Comparison of the effects of carvedilol and nifedipine in patients with essential hypertension and non-insulin-dependent diabetes mellitus. The safety and tolerability of carvedilol, a new antihypertensive agent with the combined pharmacological activities of beta-blockade and vasodilation, and of nifedipine were investigated in patients with essential hypertension and non-insulin-dependent (type II) diabetes mellitus. Twenty patients were openly randomized to receive 25 mg carvedilol once daily (five men and five women; mean age, 63 years) or 10 mg nifedipine t.i.d. (three men and seven women; mean age, 64 years) for a period of 4 weeks. Baseline mean sitting blood pressures were 168/98 and 169/95 mm Hg in the carvedilol and nifedipine groups, respectively. Baseline mean areas under the curve (AUC) of the intravenous glucose tolerance test (IVGTT) for the carvedilol and nifedipine groups were 6,136 +/- 1,195 and 6,287 +/- 1,228 mg/dl/min, respectively. Demographic and efficacy variables were not statistically different between treatment groups. After 4 weeks of therapy, mean sitting blood pressure was significantly (p less than 0.02) reduced to 144/91 mm Hg in the carvedilol group and to 149/87 mm Hg in the nifedipine group. Week 4 IVGTT AUC values of 5,735 +/- 1,464 mg/dl/min in the carvedilol group and 5,988 +/- 993 mg/dl/min in the nifedipine group, representing mean reductions of 6.14% and 3.17%, respectively, were not statistically different from baseline. Both treatments were well tolerated. No patient experienced adverse events in the carvedilol treatment group, whereas two patients in the nifedipine group reported episodes of headache (one patient) and palpitations (one patient); each episode was mild in severity and considered to be related to study medication.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of carvedilol on atrial natriuretic peptide, catecholamines, and hemodynamics in hypertension at rest and during exercise. Possible counterregulatory neurohumoral and hemodynamic responses to carvedilol (a new vasodilating nonselective beta-receptor blocker) were studied in 19 men with essential hypertension (age range, 34-59 years; mean age, 44 years). Intra-arterial pressure, cardiac output (Cardio-green), heart rate, and the vasoactive peptides norepinephrine, epinephrine, and atrial natriuretic peptide (ANP) were measured at rest supine and sitting and during 100-W bicycle exercise before and 2 h after administration of 25 mg carvedilol. The same protocol was followed after 9 months of chronic carvedilol treatment (mean dose, 52 mg/day). Carvedilol induced both acute and chronic reductions (at rest supine, 11%) in mean arterial pressure, due in part to reduction in cardiac output (5%) and in part to reduction in total peripheral resistance (5%). At rest supine, carvedilol induced a reduction in ANP (27%) that could be viewed as a counterregulatory response to decrease in cardiac output, preventing excessive blood pressure reduction. ANP decreased (18%) when the patient sat up from the supine position and increased (67%) during exercise, but no further change was seen after acute or chronic carvedilol treatment. With the patient in the sitting position, norepinephrine was 110% higher than at rest supine; during 100-W exercise, norepinephrine increased 368%. A further increase (38-86% in the three situations, respectively) was seen after the first dose of carvedilol. Epinephrine showed similar but less marked changes. Neither extracellular fluid volume nor plasma volume (isotope dilution techniques) changed significantly during the study, but the acute blood pressure response to carvedilol was directly related to changes in extracellular fluid volume.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of long-term carvedilol therapy on renal function in essential hypertension. We assessed the long-term effects of carvedilol on renal function in 10 patients with mild-to-moderate essential hypertension. After a 2- to 4-week placebo run-in period, all patients received 5 mg carvedilol once daily. If the effect was insufficient, the dosage was successively increased to 10 or 20 mg once daily. The mean +/- SEM duration of treatment was 17.3 +/- 1.0 weeks, and the final mean daily dosage was 13.5 +/- 2.2 mg/day. With treatment, systolic and diastolic blood pressures decreased significantly from 159.7 +/- 1.3 to 140.5 +/- 3.2 mm Hg (p less than 0.001) and from 98.3 +/- 1.0 to 88.2 +/- 2.7 mm Hg (p less than 0.001), respectively. Carvedilol did not cause significant changes in glomerular filtration rate, effective renal plasma flow, blood urea nitrogen, or serum creatinine. Renal vascular resistance decreased significantly from 12.7 +/- 1.4 to 11.2 +/- 1.2 dyne.s.cm-5/1.48 m2 x 10(3) (p less than 0.05). Thus, long-term carvedilol therapy was effective in reducing blood pressure in essential hypertension without causing impairment of renal function.


Functional significance of induction of differentiation in human myeloid leukaemic blasts by 1,25-dihydroxyvitamin D3 and GM-CSF. We studied the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and granulocyte-macrophage colony stimulating factor (GM-CSF) on monocytic differentiation of the U937 leukaemic cell line and blasts from patients with AML. 1,25(OH)2D3 and GM-CSF synergistically increased functional and phenotypic aspects of differentiation in the U937 cell line. In addition, the effective concentration of 1,25(OH)2D3 was reduced by up to 100 times in the presence of GM-CSF. GM-CSF alone had little differentiation-inducing effect on AML blasts. 1,25(OH)2D3 induced CD14 antigen expression in 67% AML blast populations and increased functional activation in 36%. 1,25(OH)2D3 and GM-CSF in combination cooperated to further induce CD14 antigen expression in one third of blast populations, while having no further effect on function. Failure to induce functionally effective levels of FcRII antigen on AML blasts following stimulation with 1,25(OH)2D3 and GM-CSF may account for the lack of functional activation.


Immunocytochemical investigation of normal and chronic lymphocytic leukaemia lymphocytes reveals unexpectedly frequent reactivity with some myelomonocytic associated antibodies. Information about the expression of some myelomonocytic markers in lymphocytes of patients with B-CLL is scarce. We studied the CD13, CD14, CD11c and CD68 surface antigens in 42 controls and in 38 patients with B-CLL to detect their possible reactivity. Eighty-nine percent of B-CLL expressed very strongly the CD14 antigen; on the contrary, the other myelomonocytic antigens tested were very weakly expressed. Forty-one of 42 controls showed a few CD14-positive lymphocytes with a statistical difference between normal and CLL lymphocytes. No statistical difference was recorded either between CD14 expression and Rai's staging system or Binet's stages, nor between CD14 and bone marrow involvement and doubling time or between CD14 and heavy or light chain expression. A minor B lymphocytic subset in humans coexpresses the CD14 and CD5 antigens, it being increasingly speculated that B chronic lymphocyte leukaemias originate precisely from this B CD5- and CD14-positive cells. Just as the CD5 antigen is regarded as an excellent B-CLL marker, it seems to us that a strong expression of the CD14 antigen might have the same diagnostic relevance.


Modulation of leukemic cell growth by tumor necrosis factor: action and expression in myeloid leukemia. Normal and leukemic bone marrow cells were studied in the presence of tumor necrosis factor alpha (TNF) together with granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) in clonogenic assays. Cells of four normal volunteers, three patients with chronic myeloid leukemia, 16 patients with acute non-lymphocytic leukemia (ANLL), and six patients with myelodysplastic disorders were compared. Our results show four patterns of response to TNF in the presence of G-CSF or GM-CSF: (a) increased sensitivity to inhibition by TNF relative to the response of normal bone marrow cells; (b) response indistinguishable from normal bone marrow cells; (c) refractoriness to TNF at all doses; (d) synergistic growth stimulation with both G-CSF and GM-CSF. Leukemic cells of eight additional ANLL patients were incubated in a 3H-thymidine incorporation assay, and three patterns of reactivity to TNF were observed: (a) decreased 3H-thymidine uptake in the presence of TNF; (b) no response to TNF at all doses; and (c) increased 3H-thymidine uptake in response to TNF. Leukemic cells of 26 ANLL patients of various FAB-types were examined for the production of TNF mRNA by Northern blot analysis. TNF mRNA could be detected in cells of eight patients, predominantly in the M5B FAB type. Our data show that the growth response of leukemic cells to TNF is not uniform and was not determined by FAB category.


Human recombinant stem cell factor stimulates in vitro proliferation of acute myeloid leukemia cells and expands the clonogenic cell pool. Stem cell factor (SCF) is a new growth factor acting on early hematopoietic progenitor and stem cells. In our experiments human recombinant SCF stimulated short-term proliferation of accessory cell-depleted acute myeloid leukemia (AML) cells in 13/14 cases, as determined by 3H-thymidine (3H-TdR) incorporation and cell counts. Stimulatory activity was significantly greater than in the presence of GM-CSF and was comparable to that of granulocyte colony-stimulating factor (G-CSF), interleukin 3 (IL-3), and 5637 cell line supernatant (SN). Conversely, the ability of SCF to induce primary colony formation by AML clonogenic cells (CFU-L) was lower than that of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 5637 SN in all but four cases. However, SCF potentiated the stimulatory effect of GM-CSF, G-CSF, and IL-3 on both 3H-TdR incorporation and colony formation. In a 7-day liquid culture SCF enhanced CFU-L recovery in all cases to a significantly greater extent than the other growth factors. A further increment was obtained by combinations of SCF with GM-CSF, G-CSF, or IL-3, and this was significantly more effective than 5637 SN. SCF did not induce leukemic cell differentiation. Human recombinant SCF is therefore highly efficient in stimulating AML cell proliferation and expanding the CFU-L pool. It was not, however, able to support long-term growth of AML cells (beyond 2-7 weeks) in five cases tested.


c-kit gene expression in CD7-positive acute lymphoblastic leukemia: close correlation with expression of myeloid-associated antigen CD13. Expression of human c-kit proto-oncogene and interleukin-7 receptor (IL-7R) in acute lymphoblastic leukemia (ALL) cells expressing CD7 was examined by Northern-blot analysis and reversed transcription polymerase chain reaction (RT-PCR) assay in relation to the phenotypes. Leukemic cells from four out of 12 CD7+ ALL patients, all of which fulfilled the criteria of ALL in the FAB classification, expressed c-kit genes. Surface CD3 (sCD3) was absent in all of these cases, while cytoplasmic CD3 (cCD3) was found in the two sCD3- cases. CD3 epsilon transcripts were detected in one of the sCD3- cCD3- cases. IL-7R genes were transcribed in the three cases with c-kit gene expression. In addition, there was a good correlation between c-kit gene expression and myeloid associated antigen CD13 positivity of the leukemic cells. None of the patients with c-kit gene expression had mediastinal tumor. Our results show that leukemic cells in a proportion of CD7+ ALL express receptors for cytokines that are secreted by bone marrow stromal cells. Ligands for c-kit genes and IL-7 could play an important role for the regulation of proliferation and differentiation of T-cell progenitors in bone marrow.


