combined biochemical and physiological analysis of Na+ channel structure and function.


Structure and functional expression of alpha 1, alpha 2, and beta subunits of a novel human neuronal calcium channel subtype. The primary structures of human neuronal alpha 1, alpha 2, and beta subunits of a voltage-dependent Ca2+ channel were deduced by characterizing cDNAs. The alpha 1 subunit (alpha 1D) directs the recombinant expression of a dihydropyridine-sensitive L-type Ca2+ channel when coexpressed with the beta (beta 2) and the alpha 2 (alpha 2b) subunits in Xenopus oocytes. The recombinant channel is also reversibly blocked by 10-15 microM omega-conotoxin. Expression of the alpha 1D subunit alone, or coexpression with the alpha 2b subunit, did not elicit functional Ca2+ channel activity. Thus, the beta 2 subunit appears to serve an obligatory function, whereas the alpha 2b subunit appears to play an accessory role that potentiates expression of the channel. The primary transcripts encoding the alpha 1D, alpha 2, and beta subunits are differentially processed. At least two forms of neuronal alpha 1D were identified. Different forms of alpha 2 and beta transcripts were also identified in CNS, skeletal muscle, and aorta tissues.


Omega-conotoxin GVIA blocks a Ca2+ current in bovine chromaffin cells that is not of the "classic" N type. Previous studies have identified two components of whole-cell Ca2+ current in bovine chromaffin cells. The "standard" component was activated by single depolarizations, while "facilitation" could be activated by large prepulses or repetitive depolarizations. Neither current component was sensitive to changes in holding potential between -100 and -50 mV; thus neither appeared to be carried by N-type Ca2+ channels. We now report that the facilitation Ca2+ current is insensitive to omega-conotoxin GVIA (omega-CgTx), but that the toxin blocks approximately 50% of the standard Ca2+ current. In some cells the toxin blocks all of the standard Ca2+ current, in others about half of the current, while in others it has no effect. Kinetic differences in current activation are observed after toxin application. These results suggest that the standard component of chromaffin cell Ca2+ current is composed of two pharmacologically distinct channels-one is omega-CgTx sensitive and the other is not. Two kinetically distinct types of 14 pS Ca2+ channels that may correspond to the omega-CgTx-sensitive and -insensitive components were observed in single-channel experiments. Because omega-CgTx blocked Ca2+ channels that were not inactivated by a depolarized holding potential, the commonly used Ca2+ channel categorization scheme may be inadequate to describe the Ca2+ channels found in chromaffin cells.


The effect of lysophosphatidylcholine on the activity of various mitochondrial enzymes. The influence of lysophosphatidylcholine (LPC) on H(+)-ATPase, cytochrome oxidase (COX), glycerolphosphate dehydrogenase (GPDH) and malate dehydrogenase (MDH) was followed. The activities of H(+)-ATPase and COX increased with increasing LPC concentration up to 0.5 mg/mg protein when maxima were achieved. This activatory effect is LPC-specific, because Lubrol-treated or frozen-thawed mitochondria showed lower activities of these enzymes. H(+)-ATPase was not influenced by higher concentration of LPC, while COX activity decreased with increasing amount of LPC. The activity of GPDH decreased at very low concentration of LPC and was not further modified at higher LPC concentration. In an attempt to find the concentration of LPC necessary for a complete permeabilization of inner mitochondrial membrane we followed the influence of lysolipid on the release of MDH activity from the mitochondrial matrix. The full activity of this enzyme was obtained with a concentration 0.75 mg LPC/mg protein indicating that mitochondria were completely broken. Our data indicate that LPC significantly affects activity of enzymes connected with mitochondrial membrane and can be useful for evaluation of the importance of phospholipid microenvironment for the enzyme function.


Characterization of a mitochondrial inorganic pyrophosphatase in Saccharomyces cerevisiae. We have studied a mitochondrial inorganic pyrophosphatase (PPase) in the yeast Saccharomyces cerevisiae. The uncoupler FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and the ionophores valinomycin and nigericin stimulate the PPase activity of repeatedly washed yeast mitochondria 2-3-fold. We have previously cloned a yeast gene, PPA2, encoding the catalytic subunit of a mitochondrial PPase. Uncouplers stimulate the PPase activity several-fold in mitochondria from both cells that overexpress PPA2 from a high copy number plasmid and cells with normal expression. These results indicate that the PPA2 polypeptide functions as an energy linked and membrane associated PPase. The stimulation of mitochondrial PPase activity by FCCP, but not by valinomycin and nigericin, was greatly enhanced by the presence of DTT. The antibiotics Dio-9, equisetin and the F0F1-ATPase inhibitor oligomycin also increase mitochondrial PPase activity several fold. This stimulation is much higher, whereas basal PPase activity is lower, in isotonic than in hypotonic solution, which indicates that intact membranes are a prerequisite for maximal effects.


The contribution of adenine nucleotide loss to ischemia-induced impairment of rat kidney cortex mitochondria. Adenine nucleotides and respiration were assayed with rat kidney mitochondria depleted of adenine nucleotides by pyrophosphate treatment and by normothermic ischemia, respectively, with the aim of identifying net uptake of ATP as well as elucidating the contribution of adenine nucleotide loss to the ischemic impairment of oxidative phosphorylation. Treatment of rat kidney mitochondria with pyrophosphate caused a loss of adenine nucleotides as well as a decrease of state 3 respiration. After incubation of pyrophosphate-treated mitochondria with ATP, Mg2+ and phosphate, the content of adenine nucleotides increased. We propose that kidney mitochondria possess a mechanism for net uptake of ATP. Restoration of a normal content of matrix adenine nucleotides was related to full recovery of the rate of state 3 respiration. A hyperbolic relationship between the matrix content of adenine nucleotides and the rate of state 3 respiration was observed. Mitochondria isolated from kidneys exposed to normothermic ischemia were characterized by a decrease in the content of adenine nucleotides as well as in state 3 respiration. Incubation of ischemic mitochondria with ATP, Mg2+ and phosphate restored the content of adenine nucleotides to values measured in freshly-isolated mitochondria. State 3 respiration of ischemic mitochondria reloaded with ATP recovered only partially. The rate of state 3 respiration increased by ATP-reloading approached that of uncoupler-stimulated respiration measured with ischemic mitochondria. These findings suggest that the decrease of matrix adenine nucleotides contributes to the impairment of ischemic mitochondria as well as underlining the occurrence of additional molecular changes of respiratory chain limiting the oxidative phosphorylation.


