Treatment of human hepatocellular carcinoma by fibroblast-mediated human interferon α gene therapy in combination with adoptive chemoimmunotherapy

The therapeutic effect of the fibroblast-mediated human interferon (IFN) gene therapy in combination with interleukin-2 (IL-2) activated killer cells (AK)/doxorubicin (i.e., adoptive chemoimmunotherapy) on nude mice bearing the human hepatocellular carcinoma (HCC) was investigated. A fibroblast cell clone (NIH3T3-IFN⁺) secreting 1024 U/ml human IFN was obtained from 14 positive clones by BMGNeo-INF DNA transfection, G418-resistant selection, limiting dilution and assay of IFN activity. After i.p. implantation of NIH3T3-IFN⁺ encapsulated into collagen, serum human IFN activity could be detected from 12 h to day 15 with a peak at 72 h. AK were prepared from human peripheral mononuclear cells costimulated in vitro by IL-2 and inactivated human SMMC 7721 HCC cells. When the NIH3T3-IFN⁺ cells were i.p. implanted into the HCC-bearing nude mice, the grown of HCC was inhibited and the survival time of the mice was extended. The growth of HCC was inhibited more obviously when AK was i.v. injected and IL-2 was i.p. injected after the NIH3T3-IFN⁺ cells had been implanted. The best therapeutic effect was achieved when NIH3T3-IFN⁺ cells were used in combination with IL-2/AK/doxorubicin. All these results suggested that the fibroblast-mediated human IFN gene therapy could be used to treat the human hepatocellular carcinoma effectively and that when used in combination with IL-2-based adoptive chemoimmunotherapy, the therapeutic effect would be better.Abbreviations IFN interferon - HCC hepatocellular carcinoma - IL-2 interleukin-2 - AK activated killer cells - Dox doxorubicin This research was supported by the National Natural Sciences Foundation of China (grant 39421009)     

Demonstration of a relatively hepatoselective effect of covalent insulin dimers on glucose metabolism in dogs

Summary Insulin analogues with relatively greater effect on hepatic glucose production than peripheral glucose disposal could offer a more physiological approach to the treatment of diabetes mellitus. The fact that proinsulin exhibits this property to a minor degree may suggest that analogues with increased molecular size may be less able than insulin to obtain access to peripheral receptor sites. Covalent insulin dimers have previously been shown to possess lower hypoglycaemic potencies than predicted by their in vivo receptor binding affinities. Reduced rates of diffusion to peripheral target tissues-might be an explanation for the lower in vivo potency compared to insulin. To test the relative hepatic and peripheral effects of covalent insulin dimers, glucose clamp procedures with D-[3-³H] glucose tracer infusions were used in anaesthetised greyhounds to establish dose-response curves for rates of hepatic glucose production and glucose disposal with insulin, N^B1, N^{B 1},-suberoyl-insulin dimer, and N^B29, N^{B 29},-suberoyl-insulin dimer. With N^B1, N^{B 1},-suberoyl-insulin dimer molar potencies relative to insulin were 68%, (34–133) (mean and 95% fiducial limits), for inhibition of hepatic glucose production and 14.7%, (10.3–20.9) for glucose disposal. With N^B29,N^{B 29},-suberoyl-insulin dimer potencies were 75%, (31–184) and 2.5%, (1.5–4.3), for inhibition of hepatic glucose production and for glucose disposal, respectively. The demonstration that both dimers exhibit a significantly greater effect on glucose production than on glucose disposal supports the suggestion that analogues with increased molecular size may exhibit reduced ability to gain access to peripheral target cells.Abbreviations B1-B 1D N^B1,N^{B 1},-suberoyl-insulin dimer - B29-B 29D N^B29,N^{B 29},-suberoyl-insulin dimer - Ra hepatic glucose production rate - Rd peripheral glucose disposal rate - M_r relative molecular weight - MCR metabolic clearance rate - ANOVA analysis of variance     