Development of a colonic release capsule dosage form and the absorption of insulin. Since the colon is relatively low in peptidase activity and drainage into the lymphatics is maximized, a peroral dosage form was developed to deliver insulin to the colon. Microemulsions, used as a vehicle for insulin, were gelled using Cab-O-Sil, and filled into gelatin capsules pretreated with formaldehyde vapor. The capsules were coated with Eudragit NE 30 D, Eudragit S100 and cellulose acetate phthalate polymers of pH-dependent and time-controlled release mechanisms. In vitro dissolution profiles of the capsule coating, using sodium salicylate as the marker, show that dissolution of the capsule begins at 4 h, at pH 5.5, and is completed at 8 h, at pH 7.7, simulating the gastrointestinal transit and pH profile of the dog. An in vivo crossover study in beagle dogs was carried out employing the following treatments: i.v. insulin, p.o. insulin microemulsion and colonic release capsule dosage form without insulin (CRC), were used as controls, a colonic release capsule dosage form with insulin (CRI) and additionally with sodium laurylsulfate (CRIL) or aprotinin (CRIA) as sorption promoter and enzyme inhibitor, respectively. Evaluation was done by measuring the reduction in blood glucose concentration levels. The pharmacological availability (P.A.) is the ratio of the area under the baseline curve (AUC), expressed as percent glucose reduction from baseline vs time, of the p.o. dosage forms to i.v. insulin administration, corrected for body weight and dose size. The P.A. for the p.o. microemulsion, CRC, CRI, CRIL and CRIA were 2.1, 0.4, 5.0, 2.7 and 6.2%, respectively. Insulin release occurred throughout the GI tract, with the exception of the stomach. Tmax occurred at 6.4 h for CRIA; the majority of insulin is taken up after the colonic arrival time is reached in the dog (4-6 h). Duration of the reduction in blood glucose levels occurred for 14 h with the CRIA dosage form.


Relation of neovascularisation to metastasis of non-small-cell lung cancer. The growth of a tumour beyond a certain size requires angiogenesis. We assessed whether intensity of angiogenesis correlates with metastasis of non-small-cell lung cancer by counting microvessels and grading their density within the initial carcinomas in 87 T1N0M0 patients. After radical surgery, metastases developed in 22. Both microvessel count and density grades correlated significantly with metastatic disease as well as tumour size and proliferative activity. The likelihood of metastasis increased as the vessel count increased. On multivariate analysis, the microvessel density count was the only independent predictor of metastasis.


Serum protease inhibitors promote anchorage-independent growth of transformed cells in serum-free medium. The effect of three serum serine protease inhibitors on the serum-free agar growth of an SV40-transformed 3T3 cell line was investigated. Antithrombin III, alpha-2-macroglobulin and alpha-1-antitrypsin were found to potently stimulate colony growth in a semisolid medium because of their anti-proteolytic properties. These results indicate that protease inhibitors can facilitate tumor cell growth in serum-free agar cultures and suggest that the stimulatory effect of serum on the growth of certain transformed cells in agar may at least partially be due to the high levels of protease inhibitors found in serum.


Purification of AMPA type glutamate receptor by a spider toxin. A glutamate receptor was purified from Triton X-100-solubilized bovine cerebellum membranes. The purification was carried out in two steps: affinity chromatography using a spider toxin (Joro spider toxin; JSTX) immobilized on a lysine-agarose column, and a Mono Q anion exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified active fraction showed a single band with Coomassie Blue staining, which migrated with a M(r) = 130,000. The specific [3H]amino-3-hydroxy-5-methyl-isoxazole propionate ([3H]AMPA) binding activity of the affinity-purified fraction was 2095-fold higher than that of the crude soluble fraction. Lineweaver-Burk plot analysis showed a Kd of 12.7 nM [3H]AMPA in the purified fraction. The purified fraction was examined with patch-clamp recording methods in reconstituted liposomes. A glutamate-activated channel was observed and was inhibited with JSTX. The rank order of potency of agonists inducing channel currents was AMPA = glutamate greater than quisqualate much greater than kainate greater than NMDA. Thus, there is strong evidence that the 130 kDa protein is a purified component of the native AMPA type glutamate channel of bovine cerebellum.


Insulin releasing effects of mastoparan and amphiphilic substance P receptor antagonists on RINm5F insulinoma cells. It has been proposed that mastoparan (INLKALAALAKKIL) and other mast cell secretagogues such as substance P (SP) or compound 48/80 act by direct activation of the pertussis toxin (PTX)-sensitive G-proteins in intact cells. Here we have investigated whether or not the antagonists of SP, [D-Trp7,9,10] SP1-11 and [D-Trp7,9,10, N-leu11]SP1-11, can similarly induce exocytosis from RINm5F cells. In intact cells mastoparan and the SP antagonists stimulated insulin release in a dose-dependent manner at concentrations ranging from 10 to 100 microM. The maximal effect on insulin release, of both mastoparan and the SP antagonists was comparable to that obtained with 100 microM forskolin. Pretreatment of the intact cells, for 18 h with PTX or 6 h with cholera toxin, did not change the responses induced by both mastoparan and the SP antagonists. This absence of PTX effect, despite the fact that the three PTX substrates at 41, 40 and 39 kDa were ADP ribosylated after pretreatment suggests intrinsic differences between mast and RINm5F cells. Thus the SP antagonists behave similarly to mastoparan in its ability to induce insulin release in RINm5F cells. However, the higher concentrations required with RINm5F cells compared to that needed for mast cells suggest differences either in G-proteins composition or in the phospholipid composition of the membranes.


Quantitative structure-mutagenic activity relationships of triazino indole derivatives. The mutagenicity of 3-(4'-benzylidenamino)-5H-1,2,3-triazin[5,4-b]-indol-4-one derivatives, new compounds with considerable platelet antiaggregating activity, was assayed with the Ames test using the Salmonella typhimurium strains TA97, TA98, TA100 and TA102. The adaptive least-squares method (ALS method) was used to carry out a quantitative structure-activity relationship (QSAR) analysis. Three equations, based on 10 congeners, were found for strains TA97, TA98 and TA100. The results suggest that lipophilicity of the substituent decreases the mutagenicity of the series.


Insulin and orthovanadate stimulate multiple phosphotyrosine-containing serine kinases. Using the synthetic peptide substrate Kemptide and cytosolic extracts of mouse fibroblasts transfected with a human insulin receptor cDNA construct, we have studied an insulin-sensitive serine kinase activity. This activity is rapidly stimulated by insulin (maximum within 5 min) and also by orthovanadate. During cell extract preparation, para-nitrophenylphosphate and phosphotyrosine are able to preserve the enzyme activity, while phosphothreonine and phosphoserine fail to do so. Using antiphosphotyrosine antibodies, specific immunoprecipitation of this insulin- and orthovanadate-sensitive serine kinase was obtained. We then analysed by gel filtration chromatography eluates containing tyrosine-phosphorylated proteins obtained from unstimulated, insulin- and vanadate-treated cells. We found that several activities, with molecular weights estimated to be 30 kDa and smaller, are stimulated by both, insulin and orthovanadate. As a whole, our data indicate that insulin and orthovanadate enhance the cytosolic content in at least 2 or 3 phosphotyrosine-containing serine kinase activities.


DNA strand breakage by hydroxyphenyl radicals generated from mutagenic diazoquinone compounds. The mutagenic diazoquinone compounds p-diazoquinone (p-DQ), o-diazoquinone (o-DQ) and 3-diazo-N-nitrosobamethan (D-BM) cleaved the phosphodiester bond of lambda DNA, phi X174 RFI DNA and M13mp8ss DNA. p-DQ also cleaved the phosphodiester bond of bis(p-nitrophenyl)phosphate. The breakage of the phosphodiester bond was inhibited by the antioxidant butyl hydroxyanisole (BHA), ethanol, the spin trapping agent DMPO, cysteine and 2-mercaptoethanol. While incubation of p-DQ and o-DQ alone gave p-hydroquinone and catechol, respectively, incubation of these compounds in the presence of BHA and ethanol gave phenol in large yields. Incubation of p-DQ and o-DQ with the spin trapping agents DMPO and PBN gave spin adducts assignable as p- and o-hydroxyphenyl adducts, respectively. The breakage of the phosphodiester bond of DNA by the diazoquinone compounds is suggested to be due to the hydroxyphenyl radicals generated during incubation.


Cytochrome P450 induction and mutagenicity of 2-aminoanthracene (2AA) in rat liver and gut. The aim of our study was to establish a relationship between the ability of rat liver and gut to activate 2-aminoanthracene (2AA) into mutagens and their P450 enzyme composition. Rats were orally pretreated with beta-naphthoflavone (beta NF), phenobarbital (PB), dexamethasone (DEX) or acetone (AT). Mutagenic activation of 2AA was detected in the Ames test. P450IA1, IA2, IIB1/B2 and IIE1 were immunochemically quantified by Western blots. All the results were compared to those obtained in untreated rats. In all tissues, beta NF treatment considerably increased the mutagenicity of 2AA. PB treatment significantly reduced the mutagenicity of 2AA in the liver but not in the intestine. By contrast, AT treatment significantly decreased the number of revertants in the duodenum but not in the liver whereas DEX treatment significantly decreased the number of revertants in both tissues. 2AA appears to be metabolized by various P450s in both organs. In the liver, reactive metabolites may be produced after metabolism by the P450IA subfamily. The other P450 enzyme seems to play a part in the metabolism of 2AA leading to formation of either mutagenic or non-mutagenic metabolites.


Cytogenetic mechanisms in the selective toxicity of cyclophosphamide analogs and metabolites towards avian embryonic B lymphocytes in vivo. Cyclophosphamide (CP) is selectively toxic to avian and mammalian B lymphocytes, but the mechanisms of action are incompletely understood. We used a structure-activity approach to determine the cytogenetic mechanisms underlying the selective lymphoid toxicity in chicken embryos at 18-19 days of incubation. Two doses of 5-bromo-2'-deoxyuridine (BrdU; 3 mg/200 microliters x 2) were pipetted onto the inner shell membrane to label lymphocyte DNA over 20 h. A single dose of the CP analogs or metabolites was given 1 h after the initial BrdU application. After a terminal 3-h exposure to demecolcine to block cells in metaphase, the embryos were sacrificed at hour 20, and their bursae and thymi were removed for cytogenetic processing. Microscope slide preparations of metaphases were stained by the fluorescence-plus-Giemsa technique to differentiate the sister chromatids for an assessment of sister-chromatid exchange (SCE) induction and cell cycle progression based on replication cycle-specific staining patterns. Isophosphamide (1.25-40 mg/kg), phosphoramide mustard (0.7-45.7 mg/kg), and 4-methylcyclophosphamide (1.3-42.1 mg/kg) selectively damaged B cells as shown by dose-related reductions in the mitotic activity, inhibition of cell cycle kinetics, and approximately 9-15-fold increases in the SCE frequency above control. B cells were up to 392 times more susceptible to the toxicity of these three bifunctional alkylating agents compared to T cells based on reductions in the mitotic activity. At most of the drug doses tested, the T-cell mitotic index was not depressed significantly and was usually higher than the control value by as much as 50-60%. Importantly, monochloroethylcyclophosphamide (70-245 mg/kg; monofunctional alkylation) did not induce differential lymphoid toxicity, although a 9-fold increase in the SCE frequency of B cells was observed at the highest dose. Didechlorocyclophosphamide (181-422 mg/kg; acrolein generation only) was a weak SCE inducer (approximately 1.8-fold increase) and was not selectively toxic to B cells. Our data show that selective toxicity to B lymphocytes is strongly associated with bifunctional alkylation via the chloroethyl groups rather than with monofunctional alkylation and acrolein-mediated damage. In addition, the results with phosphoramide mustard and 4-methylcyclophosphamide emphasize that aldehyde dehydrogenase activity is not the primary determinant in the relative sparing of T lymphocytes in vivo.