Blockade of sodium channels by Bistramide A in voltage-clamped frog skeletal muscle fibres. The effect of Bistramide A, a toxin isolated from Bistratum lissoclinum Sluiter (Urochordata), on the peak sodium current (INa) of frog skeletal muscle fibres was studied with the double sucrose gap voltage clamp technique. External or internal application of Bistramide A inhibited INa without alteration of the kinetic parameters of the current nor of the apparent reversal potential for Na. The steady-state activation curve of INa was unchanged while the steady-state inactivation curve of INa was shifted towards more negative membrane potentials. Dose-response curves indicated an apparent dissociation constant for Bistramide A of 3.3 microM and a Hill coefficient of 1.2 which suggested a one to one relation between the toxin and Na channel. The inhibition of INa occurred at rest, and was more important at more positive holding potentials. Bistramide A exhibited only a weak frequency-dependent effect. The toxin did not interact with the use-dependent effect of lidocaine. It mainly blocked Na channels at more depolarized holding potentials. The toxin blocked Na channels when it was internally applyed and when the inactivation gating system has been previously destroyed by internal diffusion of iodate. The data suggest that Bistramide A inhibited the Na channel both at rest and in the inactivated state and occupied a site which was not located on the inactivation gate.


Platelet membrane phosphatidylinositol kinase activity. Triton X-100 effects provide evidence for intramicellar reaction. Phosphatidylinositol (PI) kinase activity of platelet membranes was solubilized and partially purified by anion-exchange chromatography to measure the initial enzymatic rates. Kinetic studies were performed in the presence of Triton X-100 to obtain mixed micelles. The partially purified enzyme exhibited a Michaelian behaviour towards ATP, with a Km of 58 microM. The enzymatic rates were dependent upon Triton concentrations. Upon increasing its concentration, this detergent exhibited an activating effect followed by an inhibitory one. The optimal micellar Triton concentration was proportionnal to the PI concentration used in the assay. Conversely, the behaviour of the enzyme towards PI was dependent upon the Triton concentration. However, when PI concentration was expressed as its surfacic concentration within the micelles, the activity became independent of the detergent concentration, and a Km value of 0.09 mol/mol was estimated. Therefore, in vitro phosphorylation of phosphatidylinositol by PI kinase is rate-limited by an intramicellar reaction, and provides a study model for the in vivo reaction.


Proteinases inhibit H(+)-ATPase and Na+/H+ exchange but not water transport in apical and endosomal membranes from rat proximal tubule. A marked increase in water permeability can be induced in Xenopus oocytes by injection of mRNA from tissues that express water channels, suggesting that the water channel is a protein. In view of this and previous reports which showed that proteinases may interfere with mercurial inhibition of water transport in red blood cells (RBC), we examined the influence of trypsin, chymotrypsin, papain, pronase, subtilisin and thermolysin on water permeability as well as on ATPase activity, H(+)-pump, passive H+ conductance, and Na+/H+ exchange in apical brush-border vesicles (BBMV) and endosomal (EV) vesicles from rat renal cortex. H+ transport was measured by Acridine orange fluorescence quenching and water transport by stopped-flow light scattering. As measured by potential-driven H+ accumulation in BBMV and EV, proteinase treatment had little effect on vesicle integrity. In BBMV, ecto-ATPase activity was inhibited by 15-30%, Na+/H+ exchange by 20-55%, and H+ conductance was unchanged. Osmotic water permeability (Pf) was 570 microns/s and was inhibited 85-90% by 0.6 mM HgCl2; proteinase treatment did not affect Pf or the HgCl2 inhibition. In EV, NEM-sensitive H+ accumulation and ATPase activity were inhibited by greater than 95%. Pf (140 microns/s) and HgCl2 inhibition (75-85%) were not influenced by proteinase treatment. SDS-PAGE showed selective digestion of multiple polypeptides by proteinases. These results confirm the presence of water channels in BBMV and EV and demonstrate selective inhibition of ATPase function and Na+/H+ exchange by proteinase digestion. The lack of effect of proteinases on water transport by mercurials. We conclude that the water channel may be a small integral membrane protein which, unlike the H(+)-ATPase and Na+/H+ exchanger, has no functionally important membrane domains that are sensitive to proteolysis.


Parathyroid hormone stimulates ATP-dependent calcium pump activity by a different mode in proximal and distal tubules of the rat. A new technique was developed to isolate basolateral membrane vesicles individually from proximal and distal tubules of the rat cortex. This new technique enabled us to study differences in their kinetics and mechanisms of hormonal regulation of Ca pump between proximal and distal tubules. The Ca pump in distal tubule has very high affinity (42.6 nM Ca2+) and the one in proximal tubule has relatively low affinity (75.6 nM Ca2+). Parathyroidectomy (PTX) decreased the Vmax of Ca pump activity in proximal tubule (4.68 +/- 0.99 vs. 9.08 +/- 2.21 nmol 45Ca2+/min per mg protein BLMV, P less than 0.05), while it increased Km in distal tubule (93.1 +/- 11.0 vs. 35.1 +/- 16.1 nM Ca2+, P less than 0.05). Restoration of serum Ca2+ concentration by 1,25(OH)2D3 supplement could not reverse these changes by PTX in Ca pump activity in either the proximal or the distal tubule. In conclusion, this study strongly suggested that parathyroid hormone stimulated Ca pump activity by increasing the Vmax in proximal tubule and by increasing the affinity in distal tubule. 1,25(OH)2D3 does not have a direct effect on the basolateral membrane Ca pump activity.