Serum cytokines in patients with rheumatoid arthritis

Serum cytokines such as interleukin 1 (IL-1), interferon (IFN-), and tumor necrosis factor (TNF) were measured in 40 patients with rheumatoid arthritis (RA). In the 40 patients studied, serum IL-1 was detected in 5 patients, IFN- in 10 patients, and TNF in 20 patients. The IL-1-positive group showed increased values of activity indices compared to the IL-1-negative group. Values of serum IFN- correlated well with the number of peripheral blood lymphocytes and CD3⁺ cells and with the percentage of CD3⁺ CD26⁺ cells. Values of serum TNF correlated positively with the number of peripheral blood monocytes and the percentage of CD3⁺ HLA-DR⁺ and CD3⁺ CD25⁺ cells. These results indicated that serum IL-1 in RA patients reflects the activity of RA, while the serum IFN- and TNF in RA patients may be related to circulating activated lymphocytes and monocytes, respectively.     

Rapid molecular characterization of Hb H disease in Chinese by polymerase chain reaction

Summary We have developed a rapid method to molecularly distinguish different types of Hb H disease. The study depended on (a) most of the Hb H disease in Taiwan having an-thalassemia-1 of the Southeast Asia type (-SEA) in one allele and (b) the differences of X box of-globin gene cluster in the other allele. To detect the -SEA allele, we utilized the primers located on either side of the breakpoint to do PCR, then characterized the amplified products. For the other allele, we sequenced part of the X box, and found that bases –2803 to –2461 of the X box of – ^3.7 belonged to the X box of ₂ globin gene. In – ^4.2, the bases belonged to the X box of ₁ globin gene, whereas in ^cs it contained both X boxes of ₁ and ₂ globin genes. There was anMboII site at this region of the X box of ₂ globin gene. We utilized PCR to amplify this region and digested it with restriction enzymeMboII, then combined it with another PCR of different primer pairs to molecularly diagnose different types of Hb H disease. One hundred and one cases of Hb H disease from different families were studied: all of the cases had one allele of -SEA deletion, while the other allele showed that 52/101 were – ^3.7, 41/101 were ^cs , 7/101 were – ^4.2, and 1/101 was – ^G.Taichung. Of 52 cases of Hb H with – ^3.7, 47 were type-I deletion and five were type-II deletion.     

HIV-1 replication in the plasma and cerebrospinal fluid

Summary The HIV-1 RNA in plasma and CSF samples from 40 HIV-1 infected patients was measured by a polymerase chain reaction (PCR) technique. The possible implication of cytokines in HIV-1 replication was investigated by measuring the concentrations of tumor necrosis factor (TNF-), macrophage colony stimulation factor (M-CSF) and interleukin-6 (IL-6) in these fluids. HIV-1 RNA was quantified in all plasma samples and in 87.5% of the CSF samples. CSF HIV-1 RNA titers did not correlate with the stage of disease or the CD4⁺ T cell counts, unlike the plasma HIV-1 RNA titers. These results were confirmed when patients with a blood brain barrier damage, as assessed by the CSF/plasma albumin ratio, were excluded from the analysis. TNF- levels were statistically correlated with the HIV-1 RNA in plasma and CSF. These data demonstrate that HIV-1 replication in the CSF at each clinical stage can be accurately measured with PCR and, although the titers of HIV-1 RNA copies in the CSF are correlated with those in the plasma, the magnitude of HIV-1 replication in CSF is not directly linked to the stage of disease, or to the CD4⁺ T cell count. The significance of early high levels of HIV-1 RNA in CSF is now being studied prospectively.