Sensitivity of different bacterial assays in detecting mutagens in urine of humans exposed to polycyclic aromatic hydrocarbons. The urine mutagenicity and excretion of 1-hydroxypyrene (1-OH PYR) in non-smoking psoriatic patients treated topically with coal-tar-based ointments were analysed in order to find the most appropriate procedure for monitoring occupational PAH exposure. The bacterial mutagenicity assays used were the plate incorporation, macro-scale fluctuation and microsuspension tests, all on Salmonella typhimurium strain TA98 in the presence of S9 mix and beta-glucuronidase. The sensitivities of the three assays in detecting mutagenic urinary PAH metabolites were compared. The efficiencies of XAD-2 and C18 resins for concentrating PAH urinary mutagens were evaluated in the microsuspension assay. The plate and fluctuation tests on XAD-2 urine extracts were shown to be insufficiently sensitive to detect low urinary levels of mutagens, being positive on urine samples with very high PAH metabolite content, estimated as more than 30 micrograms/g of creatinine of 1-OH PYR. The microsuspension assay on XAD-2 or, even better, on C18 urine extracts was very sensitive in detecting up to 5 micrograms/g of creatinine of 1-OH PYR. It therefore seems to be applicable to the biological monitoring of most occupational low exposures to coal tar.


DNA damage induced by cigarette smoke condensate in vitro as assayed by 32P-postlabeling. Comparison with cigarette smoke-associated DNA adduct profiles in vivo. Cigarette smoke induces a multitude of bulky/aromatic DNA adducts in vivo as revealed by 32P-postlabeling assay. The formation of such adducts is thought to involve metabolic activation of aromatic chemicals especially polycyclic aromatic hydrocarbons (PAHs) present in tumor-initiating cigarette tar fractions, via cytochrome P450-associated monooxygenases. Because radicals are present in both the gas and particulate (tar) phase of cigarette smoke and in aqueous extracts of cigarette smoke condensate (CSC), we addressed the question as to whether cytochrome P450-independent, possibly free radical-mediated reactions may contribute, also, to formation of cigarette smoke-associated bulky DNA adducts. Rat-lung DNA was incubated with aqueous extracts of CSC in the absence of microsomes under various conditions and analyzed by 32P-postlabeling. Radioactively labeled bulky reaction products were found to accumulate in a time- and CSC concentration-dependent manner. The resulting chromatographic profiles resembled cigarette smoke-associated DNA-adduct patterns observed in vivo. Pretreatment of aqueous CSC extract with radical scavengers/reducing agents (ascorbic acid, glutathione) diminished adduct formation in a concentration-dependent manner. Adduct formation in vitro may involve oxygen-free radicals, which are known to be present in aqueous CSC extracts and could (i) attack DNA directly to produce bulky adducts, (ii) induce radical sites on DNA covalently binding CSC components, or (iii) convert CSC components to DNA-reactive electrophiles. In addition, DNA may react with direct-acting mutagens in CSC. Adduct fractions derived from in vitro and in vivo experiments showed similar chromatographic behavior, suggesting that metabolic activation as well as processes not involving metabolism lead to formation of smoking-induced bulky DNA adducts in vivo.


A Drosophila simulans mutant strain sensitive to benzo[a]pyrene and 2-acetylaminofluorene. We have identified a Drosophila simulans mutant, 364 yu, that is sensitive to the toxic effects of the procarcinogens B(a)P and 2-AAF. Heterozygotes obtained by crossing it to the wild resistant Turku strain (female 364 yu x male Turku) were more sensitive than heterozygotes obtained from the reciprocal cross (female Turku x male 364 yu) to both the toxic and the mutagenic effects of B(a)P in Drosophila tests that measured lethality and the induction of somatic mosaicism, respectively. The non-carcinogens pyrene, B(e)P and 4-AAF were only weakly toxic and non-mutagenic. In the Ames test B(a)P activation with S15 fractions prepared from the homogenates of Drosophila larvae and imagoes of the 364 yu strain, as well as of the more resistant D. melanogaster y ++/+ w sn3 heterozygotes, did not significantly increase the number of S. typhimurium TA100 revertants even following pretreatment with inducers of microsomal monooxygenases (B(a)P, PCB, PB). As for 2-AAF, a certain increase was observed following only PB, but not B(a)P pretreatment. Possible mechanisms of B(a)P and 2-AAF sensitivity of the 364 yu strain, and perspectives on using it for monitoring genotoxic environmental pollutants, are discussed.


Mutagenic activation of benzo[a]pyrene by human red blood cells. The induction of sister-chromatid exchanges (SCEs) and micronuclei (MN) was used as an endpoint to evaluate the cytogenetic effects of benzo[a]pyrene (B(a)P) activated by human red blood cells and S9 mix. Human erythrocytes can metabolically activate B(a)P. It was shown that both human erythrocytes and S9 mix activate B(a)P and that the resulting excess SCE and MN depend in a linear manner on the B(a)P dose. HPLC analysis suggested that quinone derivatives formed by the red blood cells are responsible for the cytogenetic abnormalities observed.


Induction of chromosomal damage by restriction endonuclease in CHO cells porated with streptolysin O. A procedure is described for the poration of living CHO cells with the bacterial cytotoxin streptolysin O (SLO) which allows the introduction into cells of the restriction endonuclease Pvu II to mimic and model the effects of ionising radiation in causing chromosomal damage. The dependence of this clastogenic effect of Pvu II on SLO concentration was measured by assaying the formation of micronuclei in cytokinesis-blocked binucleate cells. The optimum concentration was found to be 0.045 U/ml. Using the micronucleus assay, the time-course of expression of chromosome damage was investigated and found to show a biphasic kinetic with time. Using a sampling time of 30 h, a dose-effect curve for micronucleus induction by Pvu II was generated. Using this optimized SLO treatment protocol, the frequency of metaphase chromosome damage was subsequently investigated and found to be also linearly related to Pvu II concentration and total aberrations were approximately double the frequency of micronuclei. The induction and repair kinetics of DNA double-strand breaks were investigated in CHO cells treated with SLO and Pvu II using the neutral filter elution technique at pH 9.6. The data presented show that SLO can be used as an alternative method for porating cells to allow the introduction of restriction endonucleases into cells.


Photo-enhancement of the mutagenicity of 9-anilinoacridine derivatives related to the antitumour agent amsacrine. The frameshift mutagenicity of the DNA intercalating drug proflavine is known to be enhanced by photoirradiation of bacterial cultures. To determine whether this phenomenon was also present in acridine-derived antitumour drugs, cultures of Salmonella typhimurium were exposed to the antileukaemia agent amsacrine and the experimental agent N-[2-(dimethylamino)ethyl]acridine-4-carboxamide dihydrochloride (acridine carboxamide) in the presence or absence of visible light. A small increase in mutagenicity was observed with amsacrine but not with acridine carboxamide. A series of analogues of amsacrine were then tested, and a striking relationship was found between the minimum drug concentration for mutagenicity and DNA binding affinity. In each case, photoirradiation was associated with a small increase in mutagenicity. Each of the compounds showing the photo-enhancement effect was capable of reversible one-electron oxidation. It is suggested that this oxidation occurs in bacteria, and that the DNA binding constant of the resulting acridine radical species will increase because of the extra positive charge. This increased DNA binding would be sufficient to explain the photo-enhancement of mutagenicity of these drugs.


Cloning properties of T lymphocyte subpopulations after treatment with 8-methoxypsoralen and UVA irradiation. The cloning rate of PHA-stimulated T lymphocytes after treatment with 8-methoxypsoralen plus UVA irradiation described by Wunder and Reischmann (1983) gives a linear dose-effect relationship at low dosages. However, with increasing doses a flattening of the negative gradient occurs. This relationship deviates from the classical exponential curve which can be observed when fibroblasts are treated with mutagens and which is explainable by a 'recovery plateau' at lower dosages. In this study we show that some subpopulations of T lymphocytes, in particular the T-helper and T-suppressor cells, influence the overall dose-effect relationship. These isolated subpopulations exhibit varying sensitivities in comparison with their depleted cell populations. It may be assumed that heterogeneous cell populations exist within each isolated subpopulation which may be separated into further subclasses according to their specific sensitivity.


The mutagenic effects of low level sub-acute inhalation exposure to benzene in CD-1 mice. Benzene is a widely used chemical and common environmental contaminant. It is carcinogenic in man and animals and is genotoxic in mice, rats, and occupationally exposed humans at doses above one part per million. In order to evaluate the genotoxic effects of prolonged exposures to very low concentrations of benzene, we exposed CD-1 mice to benzene by inhalation for 22 h per day, seven days per week for six weeks at 40, 100 and 1000 parts per billion (ppb). Additional groups were exposed to purified air or were housed in standard plastic cages. The effects of in vivo exposure to benzene were evaluated by using an autoradiographic assay to determine the frequency of mutants which represent mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in spleen lymphocytes. At the end of the six weeks exposure period lymphocytes were recovered from the spleens of the mice and cryopreserved prior to assay. Mutant cells were selected on the basis of their ability to incorporate tritiated thymidine in the presence of 6-thioguanine. The weighted mean variant (mutant) frequencies (Vf) of female mice (three per group) were 7.2 x 10(-6) at 0 ppb; 29.2 x 10(-6) at 40 ppb; 62.5 x 10(-6) at 100 ppb and 25.0 x 10(-6) at 1000 ppb. The Vf of unexposed mice housed in standard cages was 13.2 x 10(-6). In male mice the same pattern of response was observed, but the increases in Vf in response to benzene were not as great. In both sexes of mice, the increases at 40 and 100 ppb were significantly greater than at 0 ppb (P less than 0.05). The increase in Vf with exposure to 100 ppb and the decline at 1000 ppb parallel the results observed for chromosome damage in spleen lymphocytes from the same animals (Au et al., Mutation Res., 260 (1991) 219-224). These results indicate that sub-chronic exposure to benzene at levels below the current Occupational Safety and Health Administration Permitted Exposure Limit may induce gene mutations in lymphocytes in mice.


Analysis of phage M13mp2 mutants produced from transfection of phage DNA having N4-aminocytosines at defined sequence positions. N4-Aminocytidine is mutagenic in various organisms. In the cell, this cytidine analog is metabolized into N4-aminodeoxycytidine 5'-triphosphate, which will then be incorporated into DNA and mutation will result during the replication of the DNA. To prove that the N4-aminocytosine residue in DNA is indeed the site of mutagenesis, we prepared a series of phage M13mp2 DNA samples that bear N4-aminocytosine residues at a few defined positions in the lacZ alpha region, by carrying out in vitro limited extension of primed phage DNA. We then transfected the DNAs to Escherichia coli and examined the progeny phages for the forward mutations. The M13mp2 DNAs bearing N4-aminocytosines produced mutant phages at high frequencies. Furthermore, DNA sequencing of the resulting mutants demonstrated that both AT-to-GC and GC-to-AT mutations took place at those positions where N4-aminocytosine residues were originally present.