Amphipathic interactions of cannabinoids with membranes. A comparison between delta 8-THC and its O-methyl analog using differential scanning calorimetry, X-ray diffraction and solid state 2H-NMR. The effects of (-)-delta 8-tetrahydrocannabinol (delta 8-THC) and its biologically inactive O-methyl ether analog on model phospholipid membranes were studied using a combination of differential scanning calorimetry (DSC), small angle X-ray diffraction and solid state 2H-NMR. The focus of this work is on the amphipathic interactions of cannabinoids with membranes and the role of the free phenolic hydroxyl group which is the only structural difference between these two cannabinoids. Identically prepared aqueous multilamellar dispersions of phosphatidylcholines in the absence and presence of cannabinoids were used. The DSC thermograms and X-ray diffraction patterns of these preparations allowed us to detect the strikingly different manners in which these two cannabinoids affect the thermotropic properties and the thickness of the bilayer. In order study the effects of the cannabinoids on different regions of the bilayer, we used solid state 2H-NMR with four sets of model membranes from dipalmitoylphosphatidylcholine deuterated in different sites, viz., the choline trimethylammonium head group, or one of the following three groups in the acyl chains; the 2'-methylene, 7'-methylene, 16'-methyl groups. Analysis of quadrupolar splittings indicated that delta 8-THC resides near the bilayer interface and the inactive analog sinks deeper towards the hydrophobic region. The temperature dependence of the solid state 2H-NMR spectra showed that, during the bilayer phase transition, the disordering of the choline head groups is a separate event from the melting of the acyl chains, and that amphipathic interactions between delta 8-THC and the membrane separate these two events further apart in temperature. The inactive analog lacks the ability to induce such a perturbation.


Interfacial ionization and partitioning of membrane-bound local anesthetics. Consideration of the interfacial protonation equilibria of membrane-associated amphiphiles indicates that the partition coefficients of the protonated and unprotonated species will differ considerably. The partition coefficients of the charged and uncharged forms of spin-labelled myristic acid in dimyristoylphosphatidylcholine bilayer dispersions have been measured by EPR spectroscopy and found to be approximately 140-fold higher for the protonated acid than for the dissociated salt form. This ratio of partition coefficients is found to be in good agreement with that predicted from the interfacial shift in pKa of the fatty acid on its partitioning into the membrane. The latter was determined from the changes in the EPR spectra of the membrane-associated fatty acid with pH and was found to be +2.1 pH units. The interfacial shifts in pKa for a series of spin-labelled analogues of tertiary amine local anaesthetics have been determined from the pH dependence of the partition coefficients in dimyristoylphosphatidylcholine bilayer dispersions and are found mostly to be in the range of approx. -1.0 to -1.5 pH units, corresponding to a 10- to 30-fold higher partition coefficient of the uncharged base compared with that of the charged ammonium form.


Phosphatase activity and potassium transport in liposomes with Na+,K(+)-ATPase incorporated. We have used liposomes with incorporated pig kidney Na+,K(+)-ATPase to study vanadate sensitive K(+)-K+ exchange and net K+ uptake under conditions of acetyl- and p-nitrophenyl phosphatase activities. The experiments were performed at 20 degrees C. Cytoplasmic phosphate contamination was minimized with a phosphate trapping system based on glycogen, phosphorylase a and glucose-6-phosphate dehydrogenase. In the absence of Mg2+ (no phosphatase activity) 5-10 mM p-nitrophenyl phosphate slightly stimulated K(+)-K+ exchange whereas 5-10 mM acetyl phosphate did not. In the presence of 3 mM MgCl2 (high rate of phosphatase activity) acetyl phosphate did not affect K(+)-K+ exchange whereas p-nitrophenyl phosphate induced a greater stimulation than in the absence of Mg2+; a further addition of 1 mM ADP resulted in a 35-65% inhibition of phosphatase activity with an increase in K(+)-K+ exchange, which sometimes reached the levels seen with 5 mM phosphate and 1 mM ADP. The net K+ uptake in the presence of 3 mM MgCl2 was not affected by acetyl phosphate or p-nitrophenyl phosphate, whereas it was inhibited by 5 mM phosphate (with and without 1 mM ADP). The results of this work suggest that the phosphatase reaction is not by itself associated to K+ translocation. The ADP-dependent stimulation of K(+)-K+ exchange in the presence of phosphatase activity could be explained by the overlapping of one or more step/s of the reversible phosphorylation from phosphate with the phosphatase cycle.


The role of surface charge and hydrophilic groups on liposome clearance in vivo. The effect of negative surface charge and hydrophilic groups on liposome clearance from blood was investigated in mice using liposome-entrapped 67gallium-deferoxamine as a label. The presence of negatively-charged lipids may retard or accelerate liposome clearance. Physicochemical features contributing to optimal retardation of liposome clearance include a hydrophilic carbohydrate moiety and a sterically hindered negatively-charged group. The relevance of the negative charge steric effect is suggested by the finding that phosphatidylinositol phosphate (PIP) and trisialoganglioside (GT1) are less effective than phosphatidylinositol (PI) and monosialoganglioside (GM1), respectively, in retarding liposome clearance. The need for negative charge in addition to the carbohydrate group for optimal effect on retardation of clearance is indicated by the observation that asialoganglioside (AGM1) is less effective than GM1 in this respect. The negative charge effect is observed with liposome bilayers having both low and high temperature phase-transitions. Increasing the molar fraction of negatively-charged lipid (hydrogenated PI derived from soya) from 23 to 41% resulted in a dramatic acceleration of liposome clearance. The clearance-accelerating effect of the high negative charge was specifically directed to the liver with selective reduction of spleen uptake. Increasing liposome size also had an accelerating effect on clearance but in this case it was accompanied by a non-specific concomitant increase of both liver and spleen uptake.