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HIV-1-Replikation im Plasma und Liquor cerebrospinalis

Zusammenfassung HIV-1-RNA wurde mittels Polymerasekettenreaktion (PCR) in Plasma- und Liquorproben von 40 HIV-1-infizierten Patienten quantifiziert. Um mögliche Einflüsse durch Zytokine auf die HIV-1-Replikation zu erfassen, wurden Tumornekrosefaktor- (TNF-), Makrophagen-Kolonie-stimulierender Faktor (M-CSF) und Interleukin-6 (IL-6) in diesen Flüssigkeiten ebenfalls bestimmt. Eine Quantifizierung von HIV-1 RNA war in allen Plasmaproben und in 87,5% der Liquorproben möglich. Im Gegensatz zu den HIV-1-RNA-Titern im Plasma fand sich zwischen HIV-1-RNA-Titern im Liquor und dem Krankheitsstadium oder den CD4⁺-T-Zell-Zahlen keine Korrelation. Diese Ergebnisse bestätigten sich auch bei Patienten, bei denen gemessen am Liquor-Ausschluß von Plasma-Albumin-Quotienten eine Blut-Liquor-Schrankenstörung bestand. Die HIV-1-Replikation kann in allen klinischen Stadien mittels PCR exakt quantifiziert werden. Obwohl die Liquor-Titer an HIV-1-RNA-Kopien mit den Plasmatitern korrelieren, besteht dennoch keine direkte Beziehung zum Krankheitsstadium oder zur CD4⁺-Zellzahl. In einer prospektiven Studie wird derzeit die Bedeutung frühzeitig auftretender HIV-1-RNA-Spiegel im Liquor untersucht.

     

A comparison of the effects of prostacyclin and the 15 (S), 15-methyl analogs of PGE2 and PGF2α on gastric parietal and nonparietal secretion

The mechanism by which prostaglandins protect the gastric mucosa is unclear. Although this action has been shown to be independent of the ability to inhibit parietal secretion as measured by hydrogen ion (H⁺) output, it may be related to stimulation of nonparietal secretion measured here as sodium ion (Na⁺) output. Five conscious, chair-adapted rhesus monkeys received an infusion of either prostacyclin (PGI₂; 125, 175, or 250 ng/kg/min intravenously) or the 15(S), 15-methyl analog of either PGF₂ (mePGF₂; 0, 1000, or 2000 ng/kg/min subcutaneously) or PGE₂ (mePGE₂; 0, 15, or 30 ng/ke/min subcutaneously) on different days. The output of H⁺, Na⁺ potassium (K⁺), and chloride (Cl^–) ion were determined using a dye dilution technique during a basal period and following the intragastric administration of an 80-ml water load. PGI₂ (175 and 250 ng/kg/min), mePGF₂, and mePGF₂ all significantly inhibited H⁺ output. Both basal and postload Na⁺ output were increased significantly by mePGE₂, while only postload Na⁺ output was significantly enhanced by mePGF₂. Na⁺ output was not modified by PGI₂. K⁺ output was unchanged by mePGE₂ or mePGF₂; however, postload K⁺ output was significantly inhibited by the highest dose of PGI₂. Both basal and postload Cl^– output were suppressed significantly by PGI₂ while basal Cl^– output was decreased significantly by the higher dose of mePGF₂ and postload Cl^– output was inhibited by both doses of mePGF₂. In contrast, Cl^– secretion was unaffected by mePGE₂. Thus, mePGE₂ markedly increased nonparietal secretion, while mePGF₂ mildly increased and PGI₂ did not affect nonparietal secretion. On the basis of these results, a stimulation of nonparietal secretion cannot be considered as the common mechanism for the gastric cytoprotective effect of these prostaglandins.Portions of this work have been published in abstract form and were presented at the Annual Meeting of the American Federation for Clinical Research, May 12, 1980.The experiments reported herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council, DHEW Publ. No. 78-23.The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of Defense.     