Substitution of p- and o-hydroxyphenyl radicals at the 8 position of purine nucleosides by reaction with mutagenic p- and o-diazoquinones. Incubation of 2'-deoxyadenosine (dAdo), 2'-deoxyguanosine (dGuo), adenosine, guanosine (Guo), thymidine, deoxycytidine with p- and o-diazoquinones, mutagens produced by the reaction of phenol and nitrite, at pH 7 and 37 degrees C resulted in a decrease of each nucleoside depending upon the concentration of the diazoquinones. pD-dAdo, pD-dGuo and pD-Guo were isolated from the reaction mixtures of dAdo, dGuo and Guo, respectively, with p-diazoquinone at pH 9.5, and oD-dGuo was from the mixture of dGuo and o-diazoquinone at pH 9.5. The products were identified as 8-(p-hydroxyphenyl)- and 8-(o-hydroxyphenyl)-purine nucleosides by 1H- and 13C-nuclear magnetic resonance spectra, secondary ion mass spectrum, ultraviolet absorption spectrum and elemental analysis. p- and o-Diazoquinones may be converted into p- and o-hydroxyphenyl radicals, respectively, which in turn attack the 8 position of the purine nucleosides. The mutagenicity of these diazoquinones may be partly due to the radical reactions.


Diminished genotoxicity of mitomycin C and farmorubicin included in polybutylcyanoacrylate nanoparticles. The genotoxic effects of mitomycin C (MMC) and farmorubicin (FR) in a free form and included in polybutylcyanoacrylate nanoparticles (PBCN) were studied employing the Salmonella/microsome mutagenicity assay and the micronucleus test in mouse bone marrow as well as in mouse fetal liver. The data obtained clearly indicated that MMC (0.25-2.00 micrograms/plate) was a strong mutagen in S. typhimurium TA102, while the same concentrations of this compound in PBCN were ineffective in inducing his+ revertant mutations in bacterial cells. A similar total suppression of mutagenic activity of FR (1.0-20.0 micrograms/plate) was registered in S. typhimurium TA98 when the drug was included in PBCN. Furthermore, the incorporation of MMC (2.0 or 4.0 mg/kg, i.p.) into PBCN strongly diminished or even abolished its clastogenic activity in the bone marrow of virgin and pregnant mice as well as in mouse fetal liver, respectively. In addition, a lack of genotoxic effect of PBCN only was also established. The toxic activity of MMC in mouse bone marrow was significantly reduced or completely abolished after its inclusion in PBCN. A conclusion might be drawn that the genotoxic activity of some antitumor drugs might be markedly diminished or even abolished after their incorporation in PBCN.


Photoreactivation implicates cyclobutane dimers as the major promutagenic UVB lesions in yeast. Previously we compared the mutational specificities of polychromatic UVB (285-320 nm) and UVC (254 nm) light in the SUP4-o gene of the yeast Saccharomyces cerevisiae. Striking similarities in the types and distributions of induced SUP4-o mutations were consistent with roles for cyclobutane dimers and pyrimidine(6-4)pyrimidone photoproducts in mutation induction by UVB. To assess the relative importance of cyclobutane dimers, we have now examined the effect of photoreactivation (PR), which specifically reverses these lesions, on UVB and UVC induction of SUP4-o mutations. PR reduced the frequencies of both UVB and UVC mutagenesis by approximately 75%. Collections of 138 and 158 SUP4-o mutants induced by treatment with UVB plus PR or UVC plus PR, respectively, were characterized by DNA sequencing and the results were compared to those for 208 UVB and 211 UVC-induced mutants analyzed earlier. PR decreased the frequency of UVB-induced G.C----A.T transitions by 85%, diminished the substitution frequencies at individual sites by 64% on average, and reduced the mutation frequencies at the five UVB hotspots by 87%. A more detailed examination revealed that the transition frequencies at the 3' base of 5'-TC-3' and 5'-CC-3' sequences were decreased by 90% and 72%, respectively. Finally, PR appeared to occur to the same extent on both the transcribed and non-transcribed strands of SUP4-o. Similar results were obtained for PR following UVC irradiation. Our findings indicate that cyclobutane dimers are responsible for the majority of UVB mutagenesis in yeast.


Nondisjunction induced by ethanol in Drosophila melanogaster females. The effect of ethanol on chromosomal segregation was investigated in Drosophila melanogaster females homozygous for a structurally normal X chromosome marked with the recessive mutation yellow (y/y). For chronic treatments the females were kept from eclosion in food supplemented with 10% or 15% (v/v) ethanol, mated 24 or 48 h later to wild-type males and brooded in freshly prepared ethanol food. For the acute treatments 24- or 48-h-old females were exposed for 60 min to a 75% (v/v) ethanol solution by means of soaked tissue paper placed at the bottom of regular culture vials and brooded daily after mating. The results obtained show that: (1) both treatments significantly increased the frequency of X-chromosome nondisjunction; (2) after acute treatment this effect declined in later broods; (3) the yield of malformed flies in the progeny of acutely treated females was significantly higher than control values and also declined in later broods; (4) ovary analysis showed that chronic ethanol treatments caused a cessation of egg production. The induction pattern of nondisjunction and malformed flies is interpreted as giving support to the assumption that these effects may result from a direct action of ethanol. Ethanol toxicity was assessed by exposing females of different ages to a 50% or a 75% (v/v) solution for 60 min and counting the surviving flies 24 h later. The surviving fraction decreased steeply from 1-day-old (100%) to 5-day-old females (1.8%). It is suggested that toxicity may have been due to the action of a metabolite of ethanol, probably acetaldehyde.


Unscheduled DNA synthesis in human hair follicles after in vitro exposure to 11 chemicals: comparison with unscheduled DNA synthesis in rat hepatocytes. A new method is described to investigate unscheduled DNA synthesis (UDS) in human tissue after exposure in vitro: the human hair follicle. A histological technique was applied to assess cytotoxicity and UDS in the same hair follicle cells. UDS induction was examined for 11 chemicals and the results were compared with literature findings for UDS in rat hepatocytes. Most chemicals inducing UDS in rat hepatocytes raised DNA repair at comparable concentrations in the hair follicle. However, 1 of 9 chemicals that gave a positive response in the rat hepatocyte UDS test, 2-acetylaminofluorene, failed to induce DNA repair in the hair follicle. Metabolizing potential of hair follicle cells was shown in experiments with indirectly acting compounds, i.e., benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene and dimethylnitrosamine. The results support the conclusion that the test in its present state is valuable as a screening assay for the detection of unscheduled DNA synthesis. Moreover, the use of human tissues may result in a better extrapolation to man.


Chromosomal aberration analysis in peripheral lymphocytes of radiation workers. Chromosomal aberration analyses were performed in two groups of radiation workers and in a group of healthy controls. Although the level of exposure was below the accepted annual limit of 50 mSv, the yields of chromosome fragments and of total aberrations were significantly higher in the radiation workers than in the controls. However, the frequencies of dicentric and ring chromosomes in the radiation workers were not significantly different from those in the controls.


Mutagenicity of 2-methylpropene (isobutene) and its epoxide in a modified Salmonella assay for volatile compounds. The mutagenic properties of 2-methylpropene (MP) and 2-methyl-1,2- epoxypropane (MEP) were investigated in the Salmonella assay. A simple exposure system, consisting of gastight tissue culture flasks, was used. This method has the advantage that the volatile test chemical is present during the entire incubation period and that several concentrations of the investigated compound can be tested on a single day. MP is not mutagenic in strains TA100, TA102 and TA1535, and in the latter strain not even in the presence of metabolizing S9 mix. MEP is mutagenic in all the strains tested, as demonstrated by a clear dose-response relationship. Strain TA1535 seems to be most sensitive to MEP compared with the other bacterial strains studied. For this strain, the mutagenic activity of MEP decreased significantly in the presence of S9 mix, compatible with the epoxide being inactivated by epoxide hydrolase and by glutathione S-transferase, as reported previously. From the present study it can be concluded that the parent compound MP is not mutagenic, but that its primary metabolite MEP is a mutagenic substance. However, very high concentrations are necessary to induce a mutagenic effect and the epoxide is efficiently detoxified by different liver enzymes.


Skin cytogenetic assay for the detection of clastogens-carcinogens topically administered to mice. A method for assessing the effect of clastogens on mouse skin epidermal cells was devised and applied. Toxic and mutagenic responses in epidermal cells were tested using two known mutagens and carcinogens, urethane (URE) and 7,12-dimethylbenz[a]anthracene (DMBA). Cell generation time, sister-chromatid exchanges (SCE) and chromosomal aberrations (CA) after topical and intraperitoneal (i.p.) treatment were measured in epidermal and bone marrow cells. After topical administration both tissues responded similarly, whereas after i.p. treatment skin cells were less responsive than bone marrow cells. However, the results indicate the validity of this new cytogenetic approach for the assessment of the genotoxicity of compounds applied directly to skin.


Use of a T-lymphocyte clonal assay for determining HPRT mutant frequencies in individual rats. Conditions for detection and isolation of HPRT- mutants in cloned rat T-lymphocytes from individual adult Lewis rats were determined. Similar to cloning of human T-cells, best results were obtained with lectin (PHA)-primed T-lymphocytes of rats. High cloning efficiencies, occasionally exceeding 50%, could be obtained when the target cells employed were isolated from cervical lymph nodes. Feeder cells used were splenocytes, irradiated with 40 Gy of X-rays after priming with Con A. Human interleukin-2, present in LAK supernatant, proved to be capable of inducing proliferative activity of rat T-lymphocytes and could replace conditioned medium from primed rat splenocytes. Under the conditions described in this paper, the frequency of mutants in the HPRT gene of T-lymphocytes in Lewis rats was about 80% lower than that found in human T-lymphocytes from adults. The inverse relationship between mutant frequency and cloning efficiency, clearly demonstrated for human data, could not be established for rats. Treatment of rats with N-ethyl-N-nitrosourea, a potent alkylating agent, resulted in a time- and dose-dependent induction of HPRT- mutants, demonstrating the usefulness of this system to study in vivo mutagenesis.