Partite expression of the bovine papillomavirus E1 open reading frame in Escherichia coli. Six recombinants were constructed which expressed portions of the bovine papillomavirus E1 open reading frame as OmpF/E1/beta-galactosidase tribrid fusion proteins in Escherichia coli. Rabbit sera containing E1-specific antibodies were generated against five of these six fusion proteins (which together constitute 74% of the full-length E1 open reading frame). The individual fusion proteins and their cognate antisera will be useful reagents for defining the structure and function of the BPV E1 protein(s).


Cloning of a human collagen-binding protein, and its homology with rat gp46, chick hsp47 and mouse J6 proteins. Several cDNA clones encoding a collagen-binding protein were isolated from human fibroblasts. The cDNA encoded a 417 amino acid protein, containing two potential N-linked oligosaccharide binding sites and a C-terminal RDEL sequence, which has been shown to act as an endoplasmic retention signal in other systems. The derived amino acid sequence of the protein shows close homology with gp46 from rat skeletal myoblasts, J6 protein from mouse F9 embryonal carcinoma cells and hsp47 from chick embryo fibroblasts. It also shows sequence similarity with members of the serpin family.


Formation and ultrastructure of somatic cell hybrids. Taken altogether, the EM evidence we have obtained indicates that the induced (both viral and PEG) and spontaneous (entrance of a splenocyte into a cell) fusion of mammalian somatic cells are associated with alterations in the structure of fusing cells. For example there are alterations in the structure of not only the surfaces of fusing cells but also in the nucleus envelopes and cytoplasmic organelles after PEG treatment. Also, there is long retention of cellular plasma membrane remnants in virally-induced heterokaryons. In short, for each case the alterations were unquestionably specific, in response to the imposed challenge. These specific features not only determine the efficiency and rate of fusion, but also the mode of which the hybrid nucleus is formed. This mode directly determines the fate of the synkaryon and the stability of the so formed hybrid genome. It might be thought that an increase in the inner nuclear envelope observed in some hybrids would counteract the consequences of the disproportion arising between the increase in cell volume and nuclear surface. The finger-like invaginations of the hybrid nuclei nuclear envelope, surrounded by replicatively and transcriptionally active chromatin, appear to be EM demonstrations of such counteracting mechanisms. These invaginations, by augmenting the available inner layer, most likely increase the anchorage sites for chromatin. It is noteworthy that the invaginations occur mainly in multichromosomal hybrids with little chromosome loss. It appears possible that some of the hybrids may contain particular chromosomes from the more differentiated parent cell.


Image analysis of gap junction structures. Isolated gap junction plaques contain hexagonal crystalline arrays of membrane channels called connexons which are a suitable specimen for electron crystallography. Image analysis of gap junction lattices has shown that while there is sufficient lattice order for structural analysis to approximately 25 A, there is enough disorder in both the lattice and the connexon to create a family of related images. This review is focused on how these images can be interpreted in terms of what is known about both the connexon and its constituent protein, connexin.


Continuous treatment with all-trans retinoic acid causes a progressive reduction in plasma drug concentrations: implications for relapse and retinoid "resistance" in patients with acute promyelocytic leukemia. Although all-trans retinoic acid (RA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL), all groups have described clinical relapses despite continued RA treatment. This finding suggests that resistance to the cytodifferentiating effects of the retinoid had been acquired. To investigate potential mechanisms of clinical resistance to RA, we serially evaluated the clinical pharmacology of the drug in APL patients treated with this agent. Leukemic cells from patients relapsing from RA treatment were cultured in the presence of RA and examined for evidence of morphologic maturation. We also studied messenger RNA expression of the newly described gene product of the (15;17) translocation in APL, PML/RA receptor-alpha (PML/RAR-alpha). Serial pharmacokinetic studies showed that continuous daily RA treatment was associated with a marked decrease in plasma drug concentrations at the time of relapse compared with the initial day of therapy. Doubling the RA dose in six patients failed to reinduce response at the time of relapse and also failed to significantly augment plasma RA concentrations. However, leukemic cells obtained at the time of relapse from four patients retained in vitro sensitivity to the differentiating activity of RA (10(-6) mol/L). No change was observed in the pattern of PML/RAR-alpha expression assessed by Northern blot analysis at the time of relapse compared with pretreatment in two patients who were tested. These results indicate that clinical relapse and "resistance" to continuous treatment with all-trans RA in APL is associated with progressive reduction of plasma concentrations, potentially to levels below those that sustain differentiation of leukemic cells in vivo. Long-term success of this treatment will require the development of strategies that circumvent this pharmacologic phenomenon.


Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors. Retroviral infection of bone marrow cells in long-term marrow cultures (LTMCs) offers several theoretical advantages over other methods for gene transfer into hematopoietic stem cells. To investigate the feasibility of this approach in a large animal model system, we subjected LTMCs from nine dogs to multiple infections with retrovirus containing the neomycin phosphotransferase gene (neo) during 21 days of culture. Feeder layers, cocultivation, polycations, and selection were not used. The in vitro gene transfer efficiency was 70% as determined by polymerase chain reaction amplification of neo sequences in colony-forming unit granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC cells were infused into autologous recipients with (four dogs) and without (three dogs) marrow-ablative conditioning. At 3 months posttransplant, up to 10% of marrow cells contained the neo gene. This percentage declined to 0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in individual CFU-GM, burst-forming unit-erythroid (BFU-E), and CFU-Mix progenitors derived from marrow up to 21 months postinfusion and in cultures of peripheral blood-derived T cells up to 19 months postinfusion. There was no difference in the percentage of neo-marked cells present when dogs that received marrow ablative conditioning were compared with dogs receiving no conditioning. Detection of neo-marked marrow cells almost 2 years after autologous transplantation in a large mammalian species shows that retroviral infection of marrow cells in LTMCs is a potentially nontoxic and efficient protocol for gene transfer. Further, our results suggest that marrow conditioning and in vivo selection pressure to retain transplanted cells may not be absolute requirements for the retention of genetically marked cells in vivo.