Rapid detection of −α 4.2 deletion of α-thalassemia-2 by polymerase chain reaction

Summary We sequenced part of the X boxes of-thalassemia-1 of Southeast Asia type (- -SEA) with– ^4.2,– ^3.7,– ^G-Taichung, and ^CS. We found the X box of– ^3.7 belonged to the X box of 2 globin gene and the X box of ^cs contained X boxes of both al and2 globin gene, whereas the X box of– ^4.2 and– ^G-Taichung was a hybrid of X boxes of 2 and 1 globin gene. We also found there are two types of– ^4.2 deletion; type 1 is a common type of– ^4.2 deletion and type 2 is linkage to– ^G-Taichung. We used a combination of two methods, the amplification refractory mutation system (ARMS) and the amplified created restriction sites (ACRS), to amplify the hybrids of X boxes specifically. The upstream primer for X box of2 globin gene was designed following the standard ARMS procedure to amplify the X segment of the-globin gene. The downstream primer was designed according to the ACRS method to check the specificity of PCR products. Using this approach, we can diagnose the different types of– ^4.2 deletion. This kind of approach can also be used to amplify the specific region from the cluster of highly homologous genes.     

Expression of the tumor necrosis factorα gene and early response genes by nodularin, a liver tumor promoter, in primary cultured rat hepatocytes

Nodularin is a new liver carcinogen possessing a potent tumor-promoting activity in rat liver, mediated through inhibition of protein phosphatases 1 and 2A, and a weak initiating activity. Since we previously reported evidence that nodularin up-regulated expression of the tumor necrosis factor gene (TNF) and early-response genes in rat liver after its i.p. administration, and since TNF had tumor-promoting activity in vitro, it is possible that TNF itself is involved in liver tumor promotion. We investigated whether hepatocytes themselves induce expression of theTNF gene and early-response genes in primary cultured rat hepatocytes treated with nodularin. Like nodularin, microcystin-LR, which is another liver tumor promoter belonging to the okadaic acid class, strongly inducedTNF gene expression in rat hepatocytes, as well as TNF release from those cells into the medium. On the other hand, 12-O-tetradecanoylphorbol-13-acetate, which has been reported to induce no tumor promotion in rat liver, induced no apparent expression of theTNF gene in primary cultured rat hepatocytes. As for the expression of early-response genes, 1 M nodularin or microcystin-LR induced expression of the c-jun, jun B,jun D, c-fos, fos B andfra-1 genes in the hepatocytes, and the expression of these genes was prolonged up to 24 h, suggesting mRNA stabilization induced by inhibition of protein phosphatases 1 and 2A. This paper presents new evidence that theTNF gene and early-response genes were expressed in hepatocytes treated with a liver tumor promoter.Abbreviations GST-P glutathioneS-transferase placental form - TNF tumor necrosis factor - GAPDH glyceraldehyde-3-phosphate dehydrogenase - SSC standard saline citrate - SDS sodium dodecyl sulfate - TPA 12-O-tetradecanoylphorbol-13-acetate     

Induction of epstein-barr virus early antigens by tumor promoters of the diterpene ester type in Raji cells and specific (receptor) binding as compared to irritant and promoting activities

Summary Sixteen new diterpene esters (DTE) of the tigliane, ingenane, daphnane, and 1-alkyldaphnane types were investigated in two in vitro assays: as inhibitors of specific binding of ³H-labeled 12-O-tetradecanoylphorbol 13-acetate (TPA) to protein kinase C in a receptor preparation from mouse brain, and as inducers of Epstein-Barr virus (EBV) early antigens in Raji cells. Inhibition of binding of [³H]TPA to the receptor preparation by tigliane and ingenane DTE correlates with irritant activity in vivo, while some daphnane and 1-alkyldaphnane DTE inhibit binding of [³H]TPA in a less pronounced manner but still are very irritant. Tumor-promoting activity does not correlate consistently with the receptor-binding data. To test the hypothesis that early antigen induction in Raji cells by DTE is coupled to functional DTE receptors (protein kinase C), the latter were searched on these Raji cells by a cold acetonefilter assay and shown to be present. The dependence of the early antigen induction rate on the concentration of the DTE tested was demonstrated. At a given concentration of DTE, differences in the induction rate between various DTE are seen. However, a clear quantiative correlation either between early antigen induction and receptor binding data in vitro, or early-antigen-inducing activity in vitro versus irritancy and tumor-promoting activity in vivo was not observed.Abbreviations ANPA 12-O-anthroylphorbol 13-acetate - DPH 12-deoxyphorbol 13-hexadecanoate - DPT 12-deoxyphorbol 13-tetradecanoate - EBV Epstein-Barr virus - HPA 12-O-hexadecanoyl-phorbol 13-acetate - HPA-6,7-oxide 12-O-hexadecanoyl-6,7-epoxyphorbol 13-acetate; I²⁴, irritancy calculated from ID ₂₄ ⁵⁰ - ID ₂₄ ⁵⁰ irritant dose 50 read 24 h after administration of compound - K _a ^d apparent dissociation constant - K _a ⁱ apparent inhibition constant - PDD phorbol 12,13-didecanoate - R _a ^t apparent total number of receptors - RPA 12-O-retinoylphorbol 13-acetate - TPA 12-O-tetradecanoylphorbol 13-acetate - 3-TI 3-O-tetradecanoylingenol - 4-PDD 4-phorbol 12,13-didecanoate Dedicated to Professor Dr. W. Kunz, Institute of Biochemistry, German Cancer Research Center, Heidelberg, on the occasion of his 65th birthday     