Assessment of radiation-induced DNA damage in human blood lymphocytes using the single-cell gel electrophoresis technique. The ability of the alkaline single-cell gel (SCG) electrophoresis technique to detect single-strand breaks and alkali-labile DNA damage in human cells induced by low doses of radiation was evaluated. Peripheral blood lymphocytes were irradiated with gamma-rays from a 137Cs source at doses from 0.01 to 1 Gy and exposed to alkali (pH greater than 13) for 20, 40 or 60 min and then electrophoresed at 25 V and 300 mA for either 20 or 40 min. The extent of DNA damage that was expressed and detected as DNA migration depended directly on the dose of radiation, the duration of exposure to alkali and the length of electrophoresis. At all experimental conditions tested, it was possible to detect a significant increase in DNA damage induced by a radiation dose as low as 0.05 Gy. Based on an analysis of the ratio of the range to the standard deviation for each radiation dose and experimental condition, the distribution of damage among cells for all doses was neither excessively homogeneous nor heterogeneous. Furthermore, the distribution was independent of radiation treatment. The SCG technique is rapid and sensitive, and useful for investigations concerned with effects of low doses of radiation.


Quantitative integration of the Salmonella microsuspension assay with supercritical fluid extraction of model airborne vapor-phase mutagens. Vapor-phase mutagens are potentially a major class of toxic contaminants in ambient and indoor air. These compounds are not routinely analyzed due to a lack of an established integrated methodology to quantitatively trap, extract and test the compounds in a bioassay. In a previous report, we emphasized the trapping of volatile and semi-volatile mutagens and the extraction of these compounds using supercritical carbon dioxide (CO2). In the present study, we discuss the use of a bioassay for the quantitation of the model mutagens, ethylene dibromide(EDB) and 4-nitrobiphenyl (4-NB), trapped from an airstream. The compounds EDB and 4-NB were released into a controlled airstream, trapped on XAD-4 adsorbent, and were extracted using supercritical CO2. The extract was tested in a microsuspension modification of the Ames Salmonella/microsome test adapted for volatile compounds. Linear dose-response relationships were obtained for supercritical CO2-extracted EDB using tester strain TA100 (+/- S9) and for 4-NB using tester strains TA98 and TA100 (-S9). Standard dose-response curves with known amounts of the compounds were also determined for comparison with measured amounts of the model compounds collected in an airstream. The gas chromatographic (GC)- and bioassay-determined quantities of EDB and 4-NB were highly correlated, accurate and precise. For example, bioassay-determined EDB concentrations were within 10% of the GC-determined concentrations. Our results demonstrate that the integrated methodology for vapor-phase mutagens developed in this study would be useful for quantitative analysis of these and related airborne vapor-phase mutagenic compounds.


Quantitative correlation of mutagenic and carcinogenic potencies for heterocyclic amines from cooked foods and additional aromatic amines. Aromatic amines have long been recognized as animal and human carcinogens. Recently heterocyclic aromatic amines (thermic amines) have been found in small amounts in cooked foods, primarily meats, and have proven to be potent mutagens and rodent carcinogens. Availability of quantitative databases for mutagenic potency in Salmonella and for carcinogenic potency in rodents has made possible a study of ten heterocyclic thermic amines and 24 aromatic amines. Potencies on mutagenic and carcinogenic scales were significantly correlated. By multiple linear regression analysis and multivariate analysis of variance, two descriptive structural factors were found to modulate the two modes of biological response. These factors were number of rings and methyl substitution at carbon atoms. The quantitative correlation between mutagenic and carcinogenic potencies and the modulating structural factors suggest a significant similarity of molecular mechanisms and support the utility of the short-term bacterial assay in evaluating hazard levels.


Enhanced cytogenetic detection of previous in vivo exposure to mutagens in human lymphocytes after treatment with inhibitors of DNA synthesis and DNA repair in vitro. To increase the sensitivity of cytogenetic surveillance of exposure to mutagens in the peripheral lymphocyte assay, structural chromosome aberrations (CA) were studied after inhibition of DNA synthesis and DNA repair with hydroxyurea and caffeine in culture 3 h prior to harvesting. CA and sister-chromatid exchanges (SCE) from conventional cultures from the same subjects were used for comparison. Smoking was used as exposure parameter. Thirty-two smokers and 35 nonsmokers were studied. In the inhibited cultures a significantly higher number of aberrations was found in lymphocytes from smokers than nonsmokers: chromatid breaks (20.4 vs. 11.8, p = 0.0002), chromosome breaks (4.5 vs. 1.7, p = 0.0003), and the number of cells with aberrations (18.9 vs. 12.4, p = 0.0001), when 50 cells per subject were analyzed. In conventional cultures no increase in gaps, chromatid and chromosome breaks or number of cells with aberrations was found in smokers when 100 cells from each subject were studied. Smokers showed an increased number of SCE (6.8 vs. nonsmokers 5.9, p = 0.02). A significant positive linear correlation (r = 0.39, p = 0.01) was seen between SCE and the number of cells with chromatid breaks from inhibited cultures. The present results indicate that adding hydroxyurea and caffeine to lymphocyte cultures for the last 3 h prior to harvesting may enhance the detection of cytogenetic damage from previous in vivo exposure to mutagens.


Repair of DNA damage induced by oxygen radicals in human non-proliferating and proliferating lymphocytes. Repair of DNA lesions induced by oxygen radicals, generated by xanthine/xanthine oxidase (X/XO), was studied in human peripheral blood lymphocytes and in PHA-stimulated proliferating lymphocytes from 4 healthy subjects. The lesions included DNA-strand breaks (SSB) and other lesions that are converted to SSB under alkaline conditions. The frequencies of SSB were estimated by fluorometric analysis of DNA unwinding. Maximum production of SSB occurred within 10 min of incubation with X/XO at 22 degrees C; with 0.5 mM or higher concentrations of xanthine; and with 0.1-0.5 units/ml of xanthine oxidase. Proliferating lymphocytes repaired X/XO-induced SSB about 4 times more rapidly than lymphocytes. Lymphocytes repaired X/XO-induced SSB more slowly than SSB caused by gamma-radiation. These findings are consistent with the evidence that a number of DNA-repair enzymes have greater activity in proliferating cells than in resting cells. These findings also support the view that there are differences between the DNA damage due to oxygen radicals and that due to ionizing radiation.


Radiation-sensitive irs mutants rejoin DNA double-strand breaks with efficiency similar to that of parental V79 cells but show altered response to radiation-induced G2 delay. Induction and repair of DNA double-strand breaks (dsb) was investigated in plateau phase Chinese hamster V79 cells and three radiosensitive mutant cell lines derived from them, irs-1, irs-2 and irs-3, using a pulsed-field gel electrophoresis assay, Asymmetric Field Inversion Gel Electrophoresis (AFIGE). There was no difference in the induction of DNA dsb per Gy and dalton between the radiosensitive mutant cells and wild-type V79 cells despite the wide differences in their radiosensitivity. Also, repair of DNA dsb proceeded in all cell lines with similar kinetics. In contrast to these observations at the DNA level, irradiation of exponentially growing cells showed a prolonged delay in G2 for irs-2 cells and a shortened delay in G2 for irs-1 cells, as compared to wild-type V79 cells. These results confirm previous observations suggesting that a deficiency in the rejoining of DNA dsb is unlikely to be the cause of the increased radiosensitivity of irs cells, and implicate alterations in postirradiation cell cycle progression as a possible cause for this phenomenon, although the mechanism is not known.


Excision repair influences the site and strand specificity of sunlight mutagenesis in yeast. A collection of 384 mutations recovered in a tRNA gene (SUP4-o) following exposure of isogenic excision-repair-proficient (RAD1) or deficient (rad1) strains of the yeast Saccharomyces cerevisiae to sunlight was characterized by DNA sequencing. In each case, greater than 90% of the mutations were single base-pair substitutions with events at G.C pairs constituting most of the changes. However, more than half of these substitutions were transversions in the RAD1 strain whereas transitions predominated in the rad1 strain. Tandem double substitutions were recovered in both strains and the individual changes were exclusively G.C----A.T transitions. The majority of single substitutions, and all tandem double changes, were at base-pairs where the pyrimidine(s) was part of a dipyrimidine sequence and the site specificities were consistent with cyclobutane dimers and/or pyrimidine (6-4) pyrimidone photoproducts contributing to sunlight mutagenesis. Yet, the data also pointed to an important role for lesions that form at G.C pairs and give rise to transversions. Analysis of the strand specificity of sunlight mutagenesis indicated that transitions or transversions at G.C pairs occurred preferentially in SUP4-o at sites where a dipyrimidine or a guanine, respectively, was on the transcribed strand. These biases required a functional excision-repair system.


Spontaneous and ultraviolet-induced mutations on a single-stranded shuttle vector transfected into monkey cells. The shuttle vector plasmid PCF3A, carrying the supF target gene, can be transfected into monkey COS7 cells as single-stranded or double-stranded DNA. Single strand-derived plasmid progeny exhibited a 10-fold higher spontaneous mutation frequency than double strand-derived progeny. The location of spontaneous mutations obtained after transfection of the single-stranded vector shared similarities with that for double-stranded vectors. However, the nature of base changes was very different. Single-stranded PCF3A DNA was used to study ultraviolet-induced mutagenesis. An earlier report (Madzak and Sarasin, J. Mol. Biol., 218 (1991) 667-673) showed that single-stranded DNA exhibited a lower survival and a higher mutation frequency than double-stranded DNA after ultraviolet irradiation. In the present report, sequence analysis of mutant plasmids is presented. The use of a single-stranded vector allowed us to show the targeting of mutations at putative lesion sites and to determine the exact nature of the base implicated in each mutation. Frameshift mutations were more frequent after transfection of control or irradiated plasmid as single-stranded DNA than as double-stranded DNA. Multiple mutations, observed at a high frequency in the spontaneous and ultraviolet-induced mutation spectra following single-stranded DNA transfection, could be due to an error-prone polymerisation step acting on a single-stranded template.


UV-induced hprt mutations in a UV-sensitive hamster cell line from complementation group 3 are biased towards the transcribed strand. The molecular nature of 254 nm ultraviolet light (UV)-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in UV24 Chinese hamster ovary (CHO) cells, which are defective in nucleotide excision repair, was determined. Sequence analysis of 19 hprt mutants showed that single base substitutions (9 mutants) and tandem base changes (7 mutants) dominated the UV mutation spectrum in this cell line. Sixty-five percent of the base substitutions were GC greater than AT transitions, whereas the rest consisted of transitions and transversions at AT base pairs. Analysis of the distribution of dipyrimidine sites over the two DNA strands, where the photoproducts causing these mutations presumably were formed, showed that 12 out of 14 mutations were located in the transcribed strand of the hprt gene. A similar strand distribution of mutagenic photoproducts as in UV24 has previously been found in two other UV-sensitive Chinese hamster cell lines (V-H1 and UV5), indicating that under defective nucleotide excision repair conditions the induction of mutations is strongly biased towards lesions in the transcribed strand of the hprt gene. A plausible explanation for this phenomenon is that during DNA replication large differences exist in the error rate with which DNA polymerase(s) bypass lesions in the templates for the leading and lagging strand, respectively.


Protective effects of ether, oxygen and their mixture for radiation in Drosophila melanogaster. Protective effects of ether mixed with air or oxygen against ionizing radiation damages were demonstrated in adult flies of Drosophila melanogaster. The protective effects against knock-down on the 2nd day and lethality on the 8th day after irradiation were not affected by the radiation sensitivity and DNA repair capacity of the strains. Ether (4.2%) in oxygen was more effective than ether in air for both endpoints. The protective effects may be due to damages not involving cell division, since no mitotic cells are observed in adult flies except in gonadal glands. A change in the orderliness of the cell membrane by ether is suggested to be the cause of the protective effects.