Changing antigen receptor gene rearrangements in a case of early pre-B cell leukemia: evidence for a tumor progenitor cell with stem cell features and implications for monitoring residual disease. A case of acute lymphoblastic leukemia (ALL) was encountered in which the two clonal gamma T-cell receptor gene (TCR gamma) rearrangements found in bone marrow (BM) samples at relapse both differed from the single clonal TCR gamma rearrangement present in BM obtained at diagnosis 5 years previously. In contrast, two clonal Ig heavy chain gene (IgH) rearrangements present at relapse were identical to those present at diagnosis. Comparison of the DNA sequences of the relapse TCR gamma rearrangements with that of the diagnostic TCR gamma rearrangement indicated that they must have been generated de novo from TCR gamma loci in germline configuration. By polymerase chain reaction using clonotypic N-region oligonucleotide primers (N-PCR), cells bearing the diagnosis or relapse TCR gamma rearrangements were undetectable in the sample from the opposite time point. Two BM samples obtained at different times in clinical remission were both devoid of detectable residual tumor when analyzed by N-PCR, indicating a depth of remission of less than 1 tumor cell per 4 x 10(5) BM mononuclear cells. The tumor cells showed a primitive phenotype: T-cell antigen-negative, CALLA/CD10-negative, CD20-negative, CD19-positive, and positive for the myeloid marker My9. This case, which appears to represent a tumor arising from a progenitor cell with both early B-lineage and certain stem cell features, has implications for monitoring residual ALL and possibly also for treatment of the disease.


Delta-thalassemia due to a mutation in an erythroid-specific binding protein sequence 3' to the delta-globin gene. We have previously described a family of Northern Sardinian descent in which the propositus was affected by thalassemia major resulting from compound heterozygosity for codon 39 nonsense mutation and the beta +IVS II nt 745 mutation and in which all heterozygotes for the beta +IVS II nt 745 mutation had normal hemoglobin (Hb) A2 levels. To define the reasons for normal HbA2 levels in otherwise typical beta-thalassemia heterozygotes, we cloned and sequenced the delta-thalassemia gene in cis to the beta +IVS II nt 745 mutation. The sequence analysis showed a single nucleotide substitution (G----A) at position 69 nts (delta +69) downstream to the polyA addition site. Dot blot analysis with an oligonucleotide probe complementary to the delta +69 mutation detected this mutation in several heterozygotes for the beta +IVS II nt 745 mutation from the proband's family, but failed to show it either in a group of normal individuals of the same origin or in nonrelated heterozygotes for the beta +IVS II nt 745 mutation of the same or different descent from the proband. The delta +69 (G----A) mutation may be responsible for the low delta-globin output from the beta +IVS II nt 745 chromosome or could be a silent polymorphism not affecting the function of the delta-globin gene. The normal G at position 69 is part of a sequence very similar to the core DNA (A/T)GATA(A/G) motif (GATA box) that is a binding site for the GATA-1 protein. Gel-retardation assay has shown that a DNA fragment containing the GATA motif with the G----A at position +69 has increased binding affinity for erythroid-specific DNA binding protein(s) as compared with the wild-type sequence. These findings may suggest that the delta +69 mutation is responsible for the deficient function of the in cis delta-globin gene.


Polyclonal origin of rheumatoid synovial T-lymphocytes. Nineteen T-cell clones from seven patients with RA were obtained by cloning infiltrating lymphocytes from needle synovial biopsies. Southern blot analysis of the T-cell receptor (TCR) beta-chain genes in these clones revealed that there were no T-cell clones with an identical rearrangement of the TCR beta gene. These results do not support the idea that the infiltrating T-lymphocytes in RA are of monoclonal or oligoclonal origin.


Intraductal carcinoma of major salivary gland. The clinicopathologic features of the cases of three patients with intraductal carcinoma of major salivary gland are described. As in the breast, these lesions of salivary ducts appear to represent an in situ or preinvasive phase of the disease. On follow-up, one patient had a local recurrence, and in another patient, the tumor subsequently became invasive. This experience suggests that wide surgical excision (preferably total parotidectomy) may be curative but that resections limited to grossly visible disease will result in local recurrence and/or the development of invasive ductal adenocarcinoma.


Nucleotide sequence of prothrombin gene in abnormal prothrombin-producing hepatocellular carcinoma cell lines. A protein induced by vitamin K absence or antagonist II, PIVKA-II is synthesized in the liver and possesses a structure similar to prothrombin except that ten glutamic acid residues in amino-terminal Gla domain are not completely gamma-carboxylated and are functionally inactive. This protein can be detected in the plasma of patients with hepatocellular carcinoma (HCC) and used as a new tumor marker. To analyze the mechanism of PIVKA-II production in HCC tissue, the prothrombin gene of PIVKA-II-secreting HCC cell lines was sequenced to detect the mutation in the Gla domain and carboxylase recognition site of leader sequence located on exons I and II that may cause the inhibition of carboxylation. Exons I and II and donor and acceptor site of intron I of the prothrombin gene in two HCC cell lines, PLC/PRF/5 and huH-2, were analyzed by polymerase chain reaction (PCR), and the product was sequenced directly. In addition, RNA samples of these cell lines were used for complementary DNA synthesis, followed by PCR and sequencing. The nucleotide sequences of the Gla domain in both HCC cell lines were conserved. One nucleotide change was detected at nt.554 (adenine to guanine), but this did not influence the amino acid sequence. Splicing sites between exons I and II, the leader sequence of the precursor prothrombin, and protease target sites also were conserved as the reported prothrombin gene, and mutations reported for other des-gamma-carboxy coagulation factors were not detected. These results also were confirmed by DNA analysis of seven human fresh-frozen samples (three PIVKA-II-positive HCC samples and four control specimens). The mechanism of PIVKA-II production in HCC is still unclear, but it is not caused by mutation in the prothrombin gene.