Comparison of the effects of three different treatment regimens of recombinant interferons (r-IFN α, r-IFN γ, and r-IFN α +cimetidine) in disseminated malignant melanoma

Summary A total of 30 patients with progressively growing visceral and/or cutaneous malignant melanoma metastases were entered in a prospective phase II trial comparing three different therapeutic regimens of recombinant interferons (r-IFN). The first group of 12 patients received r-IFNA 9–36 IU/day i.m. on 5 consecutive days/week. A second group of 11 patients was treated with r-IFNA and oral cimetidine, 1000 mg/day. The third group of 7 patients had i.v. infusions of r-IFN 0.25–0.5 mg/m² on 3 days/week. of the 12 r-IFNA-treated patients, 1 responded (complete response, CR), 5 patients exhibited no change (NC), 3 patients had progressive disease (PD), and 3 patients could not be evaluated after therapy. In the group treated with r-IFNA plus cimetidine 3 patients responded (1 CR, 2 partial responses) and 3 exhibited NC. The remaining patients showed PD. Treatment responses were found exclusively in patients with cutaneous and/or lymph node metastases. In contrast, none of the r-IFN-treated patients responded to therapy. Known IFN side effects of varying degrees, sometimes severe, were observed in all patients. Despite the small numbers of patients treated, our preliminary data indicate that r-IFNA therapy seems (1) to be of some therapeutic value in the treatment of cutaneous melanoma metastases, (2) to be superior to r-IFN therapy, and (3) that overall response rates improve with the addition of oral cimetidine to r-IFNA treatment.     

Synergistic cytotoxicity of human recombinant tumour necrosis factor α combined with microtubule effectors

Summary Combinations of human recombinant tumour necrosis factor (rhTNF) with each of four different agents disturbing the microtubule system of the cellular cytoskeleton were tested for synergistic cytotoxic action against murine melanoma B16K and L-M(S) cells. In addition to the known microtubule effectors colchicine, vincristine, and taxol, the influence of the fluorenone-azomethine derivative-diphenylene-N-{p-[bis-(-hydroxyethyl)-amino]-phenyl}-nitrone (DHPN) on the rhTNF cytotoxicity was studied. Applying a novel computerbased isobole method [Suehnel J (1990) Antiviral Res 13:23–40] concentration ranges of synergistic, zero, and antagonistic interaction were found after in vitro combination of rhTNF with each of the drugs tested in a 72-h cytotoxicity assay. In contrast, a 24-h exposure of B16K cells to these combinations still did not inhibit in vitro colony formation to a greater extent than either drug alone. A preliminary in vivo experiment revealed an increased antitumour effect after treatment of established subcutaneous melanoma B16 tumours with a combination of rhTNF and DHPN.Abbreviations rhTNF human recombinant tumour necrosis factor - DHPN -diphenylene-N-{p-[bis-(-hydroxyethyl)-amino]-phenyl}-nitrone     