Phenotypes conferred by the Bacillus subtilis recM13 mutation and the din23 fusion. The din23 fusion encodes a B. subtilis SOS-inducible regulatory region fused to the E. coli lacZ gene (Love et al., 1985). A strain encoding the din23 fusion and a recM13 allele showed low-level constitutive beta-galactosidase expression, was induced for beta-galactosidase production by DNA gyrase inhibitors but not by DNA-damaging agents, and was slightly induced by a variety of agents which do not normally induce the SOS regulon. The din23 fusion itself resulted in high levels of spontaneous prophage induction in wild-type, recM- and recA-hosts, despite the fact that the din23recM13 strain was not induced for beta-galactosidase production by DNA-damaging agents. The results suggest that the recM gene may be involved with the regulation of the RecA protease-mediated SOS response, while the din23 gene may be involved with the regulation of an alternative function which results in the cleavage of prophage repressor.


Absence of strand-specific repair of cyclobutane pyrimidine dimers in active genes in Drosophila melanogaster Kc cells. Strand-specific excision repair of UV-induced cyclobutane pyrimidine dimers was investigated in three genes: Gart, Notch and white in the permanent Kc cell line derived from wild-type Drosophila melanogaster embryonic cells. In this cell line Gart and Notch are transcriptionally active, whereas white is not expressed. Cells were irradiated with 10 or 15 J/m2 ultraviolet (UV) light (predominantly 254 nm). In all three genes, cyclobutane pyrimidine dimers were removed from the non-transcribed strand at the same rate and to the same extent as from the transcribed strand, indicating the absence of strand-specific repair in permanent Drosophila embryonic cell lines.


Inhibition of transcription and strand-specific DNA repair by alpha-amanitin in Chinese hamster ovary cells. Recent studies have shown preferential repair of UV-induced cyclobutane pyrimidine dimers (CPD) in the transcribed strand of the expressed dihydrofolate reductase (DHFR) gene in human and rodent cells. We have tested the hypothesis that the strand-specific repair of such transcription-blocking lesions is dependent upon concurrent transcription. Chinese hamster ovary (CHO) B11 cells with an amplified DHFR gene were treated with alpha-amanitin before irradiation with UV (254 nm) and during post-irradiation incubation. Nuclear run-off analysis verified inhibition of transcription in the DHFR gene. CsCl density gradient analysis showed that alpha-amanitin at the levels used does not significantly interfere with overall genomic repair replication or semiconservative replication. However, we did observe a dramatic reduction in the removal of CPD from the transcribed strand in the 14 kb KpnI fragment within the DHFR gene in treated cells. We conclude that strand-specific repair of an active gene in CHO cells is dependent upon the activity of the transcribing RNA polymerase. Our results support the model that transcription complexes stalled at CPD signal the repair machinery to achieve efficient repair of the transcribed strand in active genes.


Mapping of a B-cell epitope present in the neuraminidase of Trypanosoma cruzi. We have previously shown that a polyclonal (rabbit anti-TCNA) and a mouse monoclonal antibody (TCN-2) against the neuraminidase of Trypanosoma cruzi (TCNA) inhibit enzyme activity, immunoprecipitate active enzyme, enhance in vitro infection, and identify a subpopulation of extracellular trypomastigotes. We now report on the identification of a synthetic peptide that contains the epitope recognized by these antibodies. The synthetic peptide (TR) is a dodecamer (D-S-S-A-H-G-T-P-S-T-P-A) deduced from the DNA sequence of the long tandem repeat (LTR) domain present in the TCNA carboxyterminus. By ELISA, rabbit anti-TCNA bound to TR coupled to ovalbumin, and the binding was inhibited by soluble TR but not by BR (Y-S-V-D-D-G-E-T-W-E), a peptide derived from the N-terminal domain of the enzyme. TCN-2 recognized TR, and this reaction as well as TCN-2 binding to endogenous TCNA could be inhibited by soluble TR but not by BR. These results indicate that the rabbit anti-TCNA and TCN-2 react with the LTR region of TCNA. Antibodies to TR reacted by immunoblot with the TCNA of the Silvio X-10/4, MV-13 and Y-H6 strains, identifying the same molecular polymorphism previously observed with the rabbit anti-TCNA and TCN-2. Furthermore, anti-TR antibodies immunoprecipitated active enzyme and immunofluorescence analysis revealed that anti-TR and TCN-2 antibodies detected equally well the differential expression of their epitopes in intra- and extracellular trypomastigotes. Moreover, expression of TR and TCN-2 epitopes on the different stages of T. cruzi paralleled the stage-specificity of TCNA activity. TCN-2 prevented desialylation by TCNA of intact cells but not of soluble glycoconjugates, indicating that TCN-2 epitope is probably not associated with the enzyme catalytic site, in agreement with the predicted sequence of the TCNA gene. Finally, analysis of the humoral response of a Chagasic patient to different areas of the TCNA molecule indicated that the antibody response is predominantly against TR suggesting that the tandem repeat is the immunodominant domain of TCNA.


Labelling and recording from dissociated target-specific rat superior cervical ganglion neurons. A population of neurons was retrogradely labelled in the superior cervical ganglia (SCG) of the adult rat following the injection of the fluorescent dye Fast blue into the submandibular salivary glands (SMG). The neurons retained the fluorescent label following dissociation and culture. Electrical and chemosensitive properties of the labelled neurons were studied with the whole-cell patch-clamp technique.


Isolation and identification from Salvia officinalis of two diterpenes which inhibit t-butylbicyclophosphoro[35S]thionate binding to chloride channel of rat cerebrocortical membranes in vitro. Ethanolic extracts from dried leaves of sage (Salvia officinalis) showed inhibition of [35S]tertiary-butylbicyclophosphorothionate ([35S]TBPS) binding to rat brain membranes in vitro. This ligand is considered to bind to the chloride channel of the GABA/benzodiazepine receptor complex in brain tissue. Substances having inhibitory activity were purified and their chemical structure identified as the diterpenes carnosic acid and carnosol (IC50 values of 33 +/- 3 microM and 57 +/- 4 microM, respectively). The two compounds did not affect binding of the ligands [3H]muscimol and [3H]diazepam to the GABA/benzodiazepine complex in vitro. Saturation experiments of [35S]TBPS binding indicated that carnosic acid decreases the binding affinity.


Young CA1 pyramidal cells of rats, but not dentate gyrus granule cells, express a delayed inward rectifying current with properties of IQ. In hippocampal CA1 pyramidal cells (CA1PC) and dentate gyrus granule cells (DGGC) we compared the expression of currents which could cause differences in discharge behaviour. Negative current injections cause a uniform hyperpolarization in DGGC whereas in CA1PC the initial hyperpolarization is followed by a repolarization towards resting membrane potential. The underlying inward current can be classified as IQ. It is sensitive to CsCl, activated at -80 mV, and it has a mean amplitude of -109.8 pA and a mean activation time constant of 187 ms with voltage jumps from -40 to -120 mV. We conclude that some of the differences in response properties of DGGC and CA1PC upon repetitive stimulation can be attributed to differences in the expression of IQ.


NMDA treatment and K(+)-induced depolarization selectively promote the expression of an NMDA-preferring class of the ionotropic glutamate receptors in cerebellar granule neurones. Growth conditions which promote the survival of cerebellar granule cells in culture, such as high K+ or N-methyl-D-aspartate (NMDA) treatment, also promote the functional expression of an NMDA-preferring subtype alone of the ionotropic glutamate receptors. The selective regulation of NMDA receptors detected electrophysiologically in individual cells, using the whole cell patch clamp technique, is characteristic of granule cells in general: NMDA-induced 45Ca2+ influx increased several-fold in cultures treated with either high K+ or NMDA. The increase in NMDA receptor activity was correlated with an increase in the expression of an NMDA receptor protein. Since the effect of these 'trophic' conditions is mediated through Ca2+, the induced increase in the density of NMDA receptors (which gate a Ca2+ conductance) provides a positive feedback for strengthening the trophic influences.


A sexually dimorphic population of galanin-like neurons in the rat lumbar spinal cord: functional implications. The rat lumbar spinal cord contains a population of galanin- and cholecystokinin-containing neurons which are located dorsolateral to the central canal and project to the thalamus. New data are presented herein which reveal that the number of these neurons, as shown by galanin-like immunostaining, is sexually dimorphic with males containing 62% more of these neurons than females. This is the first demonstration of a sexually dimorphic population of intraspinal neurons which projects to higher CNS centers rather than to peripheral targets.


Nitric oxide may act as a messenger between dorsal root ganglion neurones and their satellite cells. The distribution of NADPH-diaphorase and cyclic GMP in neonatal dorsal root ganglia in vitro has been investigated under control conditions and in response to incubation with either sodium nitroprusside or N-methyl-D-aspartate. NADPH-diaphorase activity which reveals the distribution of nitric oxide (NO) synthase in neurons was found to be intense in some dorsal root ganglion neurones and present at a lower level in the majority. Basal levels of cGMP were found to be low but when stimulated by sodium nitroprusside were found to be selectively increased in satellite cells. The results suggest that NO may function as a signalling system between neurones and satellite cells in sensory ganglia.


Regulation of rodent myelin proteolipid protein gene expression. The regulation of rodent proteolipid protein (PLP) gene expression was studied during rat development and in cultured cells. Nuclear run-on assays demonstrate a strong transcriptional component associated with the developmental regulation of the PLP mRNA. Transcription rates of the PLP and MBP genes parallel their respective steady-state mRNA levels throughout rat brain development. In addition, a moderate 25-h half-life was measured for PLP mRNA in 37-day-old cultured oligodendrocytes, suggesting that regulation of PLP expression occurs predominantly at the transcriptional level. Finally, 5400 and 1400 bp of mouse PLP 5'-flanking sequence demonstrate transcriptional activity 13-fold and 5-fold above background, respectively, in hamster glial cells. Far upstream elements are clearly involved in transcription of the PLP gene. The 5400 bp sequence demonstrates no more activity than the 1400 bp in a mouse hepatoma cell line suggesting that elements involved in the glial cell-specific expression of PLP lie between 1400 and 5400 bp upstream of the gene.


Nicotine- and capsaicin-, but not potassium-evoked CGP-release from cultured guinea-pig spinal ganglia is inhibited by Ruthenium red. In the present study we have investigated the effects of nicotine, capsaicin, potassium, glutamate and aspartate on release of calcitonin gene-related peptide (CGRP)-like immunoreactivity (-LI) from guinea-pig dorsal root ganglion (DRG) cultures. In addition the possible influence of Ruthenium red (RR), neuropeptide Y (NPY) and noradrenaline (NA) on the CGRP-LI outflow has been evaluated. Nicotine, capsaicin and potassium, but not glutamate or aspartate, evoked a Ca(2+)-dependent increase in the culture medium, suggesting release of CGRP-LI. RR inhibited the effect of both capsaicin and nicotine but did not influence potassium-induced CGRP-LI release. Furthermore, the nicotine- but not capsaicin-evoked CGRP-LI release was inhibited by NPY. It is concluded that DRG cultures represent an experimental model where regulation of CGRP-LI release can be studied. The ability of RR to inhibit not only capsaicin but also nicotine effects indicate that the proposed selectivity of RR may depend on the agent used to evoke peptide release and/or concentrations used.