Intraductal carcinoma of the pancreas.

Intraductal carcinoma of the pancreas. Four multicentric intraductal papillary carcinomas arising in the main pancreatic duct are presented. Three of the neoplasms showed stromal invasion and metastasized to regional lymph nodes. Three patients had a long history of epigastric pain, confirming the progressive slow growth and less aggressive nature of this clinicopathologic entity. Histologically, all tumors were papillary, and three also showed a pseudocribriform pattern. Individual cells exhibited a range of atypia from mild to overt malignant change. Focal intestinal differentiation was recognized in two tumors. Despite the well-differentiated appearance of these tumors, two patients died within 1 year of surgery. One patient with an entirely intraductal carcinoma is alive and well 3 years after surgical treatment. The fourth patient who had lymph node metastasis is alive 6 months after a Whipple's procedure.


Etoposide, ifosfamide, and cisplatin in extensive small cell lung cancer. From December 1987 through April 1989, 40 patients with extensive-stage small cell carcinoma of the lung were enrolled in a Hoosier Oncology Group (HOG) trial using etoposide, ifosfamide, and cisplatin (VIP). Patients with extensive disease were eligible if they had not received prior chemotherapy, had a Karnofsky performance status of 50 or more, and had adequate renal function (creatinine, less than 1.5 mg/dl) and bone marrow reserve (granulocyte count, greater than or equal to 2500/microliters; platelets, greater than or equal to 125,000/microliters). Doses of therapy were: etoposide 75 mg/m2/day on days 1 to 5, ifosfamide 1.2 g/m2/day on days 1 to 5, and cisplatin 20 mg/m2/day on days 1 to 5. The first 11 patients received a 5-day course; this was repeated every 21 days for four cycles, but therapy was shortened to 4 days when unacceptable toxicity was noticed in these patients. Overall, 14 (37%) had a complete remission (overall response rate, 71.1%) with a median survival of 42 weeks (28 weeks on 5-day regimen and 45 weeks on 4-day regimen). There were five early deaths. Although toxic, VIP produces a high complete remission rate in patients with extensive disease and warrants further evaluation. A prospective randomized trial comparing cisplatin and etoposide to the VIP regimen is underway through HOG.


Comparison of OK-432 and mitomycin C pleurodesis for malignant pleural effusion caused by lung cancer. A randomized trial. A prospective randomized study to compare the effectiveness of pleurodesis by two new sclerosing agents: OK-432 and mitomycin C were conducted in 53 patients with malignant pleural effusion caused by lung cancer. None of the patients received concomitant systemic chemotherapy or radiation therapy during the study. After complete drainage of pleural fluid, the patients were allocated randomly to receive 10 Klinische Einheit units of OK-432 or 8 mg of mitomycin C by intrapleural injection at weekly intervals. The treatment was terminated if the pleural effusion disappeared or the patients had received four consecutive procedures. There were 26 patients who received pleurodesis with OK-432 and 27, with mitomycin C. Patient characteristics in the two treatment groups (age, sex, histologic type, performance status, and prior treatment before pleurodesis) were compatible. These results showed that pleurodesis with OK-432 achieved a higher complete response rate (73%) than that of mitomycin C (41%). The rates of objective treatment response (complete response plus partial response) were comparable in both groups (88% for OK-432 and 67% for mitomycin C). The average number of intrapleural injections needed to achieve complete response was fewer in the OK-432 group (1.9 +/- 0.9) than in mitomycin C group (2.8 +/- 0.9). There was no significant difference in the median survival of the patients who received pleurodesis with OK-432 (5.8 months) or mitomycin C (5.1 months). However, the effusion-free period in the OK-432 group was significantly longer than that in the mitomycin C group (7.0 months versus 1.5 months). Patients who underwent OK-432 pleurodesis had a higher complication rate (80%) than did those in the mitomycin C group (30%). Transient febrile reaction was the most common reaction encountered. The immunologic study in OK-432 group showed an increase in peripheral leukocyte count and decrease in the OKT4/OKT8 ratio. The mitomycin C group had a mild reduction in peripheral blood leukocyte count and no significant change in the OKT4/OKT8 ratio. It was concluded that pleurodesis with OK-432 is an effective alternative treatment for malignant effusion in patients with lung cancer.


Occurrence of human papillomavirus DNA in primary lung neoplasms. The occurrence of human papillomavirus (HPV) DNA in primary lung carcinomas and in squamous metaplasia of the bronchus was studied using in situ hybridization techniques and commercially available biotinylated DNA probes to HPV subtypes 6/11, 16/18, and 31/33/35. The authors found HPV DNA in six of 20 cases of squamous cell carcinoma and one of six cases of large cell undifferentiated carcinoma. There were two cases each of the 6/11 serotypes and the 16/18 serotypes and three cases of the 31/33/35 serotypes. Infected cells of the squamous carcinomas uniformly showed koilocytosis. No case of adenocarcinoma, bronchioloalveolar carcinoma, or small cell carcinoma was positive (of 32 cases). Areas of squamous metaplasia in infected tumors showed similar HPV DNA expression in 15% of cases, especially in those with condylomatous atypia. In 5.8% of random bronchial biopsies of squamous metaplasia, HPV DNA was identified. The relationship of HPV infection to the development of upper and lower respiratory tract carcinomas is discussed.