Interferon-α induction of lymphocytes containing parallel tubular structures

Summary The induction by IFN- in peripheral blood lymphocytes of parallel tubular structures (PTS) and/or electron-dense granules occurring in a minority of peripheral blood lymphocytes was examined. IFN reportedly augments natural killer (NK) cell activity of large granular lymphocytes (LGL); these cells contain PTS and/or electron-dense granules. Normal peripheral blood mononuclear cells were incubated with IFN- and surface antigen expression was measured by means of indirect immunofluorescence and, at the ultrastructural level, using gold labelled monoclonal antibodies. Surface antigen reactivity with the monoclonal antibodies OKT 3, 4, 8 and Anti-Leu-7 (HNK-1) showed no difference between the IFN- incubation and non-IFN- groups. However, electron microscope investigation revealed significant absolute increases in the percentage of OKT 8⁺ and Anti-Leu-7⁺ cells which were PTS-positive after IFN- treatment compared with the control groups. The cytotoxicity assay using the K 562 cell line showed enhanced lytic activity. Our results suggest that cells coexpressing the OKT 8 and Leu-7 antigens may be responsible for a minor proportion of the increase in PTS but that IFN- mainly induces PTS and/or associated structures in cells which express the OKT 8⁺ antigen. These PTS⁺/OKT 8⁺ cells may contribute to enhanced cell cytotoxicity.     

Activation of Tumor Necrosis Factor-α System in Chronic Hepatitis C Virus Infection

Tumor necrosis factor- (TNF-)plays a central role in the host's immunomodulatoryresponse to infective agents. To evaluate theTNF- system in patients with chronic hepatitis Cvirus (HCV) infection, plasma, serum, and peripheral bloodmononuclear cells (PBMC) were prospectively collectedfrom 53 patients and 33 healthy control subjects.Circulating TNF- and TNF receptors were assayed by their respective enzyme immunoassays. Inaddition, TNF- mRNA was quantitated in PBMC usinga branched DNA assay, and production of TNF- byPBMC with and without lipopolysaccharide was also assessed. Patients with chronic HCV infectionhad a higher level of circulating TNF- comparedto healthy control subjects (9.62 ± 6.01 vs 3.66± 1.23 pg/ml, P < 0.001). They also had highercirculating levels of TNF receptors compared to control(CD120a: 3323 ± 1267, pg/ml, N = 49 vs 1855± 422 pg/ml, N = 33, P < 0.001; CD120b: 1290± 650 pg/ml, N = 51, vs 863 ± 207 pg/ml,N = 33, P < 0.001). Plasma TNF- level correlated with circulatingCD120a (r = 0.52, N = 49, P < 0.001) and weakly withCD120b (r = 0.32, N = 51, P = 0.02). Plasma TNF-also correlated with markers of hepatocellular injury, including ALT (r = 0.34, N = 53, P = 0.01) and-GST (r = 0.31, N = 43, P = 0.042), but not withserum HCV RNA levels. There was no difference in theTNF- mRNA levels in PBMC between patients with chronic HCV infection (1.4 ± 1.9units/106 cells, N = 8) and healthy control subjects(2.1 ± 1.4 units/106 cells, N = 8, P = NS). Therewas also no difference in the spontaneous production ofTNF- by PBMC (1 × 10⁶ cells/ml)between patients with chronic HCV infection (14.2± 36.5 pg/ml, N = 11) and healthy subjects (11.9± 14.0 pg/ml, N = 14, P = NS). However, patientswith chronic HCV infection produced more TNF- upon stimulation withlipopolysaccharide compared to healthy control subjects(1278 ± 693 pg/ml, N = 11, vs 629 ± 689pg/ml, N = 14, P < 0.05). These data indicate thatthe TNF- system is activated in patients with chronicHCV infection.     