Evidence that pharmacological manipulations of central L-arginine-NO pathway influence blood pressure and heart rate in rats. In the present study we have investigated whether pharmacological manipulations of central L-arginine-nitric oxide (L-Arg-NO) pathway could affect blood pressure (BP) and heart rate (HR) in normotensive rats either untreated or pretreated with E. coli lipopolysaccharide (LPS). The intracerebroventricular injection (i.c.v.) of N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthesis, caused a fall of BP and HR in LPS-treated but not in control rats. Furthermore, the pressor responses to i.c.v. injection of N-methyl-D-aspartate (NMDA) were enhanced by L-Arg or LPS treatment and, in both cases, this potentiation was blocked by L-NAME. The present results show that in some experimental conditions, such as activation of NMDA receptors or LPS pretreatment, the central microinfusion of drugs affecting the L-Arg-NO pathway may interfere with BP and HR.


Prediction of response to adjuvant chemotherapy in premenopausal lymph node positive breast cancer patients with morphometry, DNA flow cytometry and HER-2/neu oncoprotein expression. Preliminary results. The value of morphometry, DNA flow cytometry and HER-2/neu oncoprotein expression for prediction of response to adjuvant chemotherapy in premenopausal lymph node positive breast cancer patients was evaluated in a group of CMF treated patients and controls with long-term follow-up. In the treated group, the Morphometric Prognostic Index (cutpoint 1.1) was the best prognosticator (p less than 0.0001, MC = 16.9), followed by the Mitotic Activity Index, the volume percentage epithelium and the number of positive nodes. For the controls, only the % HER-2/neu oncoprotein expression revealed significant differences (p less than 0.0001, MC = 16.3). When directly comparing treated patients and controls stratified for a certain parameter, no significant differences were obtained, although a trend towards improved survival in the treated group was present for some of the subgroups for several parameters. These preliminary results indicate that morphometric features and quantitative HER-2/neu oncoprotein expression may be important factors for identifying cases that will or will not respond to adjuvant chemotherapy.


X-ray induction of micronuclei in human lymphocyte subpopulations differentiated by immunoperoxidase staining. In this study we sought to confirm the radiosensitivity of human peripheral blood lymphocyte subpopulations using a micronucleus assay. Mononucleated cells isolated from peripheral blood were irradiated with X rays. After being cultured for 3 days, cells were fixed and stained using the immunoperoxidase staining technique. Lymphocyte subpopulations were characterized by means of the monoclonal antibodies Leu4 (CD3), Leu2a (CD8) and Leu19 (CD56). Dose-response curves were obtained by scoring the number of micronuclei in binucleated cells that reacted with a specific antibody and were then stained. The dose response of CD8+ (suppressor/cytotoxic) cells was quite similar to that of CD3+ (pan T) cells. In comparison, CD56+ (natural killer) cells were significantly less sensitive, although scorable binucleated CD56+ cells made up less than 4% of the total number of binucleated cells.


Antiribosomal antibodies in systemic lupus erythematosus. ARA occur in approximately 10% of randomly selected SLE patients but in up to 40% of patients with active disease. Anti-P antibodies appear to be a highly specific diagnostic marker for SLE because they are rarely detected in other multisystem autoimmune disorders. ARA are most frequently directed against the P proteins, and the shared conserved C-terminus of the P proteins is immunodominant in almost all sera tested. Anti-P antibodies increase in titer in patients with active disease and have been reported to be detected more frequently in patients with severe behavioral disturbances. This may be particularly true of patients with affective disorders. The clinical utility of serologic tests for anti-P in central nervous system lupus must await large, prospective studies. Other ARA antibodies have been detected in patients with SLE. These antibodies include anti-28S rRNA, anti-S10, and anti-L12. In all cases, the frequency with which these antibodies are detected is increased in sera containing anti-P. The P proteins and the 28S rRNA epitope play essential, but as yet undefined, roles in GTPase activity on the ribosome. The L12 protein is the mammalian homologue of the E. coli and yeast proteins known to bind to the 28S rRNA epitope. These findings indicate that some SLE patients produce autoantibodies against multiple components of a functionally related domain of the ribosome. This, in turn, supports the notion that the ribosome initiates and/or maintains autoantibody production. Despite these findings, attempts to induce anti-P antibodies by immunization with autologous ribosomes in the autoimmune strain of mouse, MRL, have been unsuccessful. It therefore seems likely that the ribosomal components must be altered to break tolerance or that other abnormalities of the immune system are necessary for autoantibody production.


Autoantibodies in polymyositis.

Autoantibodies in polymyositis. A group of autoantibodies have been identified that are found almost exclusively in patients with polymyositis and dermatomyositis (myositis-specific antibodies). Most have been associated with characteristic clinical subgroups. Five of the myositis-specific antibodies are directed at aminoacyl-tRNA synthetases and have been associated with a similar clinical syndrome marked by myositis, interstitial lung disease, arthritis, and Raynaud's phenomenon (antisynthetase syndrome). Myositis-specific antibodies can help with patient diagnosis, subgroup classification, and possible prognosis. Their role in the pathogenesis of myositis remains to be defined, but their production is genetically influenced and appears to be linked to fundamental etiologic factors.


[Role of the hypothalamo-hypophyseal neurosecretory system in the accomplishment of the antistress effect of substance P]. Light and electron microscopy and spectrophotometry were employed in study of the morphofunctional condition of the hypothalamo-hypophyseal neurosecretory system (HHNS) of intact rats after exposure to stress in injection of substance P. Injection of 12.5 mcg/100g of substance P decelerates the outflow of the neurosecretory substance in intact animals and inhibits the processes of biosynthesis in the neurocytes of the hypothalamic supraoptic nucleus. Injection of substance P prior to exposure of the organism to the stress effects leads to diminution of the HHNS response of them and prevents the development of dystrophic changes in the gastric mucosa.


[Subjectivity and objectivity in transperineal biopsy and transrectal prostatic aspiration cytology]. Transperineal biopsy (TPB) together with transrectal aspirative cytology (TRAC) was performed in 50 prostates, 28 carcinomas and 22 no malignant pathologies. Both techniques were compared both objectively as subjectively. With TPB in 3.5% of carcinomas and in 13.6% of benign pathologies not enough sampling material was obtained to perform the diagnoses. With TRAC these figures were respectively 3.5% and 9%. With the viable samples TPB sensibility was 100% being TRAC 96.3%. Specificity was 100% for both techniques. With TPB, 40% of the biopsies labelled as "dubious obtention of enough material to perform diagnoses", were finally insufficient; on TRAC this rate was 11%. In the three cases of insufficient sampling material with TRAC only aspirative punction had been performed. We think it is necessary to perform more than one punction in TRAC. TRAC is, for its lack of complications, efficacy, and because it is easy to perform, the preferred technique versus TPB.


Sodium nitroprusside increases cyclic GMP in fetal rat bone cells and inhibits resorption of fetal rat limb bones. To elucidate the role of cGMP in bone resorption, the nucleotide was measured in bone and bone cells in response to several agents including stimulators of bone resorption. In other experiments, sodium nitroprusside (SNP), which elevates bone cGMP, was tested for effects on resorption. In cells from 20-day fetal rat calvaria, cGMP was 52.4 +/- 8.4 fmol/10(6) cells; cAMP was 5.3 +/- 0.3 pmol/10(6) cells. SNP, 0.1 mM, in the presence of IBMX, increased cGMP 74% with no significant effect on cAMP. Parathyroid hormone (PTH), 0.5 microM, did not significantly affect cGMP, but increased cAMP 711%. Calcitriol did not affect either nucleotide. In bone resorption studies, 0.1 mM SNP inhibited the effects of PTH and calcitriol. Lower concentrations of SNP (0.001, 0.01 mM) had no effects on hormone-stimulated resorption. Unstimulated control bones were not affected by 1 nM-1 mM SNP. The results suggest that elevated cGMP could result in inhibition of bone resorption.


A new restriction fragment length polymorphism of the human alpha-fetoprotein gene in Japanese individuals: detection of loss of heterozygosity in human hepatocellular carcinoma. By using the cDNA probe for the human alpha-fetoprotein (AFP) gene, one restriction fragment length polymorphism (RFLP) was discovered in Japanese patients. A variety of combinations of restriction enzymes and probes were evaluated in DNA samples from 25 Japanese individuals who were genetically unrelated. As a result, AvaII identified one invariant band of 8 kb and polymorphic bands at either 2.8 or 2.0 kb with the 5'-region probe from a cDNA clone, pHAF7. With the AvaII/pHAF7 combination, we examined the loss of heterozygosity in 14 patients with hepatocellular carcinoma (HCC). Of 6 informative specimens, 2 showed loss of heterozygosity. These data suggest that the AFP cDNA probe could be useful for analyzing loss of heterozygosity on chromosome 4q in human HCC.


Plasma levels of vasoactive regulatory peptides in patients receiving regular hemodialysis treatment. The fasting plasma levels of 10 vasoactive regulatory peptides were measured by radioimmunoassay in 23 stable patients with chronic renal failure receiving regular hemodialysis treatment (RDT) and compared with those of healthy controls. The plasma concentrations of arginine vasopressin, atrial natriuretic peptide, beta-endorphin, methionine-enkephalin, motilin, neuropeptide Y, substance P, and vasoactive intestinal peptide were increased. The plasma level of calcitonin gene-related peptide was not statistically different from that of the controls. The plasma concentration of gamma 2-melanocyte-stimulating hormone was lowered in the RDT-patients. The arterial blood pressure correlated with the plasma levels of motilin and neuropeptide Y. We conclude that patients with chronic renal failure receiving RDT have increased concentrations of 8 out of 10 measured vasoactive regulatory peptides. The elevated levels of vasoactive peptides may contribute to the adaptation of the cardiovascular system to impaired renal function.


Long term complications of the intraprostatic spiral. Case report. A 76-year-old man had an intraprostatic spiral inserted to relieve bladder outlet obstruction that was caused by benign prostatic hypertrophy. After 30 months numerous complications had arisen including severe encrustations, urethral stricture, and sclerosis of the bladder neck. Regular replacement of the spiral or close monitoring of the development of encrustations seem necessary. The distal tip and the plating of the spiral require improvement.