Characterization and comparison of two newly established Epstein-Barr virus (EBV)-negative and EBV-positive Burkitt's lymphoma cell lines. EBV-negative cell line with a low level of expression of ICAM-1 molecule and EBV-positive cell line with a high level of expression of ICAM-1 molecule. Two human Burkitt's lymphoma cell lines (HBL-4 and HBL-5) were established individually from two patients with small noncleaved cell lymphoma (Burkitt's type). The HBL-4 cell line is Epstein-Barr virus (EBV)-negative, and the HBL-5 cell line is EBV-positive. Cytogenetically, both cell lines had the same chromosomal translocation, t(8;14)(q24;q32) as those observed in the primary malignant cells from individual patients. Morphologic, immunophenotypic, cytogenetic, and molecular studies confirmed that both cell lines were derived from the primary lymphoma cells in vivo. HBL-4 cells lacked CD23(H107), CD11a(LFA-1), and latent membrane protein (LMP) but expressed CD54(ICAM-1) at low levels, whereas HBL-5 cells showed the high level of expression of CD54 and faint expression of LMP but lacked CD11a. In addition, the EBV-positive lymphoblastoid cell line (LCL) expressed CD11a, CD23, CD54, and LMP at high levels. Therefore, an HBL-5 phenotype with expression of CD54 and LMP tends toward an LCL phenotype, and the augmentation of CD54 on the HBL-5 cells in comparison with primary lymphoma cells is likely to be upregulated by LMP, probably resulting from the EBV infection. There was little difference in the BrdUrd uptake in vivo and in vitro, doubling time, tumorigenicity, and dynamics of tumor growth in athymic nude mice between both cell lines. These findings indicate that the potentiality of cell growth and tumorigenicity of these two cell lines are unlikely to be related with EBV.


Treatment of metastatic chemodectoma.

Treatment of metastatic chemodectoma. Six patients were treated for metastatic chemodectoma at Memorial Sloan-Kettering Cancer Center from 1971 through 1988. Four patients' primary tumors arose in the cervical region, and two arose in the retroperitoneum. Four patients received a total of eight different chemotherapeutic regimens, including cisplatin, doxorubicin, cyclophosphamide, and dacarbazine. Metastatic sites treated included bone, liver, lung, and retroperitoneum. No patient had a response to chemotherapy. Four patients received a total of nine courses of radiation therapy for palliation of bone metastases. Pain relief was complete in eight patients and partial in one. One patient was irradiated for a mass in the left psoas muscle, with stabilization of disease for 6 months after treatment. One patient was irradiated for epidural compression at T6, with resolution of neurologic symptoms and 50% clearing of the spinal block on follow-up myelogram. Recurrence or progression of disease in a previously irradiated site occurred in one patient 2 years after treatment. One patient was lost to follow-up 3 months after radiation therapy for epidural compression. The other five patients died of widespread metastatic disease 6 months to 9 years after initial treatment for their metastatic disease.


The use of nuclear morphometry to predict response to therapy in Wilms' tumor. Using nuclear morphometric analysis, a retrospective study was made of 27 patients with Wilms' tumor. The blinded group consisted of 17 patients with Stage I and II favorable-histology Wilms' tumors who did not respond to therapy and ten patients with Stage III and IV favorable-histology Wilms' tumors who did respond. In this complex group of patients, multivariate analysis (with several morphologic descriptors) was used to predict which patients responded to therapy. No single-shape descriptor predicted the response to therapy (P greater than 0.5). However, three shape descriptors: maximum ellipticity (ME), standard error of bending energy (SEBE), and the range of chain code-maximum peak (RCCM), with the multivariate formula of (1131 x SEBE) + (-7 x ME) + (50 x RCCM) + 7.18, separated the two groups (P less than 0.004). This result was statistically significant. Using this multivariate equation and a cutoff value of 0.6 units, this test yielded a sensitivity of 94% and a specificity of 70%. This result, in a complex group of patients, suggests that nuclear morphometry may be useful in the initial assessment of patients with Wilms' tumor and warrants further analysis.


Human herpesvirus-6: tumorigenicity and tumor infiltrating lymphocytes. We previously reported that human herpes-virus-6 (HHV-6) genome (strain GS) and a cloned subfragment (pZVH14) transfected NIH 3T3 cells, induced foci of transformation with a frequency significantly above the background level. The transformed cells produced tumors in nude mice and immunocompetent (Swiss) mice. In the current study, nude mice tumors were passed into Swiss mice and more aggressive tumors (G-2TS and 14-2TS derived from HHV-6 genome and pZVH14 DNA, respectively) were produced. 14-2TS tumors caused lung metastasis upon intravenous injection. In the case of subcutaneously growing aggressive tumors, tumor infiltrating lymphocytes (TIL) were isolated and characterized as T cells but lacked tumor specific killing as monitored by 51Cr-release assays. TIL lost activity at day 52 in a 4 h assay (but not in a 19 h assay) against the autologous tumor, but not a Maloney virus induced tumor (Yac-1). These studies indicate that an established nontumorigenic fibroblast cell line, when transfected with pZVH14 DNA of HHV-6, acquires both tumorigenic and metastatic potential and tumor bearing hosts mount immune response against such tumors.


Pristane induced gene activation.

Pristane induced gene activation. Studies were performed to examine the effects of 2,6,10,14-tetramethyl pentadecane (pristane) versus 12-O-tetradecanoylphorbol 13-acetate (TPA) on the activation of the CAT gene under the regulatory control of viral promoter/enhancer elements transfected into NIH-3T3, CV-1 and COS-7 cells. The results of these studies demonstrated that (1) pristane or TPA induced trans-activation of SV2cat, HIVcat, RSVcat and MMTVcat in cells transfected with each respective plasmid construct, (2) only pristane induced activation of pA10cat and pOSP/11 and (3) neither TPA nor pristane trans-activated pSV0cat. Furthermore, treatment with either pristane or TPA elicited changes in the morphology of each of the cell lines. Collectively these results indicate that pristane is a potent inducer of gene expression and exhibits similar characteristics as the tumor promoter, TPA.