Iloprost antagonizes the increase in internal calcium concentration induced by α-thrombin in human platelets: A study of desensitization

Summary We studied the interaction between the synthetic prostacyclin analog iloprost and the aggregating agent -thrombin by measuring the internal calcium ion concentration ([Ca²⁺]i) of human fura-2-loaded platelets. Iloprost (0.003–100 µg/l) did not modify the resting calcium level; when added 2 minutes before exposure of the platelets to a submaximally active concentration of -thrombin (10 U/l), iloprost dose-dependently antagonized the increase in [Ca²⁺]i. To evaluate if iloprost retained this antagonistic effect even after a prolonged contact, which is well known to cause a desensitization phenomenon, platelets were prein-cubated with iloprost (35 µg/l) for 3 hours. After washout, the effect of newly added iloprost (0.01–100 µg/l) on the -thrombin-induced increase in [Ca²⁺]i was tested. Iloprost was still able to antagonize the increase in [Ca²⁺]i induced by -thrombin in desensitized platelets; however, the dose-inhibitory response curve was significantly shifted to the right when compared with that obtained in control platelets (i.e., platelets preincubated for 3 hours with iloprost's solvent), and the resulting IC₅₀ was significantly higher: 1.78 versus 0.2 µg/l (p<0.001). Since the maximal inhibitory effect of iloprost could also be reached under these experimental conditions, we conclude that iloprost retains its ability to antagonize the increase in [Ca²⁺]i induced by -thrombin in desensitized platelets.     

Bone marrow and peripheral blood globin chain biosynthesis in iron deficiency

Summary Globin chain synthesis was studied in 13 iron-deficient patients. The mean whole-cell globin / ratio in the peripheral blood of 11 patients was 1.05±0.06 which is similar to the value 0.99±0.08 obtained for 10 controls. The ratios odtained for stroma-free globin were not significantly different from those of whole cell preparations. In contrast, the / ratio of bone marrow was 0.73±0.14 in 10 iron deficient patients, which is significantly lower than that of controls. Two other patients had decreased / ratios in the peripheral blood, probably because of the presence of an -thalassemia gene. These results demonstrate a reduced rate of synthesis of chains relative to that of chains in the bone marrow of iron-deficient patients that is not demonstrable in the peripheral blood.This work was partly supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil     

Benzo[a]pyrene-diol-epoxide-induced anchorage-independence in diploid human fibroblasts

Summary Treatment of diploid human fibroblasts with stereoisomeric benzo[a]pyrene anti and syn diol epoxides has been shown to induce anchorage-independent clones of cells with a dose dependence and frequency [(0.5–12)×10^-4] not significantly different from mutations at the hypoxanthine-guanine phosphoribosyltransferase locus [(1–8)×10^-4] in these cells. The majority of the anchorage-independent clones that were picked retained their mutagen-induced, anchorage-independent phenotype through at least 20 generations of expansion in monolayer culture. No variant cells showing extended life-span were detected among survivors in any of the mutagen treatment groups (<1.6×10^-7 frequency). Extensive analysis of a pool of 15 cellular protooncogenes (Ha-ras, Ki-ras, N-ras, mos, fos, fes, myc, abl, sis, myb, erbA, erbB, src, raf, N-myc), using Southern and northern blot analysis, was done to determine whether mutagen-induced rearrangement, amplification or overexpression of any of these genes was responsible for the mutagen-induced, anchorage-independent phenotype. We found no evidence that the genomic arrangement or expression level of any of these genes had been altered, thus indicating that an alternative form of mutation, or an alternative gene not included in this screening was responsible for the mutagen-induced, anchorage-independent phenotype.Abbreviations used s⁶G^r 6-thioguanine-resistant - anti diol epoxide, racemic mixture of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - syn diol epoxide, racemic mixture of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene - DME Dulbecco's modified Eagle medium - hprt hypoxanthineguanine phosphoribosyltransferase     