Immunoreactivity, epitope mapping and protection studies with anti-conotoxin GI sera and various conotoxins. Goat or rabbit anti-conotoxin GI sera recognized native or dithiothreitol-reduced alpha-conotoxins GI, MI and SI in an enzyme-linked immunosorbent assay (ELISA). Native mu-conotoxin GIIIA or omega-conotoxin GVIA did not react with either anti-conotoxin GI serum in the assay. The goat anti-conotoxin GI serum neutralized 2.5 LD50S of alpha-conotoxins GI or MI in mouse lethal assays, while the rabbit antiserum had little protective capabilities. Epitope mapping of synthesized conotoxin peptide fragments revealed that both anti-conotoxin GI sera recognized linear sequences from five different alpha-conotoxins: GI, GIA, GII, MI and SI. The CCNPAC sequence was optimally recognized by both antisera.


Variation in the antigenic characteristics of venom from the Mojave rattlesnake (Crotalus scutulatus scutulatus). Venoms from 31 specimens of the Mojave rattlesnake (Crotalus scutulatus scutulatus) were examined to further characterize reported differences among venoms of this species. Twenty-two venoms were recognized by a monoclonal antibody to Mojave toxin, CSS12. Nine venoms were recognized by CA-P-8, a monoclonal antibody produced against the hemorrhagic venom of C. atrox. Seven of these produced strong hemorrhage in mice and were also recognized by polyclonal antibodies (anti-F5) produced against a fraction of Mojave rattlesnake venom that inactivates serum complement. Fractionated venom revealed that CA-P-8 and anti-F5 recognized different proteins. Two of the venoms recognized by CA-P-8 were not recognized by anti-F5 and produced minimal hemorrhage in mice. This suggests that more than one factor may be necessary to induce strong hemorrhage.


Effects of FK506 on rat thymic epithelial cells; immunohistochemical study. The effects of FK506, a new immunosuppressive agent, on the rat thymus were investigated using the immunoperoxidase technique and flow cytofluorometry using monoclonal antibodies. Flow cytometric analysis of the thymus revealed that the proportion of cells labelled positively with OX7 (Thy-1 antigen), OX8 (CD8, T cytotoxic/suppressor cells) and W3/25 (CD4, T helper cells and macrophages) increased following treatment, with FK506, 1 mg/kg body weight for 14 days. A marked reduction of the thymic medulla following treatment was clearly demonstrated by staining with OX18 (MHC class I) and OX6 (MHC class II). Changes produced by FK506 were also observed in the cortical area of the thymus, being especially marked in the subcapsular area and around the blood vessels by staining with OX6, PKK-1 (alpha-cytokeratin), AB-1040 (type IV collagen), and AB-1220 (laminin). Eventually FK506 treatment resulted in patchy reduction of OX-6, PKK-1, AB-1040 and AB-1220 positive area in the cortex. This evidence suggests that FK506 may impair the thymic microenvironment and subsequently disturb the thymocyte maturation.


Inhibition of the classical activation pathway of complement-mediated lysis by monoclonal antibodies to complement components C3c and C3d. Eight epitope-mapped monoclonal antibodies (MoAbs) to complement component C3d and five to complement component C3c were investigated to determine whether they could inhibit the classical activation pathway of complement-mediated lysis (CML) by using blood group AB red cells sensitized by A or B MoAbs. Three IgM C3d MoAbs and one IgG1 C3c MoAb were able to inhibit CML in a dose-dependent manner. In the presence of excess complement, no inhibition was observed. The greatest inhibition was observed with two high-affinity IgM antibodies that were specific for epitope 1 on the C3d component. Some inhibition was observed with a high-affinity IgM antibody specific for epitope 3 of the C3d component and also with a lower-affinity IgG antibody specific for epitope 1 of the C3c component. The results indicate that some complement MoAbs have the capacity to distinguish between conformationally and/or functionally different forms of red cell-bound C3.


Distinct hemagglutinin and neuraminidase epitopes involved in antigenic variation of recent human parainfluenza virus type 2 isolates. A panel of fourteen neutralizing anti-HN monoclonal antibodies (mAbs) to the prototype Greer strain of human parainfluenza virus type 2 (PI2) was used to determine the extent of antigenic variation in recent virus isolates. Competitive binding analysis with the mAbs indicated the presence of at least five distinct antigenic sites (I to V) on the HN glycoprotein molecule. MAbs recognizing different antigenic sites were found to be associated with the hemagglutinin (sites I, IV and V), hemagglutinin and neuraminidase (site II), or neuraminidase (site III) activities. The location of two distinct epitopes identifying the neuraminidase sites (II and III) was further verified from the generation of escape mutants. Antibodies directed to sites I and III failed to show any detectable binding or neutralizing activity against a number of natural PI2 virus isolates collected in Texas between 1986 and 1987. Interestingly, these natural variants, unlike the prototype virus, did not show any detectable neuraminidase activity with fetuin as a substrate and the enzyme activity was only detected with N-acetylneuramin-lactose as an alternative substrate. Despite the observed variation in the antigenic sites, primary infection with the prototype virus or the natural variants generated a protective immune response against challenge infection with the other virus strains.


Sendai virus M protein is found in two distinct isoforms defined by monoclonal antibodies. The use of a monoclonal antibody defines a subset of Sendai virus M protein representing about 30% of total. This M protein acquires, during the hour following synthesis, an epitope not present on the bulk of M. This epitope maturation is observed in acutely as well as in persistently infected cells. It takes place in vivo in absence of other viral proteins, but it is not observed when the protein is synthesized in a reticulocyte lysate. Epitope maturation does not appear to result from phosphorylation, acylation or disulfide bond formation. If immunofluorescent staining seems to indicate a preferential association of this subset of M protein with nucleocapsids, this is not confirmed by immunogold staining or by nucleocapsid isolation. Incubation of cytoplasmic extracts or of purified M protein in conditions which do not favor M to M protein association results in a relative increase of M protein carrying the maturing epitope. It is concluded that M protein exists in two distinct isoforms.


From records to self-description: the role played by RNA in early evolutive systems. We study the appearance of genetic information starting from a system where self-reproductive and enzymatic functions are supported by the same sort of molecules. In a first phase, the information must have arisen in the form of rate independent sequences as records of enzymatic functions. Although this stage must have played an important role in evolution, it will be shown how its evolutive capacities were blocked by the impossibility of appearance of geno/phenotype duality. Finally, a logical scheme is proposed for a transition process toward a system with a code offering a simplification of the conditions required from the assumption of a maximum use of the double RNA capacity, both reproductive and enzymatic.


Influence of chromatin molecular changes on RNA synthesis during embryonic development. Two aspects of the chromatin repeat length (rl) are discussed: (i) Why is rl longer for slowly dividing cells than in rapidly dividing cells?, and (ii) Why is the temporal evolution of rl a decreasing function of time (t) in mammalian cortical neurons, whereas it is an increasing function of t for granule cells around the time of birth? These questions are discussed in terms of a hypothesis which assumes a correlation between deoxyribonucleic acid (DNA) packaging, transcription, and replication.


Differences in the prevalence of hypertension by ethnic origin and age at immigration in a cohort of 5,146 Israelis. Marked ethnic differences in hypertension prevalence have been described in Jewish immigrants to Israel. The extent to which this phenomenon has persisted after a long period of living in the same country, and whether native-born descendants exhibit similar patterns, is not clear. The aim of this study was to determine the prevalence of hypertension in immigrants to Israel and native-born Israelis by region of origin and age at immigration. Complete data were available for 5,146 subjects (3,607 men and 1,539 women) aged 20-64 years who were employed in Israeli industries and were examined during 1985-1987. In both sexes, Jews originating in the West (Europe and the Americas) had higher blood pressures and a significantly higher prevalence of hypertension than those from northern Africa or Asia, particularly in the age group 20-44 years (17% vs. 9% and 8% in men, respectively, and 9% vs. 3% and 5% in women). There was a significant positive association between the prevalence of hypertension and age at immigration (p less than 0.001) in both sexes, and this finding was present in all ethnic groups. In multiple logistic regression analysis, the associations of hypertension with ethnic origin and age at immigration were only partly explained by variations in body mass index, after controlling for other potentially confounding variables. These findings suggest that despite these subjects' having shared a relatively similar physical environment for many years, ethnic differences in the prevalence of hypertension persist. Immigration at an older age was associated with a higher prevalence of hypertension for both subjects originating in industrialized countries and those originating in nonindustrialized countries, suggesting that the process of immigration itself may adversely affect blood pressure.


Carcinoma of the body and tail of the pancreas. Recently, several institutions have reported improved results in the treatment of patients with carcinoma of the head of the pancreas. In an attempt to determine whether similar trends could be demonstrated for patients with carcinoma of the body and tail of the pancreas, the records of all 113 patients with an adenocarcinoma of the body or tail of the pancreas treated at The Johns Hopkins Hospital between 1972 and 1989 were reviewed. The patients were divided into two groups: those diagnosed between 1972 and 1982 (41 patients) and those between 1983 and 1989 (72 patients). No significant differences in tumor stage were observed between the two groups. The proportion of patients who underwent surgery decreased from 68% to 47% (p = 0.02). The number of patients who had bypass operations (15% versus 17%) or pancreatic resection (5% versus 10%) was similar in the two groups, but the proportion of patients who underwent exploratory laparotomy with biopsy only decreased from 49% to 21% (p = 0.002). The postoperative 30-day mortality (7% versus 3%), postoperative morbidity (18% versus 21%), median survival (4 months versus 3 months), and the 1-year survival (8% versus 9%) did not differ significantly between the two groups. One patient survived for 6 years after resection, and another patient is still alive 3 years after resection. Thus, unlike adenocarcinoma of the head of the pancreas, it appears that treatment results for patients with adenocarcinoma of the body or tail of the pancreas have not improved in recent years, the only change being a decreased need for exploratory laparotomy with biopsy only.


Age-related occurrence of signs and symptoms in the Rett syndrome. The occurrence of signs and symptoms in the Rett syndrome (RS) was analyzed in a series of females born 1945-87 (median age 17 1/2 years) and fulfilling the diagnostic criteria for classic RS. For general information, data from 91 girls and women were used (group A), while the more detailed analyses were based on three age related subgroups: the youngest 20, born 1980-87 (group B); the 34 girls born 1970-79 (group C); and the oldest 37, born 1945-69 (group D). Data from group A indicated a developmental stagnation (stage I) at median age 11 (5-24) months and loss of ability to use acquired skills (stage II) at 19 (12-36) months. Group B displayed subtle prodromes in the first months of life, and later in infancy gross motor delay with insufficient equilibrium control. Development invariably came to a definite break at a crucial stage of maturation and was followed by a remarkable "awakening" and return of interest to act and interact (stage III). Loss of skills belonged to the triad contact/communication, hand use/skill, and babble/words. By age 2 1/2 years, apraxia and involuntary and stereotyped movements, were found in all. Seizures, hyperventilation and spells of screams and laughter were more frequent in group C (94%, 65% and 71%, respectively), and breathholding, bloating and drooling in group D (73%, 43% and 81%, respectively). Plantar flexion and abiotrophy of feet, as well as peroneal weakness and scoliosis, increased with age and was found in 89%, 86%, and 89%, respectively. The sequence of events described, emerged as subtle insufficiency and more or less abruptly turned into loss. Conversely to known progressive encephalopathies, the deterioration was followed by excess of activity, only years later to turn into restriction.