Cigarette smoke-induced DNA damage in cultured human lung cells: role of hydroxyl radicals and endonuclease activation. Cigarette smoke can cause DNA single strand breaks in cultured human lung cells (T. Nakayama et al., Nature, 314 (1985) 462-464) but the mechanisms behind this DNA damage have not been clearly elucidated. In the present study we have investigated the possibility that one of the major constituents in cigarette smoke, hydroquinone, may be important for mediating smoke-induced DNA damage in the human epithelial lung cell line, A 549, and the mechanisms behind this damage. Cells were exposed to cigarette smoke, hydrogen peroxide, or hydroquinone, in the absence and presence of different inhibitors, and the resulting DNA damage was assessed either as DNA single strand break formation or formation of the oxidative DNA adduct, 8-hydroxydeoxyguanosine. It was found that (i) exposure to cigarette smoke, hydrogen peroxide or hydroquinone causes a rapid decrease in the intracellular thiol level and a considerable DNA single strand break formation, (ii) the formation of DNA single strand breaks in cells exposed to cigarette smoke is inhibited by catalase, dimethylthiourea, and o-phenantroline, suggesting that hydroxyl radicals generated from iron-catalyzed hydrogen peroxide dissociation are involved in the DNA damage, (iii) hydroquinone causes considerable DNA strand break formation that is blocked by aurintricarboxylic acid, an inhibitor of endonuclease activation, and by BAPTA, an intracellular calcium chelator, (iv) addition of hydroquinone to a smoke condensate greatly enhances its ability to cause DNA single strand breaks, and (v) smoke, but not hydroquinone, causes formation of 8-hydroxydeoxyguanosine, a DNA damage product induced by the action of hydroxyl radicals on the DNA base, deoxyguanosine. These findings suggest that the ability of cigarette smoke to cause DNA single strand breaks in cultured lung cells is due to mechanisms involving hydroxyl radical attack on DNA and endonuclease activation. They also suggest that hydroquinone is an important contributor to the DNA damaging effect of cigarette smoke on human lung cells.


Inhibition of microsomal glucose 6-phosphatase by unsaturated aliphatic aldehydes and ketones. Aldehydes and ketones with one double bond conjugated to the carbonyl group inhibited the enzyme glucose 6-phosphatase, which is embedded in the microsomal membrane. The Michaelis constant, Km and the maximal rate of reaction, V, were affected in a way dependent on the inhibitor's chain-length: trans-2-pentenal and 1-penten-3-one increased Km linearly with concentration and had almost no effect on V, whereas trans-2-nonenal caused a large increase in V but only a small and non-linear change in Km. The effect of the short-chain aldehydes on the kinetic parameters increased with chain-length, but pentenone increased Km more than did trans-2-heptenal and conjugated dienals did not act as inhibitors. Therefore, sterical effects apparently are of importance. Washing the microsomes after incubation with hexenal or heptenal did not substantially decrease the inhibition, but with nonenal the inhibition was reduced by washing. Inhibition by the SH-group blocking reagent p-hydroxymercuribenzoate was competitive to inhibition by the alkenals. It is concluded that the alpha-beta unsaturated oxo-compounds inhibit glucose 6-phosphatase by binding covalently to an important mercapto group and that perturbation of the enzyme's membrane environment also plays a part in the inhibition.


Embryolethality and induction of 7-ethoxyresorufin O-deethylase in chick embryos by polychlorinated biphenyls and polycyclic aromatic hydrocarbons having Ah receptor affinity. The lethality and 7-ethoxyresorufin O-deethylase (EROD)-inducing potency of some individual polycyclic aromatic hydrocarbons (PAHs) and coplanar polychlorinated biphenyls (PCBs) in chick embryos were measured in order to compare the mechanisms of action of these compounds. In previous studies it was found that coplanar PCBs and certain PAHs have a high embryolethality in the chicken and that they induce embryonic EROD activity. Although the most potent PAHs were almost as embryolethal as the PCBs when injected into hens' eggs 72 h prior to measurement, they were considerably less potent EROD inducers. In the present study, three coplanar PCBs (3,3',4,4'-tetrachlorobiphenyl (TCB), 3,3',4,4',5-pentachlorobiphenyl (PeCB) and 3,3',4,4',5,5'-hexachlorobiphenyl (HCB)) and four of the most toxic PAHs (benzo[a]anthracene (BaA), benzo[k]fluoranthene (BkF), indeno[1,2,3-cd]pyrene (IP) and dibenzo[a, h]-anthracene (DBahA] were administered to chick embryos in different ways, including co-administration. Additive embryolethality was found when BkF and PeCB were co-administered as well as when BaA and DBahA were given simultaneously. The PAHs were more effective as EROD inducers when injected on day 9 (24 h prior to measurement) than when injected on day 7 (72 h prior to measurement). The opposite was found for PeCB and HCB, whereas no difference in potency was noted when comparing TCB injected 24 and 72 h before EROD determination. These substance-related differences were probably due, at least partly, to differences in biotransformation rates. EROD activities found after treatment with high doses of BkF, IP, or DBahA on day 9 were similar to those measured after treatment with PeCB in doses high enough to give maximal induction. Co-administration of high doses of BkF and PeCB did not further increase the activity, indicating that the PAHs and coplanar PCBs induce EROD to a common maximal value. To decrease the influence of metabolization of the PAHs on their EROD-inducing potency, EROD was determined early in development (day 8) and soon after treatment (24 h) in one experiment. In that experiment, the PAHs proved to be only a few times less potent EROD inducers in relation to their embryolethalities compared with the PCBs. The results of the present study, a previously observed similarity in pathology between chick embryos treated with PAHs and embryos treated with coplanar PCBs, and the fact that the most toxic PAHs also are the most avid Ah receptor binders suggest that the coplanar PCBs and the PAHs largely exert their toxicity in chick embryos via an Ah receptor-mediated mechanism. The differences between the compounds in their EROD-inducing potency/embryolethality ratios could probably be explained by their different rates of biotransformation.