Na,K-ATPase isoform gene expression in normal and hypertrophied dog heart

Objectives The catalytic subunit of the sodium-potassium ATPase, the target of digitalis glycosides, has three isoforms is tissuespecific and developmentally regulated. While the effect of pressure overload on Na, K-ATPase isoform expression has been studied in rodent heart, there are no systematic data on this question in hearts of larger animals, which differ from those of rodents both in isoform composition and in glycoside sensitivity. Thus, we investigated the expression of Na, K-ATPase isoforms in normal dog heart; we also examined the effect of experimental left ventricular hypertrophy on isoform expression.Methods hypertrophy was produced by aortic banding. Expression was assessed by quantitative Northern and Western blotting, immuno-fluorescence, and³H-ouabain binding.Results RNA blotting indicated that the 3 isoform represented 11% of Na, K-ATPase mRNA in normal dog LV. Normal dog LV expressed 1 and 3 protein, but no detectable 2; immunoreactive 1 and 3 protein were also present in Purkinje fibers. There was a statistically significant decrease in total expression of all isoform mRNA's in hypertrophied dog LV, resulting in a greater proportion of 1. The expression level of the 3 isoform mRNA and protein was lower in hypertrophied hearts.Conclusions These results indicate a greater proportion of 1 isoform pumps in experimental canine hypertrophy. Thus, shifts in Na, K-ATPase isoforms occur in pressure-overloaded heart in large animals as well as rodents.     

Mutation of E1α gene in a female patient with pyruvate dehydrogenase deficiency due to rapid degradation of E1 protein

Summary A mutation of an insertion of 4 bp in the gene for the subunit of pyruvate dehydrogenase (E1) was found in a female with pyruvate dehydrogenase deficiency due to the rapid degradation of and subunit proteins of pyruvate dehydrogenase. This mutation caused a frameshift that altered the amino acid sequence and created a premature stop codon. This 4-bp insertion has been found in an unrelated female patient with E1 deficiency. It is rare that the same mutation is found in unrelated patients with this rare inborn error of metabolism. Furthermore, short deletions or duplications in the E1 gene of patients with E1 deficiency have been found only in exons 10 and 11. These exons may be hot spots for the mutations by the recombinational processes. This patient was heterozygous for the normal and a mutant allele. However, in most of the cultured skin fibroblasts from this patient, the mutant allele was expressed. These observations suggest that the X chromosome containing the normal allele was predominantly inactivated so that she developed lactic acidaemia and neurological abnormalities despite being heterozygous. The mutant subunit protein failed to form a stable structure of pyruvate dehydrogenase, so that both and subunit proteins were degraded rapidly.     

Mutation of the 97-197-197-1subunit of the pyruvate dehydrogenase complex,in relation to heterogeneity

Summary Pyruvate dehydrogenase complex was studied using bio- and immunochemical methods in cultured cells derived from two patients with the severe type and one patient with the mild type of pyruvate dehydrogenase complex deficiency. In patients 1 and 2, enzyme activity was all but undetectable and associated with the absence of E₁ subunit of the complex. Patient 3 had a slightly reduced level of enzyme activity, and this was associated with a larger form of E₁ subunit. The amount and size of E₁ mRNA in the three patients was similar to that of control samples. Thus, the severity of E₁ deficiency in these three patients is likely to depend on the type of mutation in the pyruvate dehydrogenase E₁ subunit and the synthesis and degradation rate of the subunit.     

Effect of urapidil on the action potentials in the guinea-pig ventricular myocardium

Summary The electrophysiological effects of urapidil, a new ₁-adrenoceptor antagonist, were assessed in the reserpinized guinea-pig ventricular myocardium. Urapidil suppressed the maximal rate of rise (_max) of steady-state action potentials elicited by the fast responses at high concentrations independently of blockade of myocardial -adrenoceptors, but not the _max of Ca²⁺-dependent slow action potentials of partially depolarized muscles in concentrations tested (up to 1.1 mM). Urapidil at high concentrations prolonged the action potential durations of the fast and slow responses in a manner similar to the quinidine-like antiarrhythmic drugs. These results suggest that the inhibitory effect of urapidil on the slow inward Ca²⁺ current and the Na⁺ current is in practice negligible.