Expression of human hypoxanthine phosphoribosyl transferase in Chinese hamster cells treated with isolated human chromosomes.

Chinese hamster cells deficient for the enzyme hypoxanthine phosphoribosyl transferase (HPRT) were incubated with isolated human metaphase chromosomes and 21 colonies were isolated in HAT medium. Three different types of cell lines were established from these clones. First, 4 cell lines had 10-30% of normal Chinese hamster HPRT activity with the same electrophoretic mobility as human HPRT. This HPRT activity remains detectable during at least 8 weeks of growth of the cells in nonselective medium. Second, 3 cell lines also had human-like HPRT with the same activity as the first type. This HPRT persists only if the cells are grown in HAT medium and disappears during 8 weeks of growth in nonselective medium. Third, other clones survived in HAT medium as well as in medium with 8-azaguanine. These cells had no detectable HPRT activity. Using differential chromosome staining techniques no recognizable human chromosome fragments were found in any of the cell lines.     

Cloning and functional expression of a human heparanase gene.

We have cloned a gene (HSE1) from a human placental cDNA library that encodes a novel protein exhibiting heparanase activity. The cDNA was identified through peptide sequences derived from purified heparanase isolated from human SK-HEP-1 hepatoma cells. HSE1 contains an open reading frame encoding a predicted polypeptide of 543 amino acids and possesses a putative signal sequence at its amino terminus. Northern blot analysis suggested strong expression of HSE1 in placenta and spleen. Transient transfection of HSE1 in COS7 cells resulted in the expression of a protein with an apparent molecular mass of 67-72 kDa. HSE1 protein was detectable in conditioned media but was also associated with the membrane fraction following cell lysis. The HSE1 gene product was shown to exhibit heparanase activity by specifically cleaving a labeled heparan sulfate substrate in a similar manner as purified native protein.     

Purine nucleotide reutilization by human lymphoblast lines with aberrations of the inosinate cycle

A purine nucleotide (inosinate) cycle is demonstrated with human lymphoblasts. The lymphoblast requires approximately 50 nmol of purine/10(6) cell increment. When the inosinate cycle is interrupted by the genetic, severe deficiency of either or both purine nucleoside phosphorylase (PNP) or hypoxanthine phosphoribosyltransferase (HPRT), purine accumulates in the culture medium as inosine, guanosine, deoxyinosine, and deoxyguanosine (PNP deficiency or PNP, HPRT deficiency) or hypoxanthine and guanine (HPRT deficiency). This accumulation represents an additional 25 to 32 nmol of purine which must be synthesized per 10(6) cell increment. PNP-deficient lymphoblasts have PPRibP contents characteristic of normal lymphoblasts, about 20 to 25 pmol/10(6) cells. HPRT-deficient lymphoblasts have four times higher PPRibP contents. The lymphoblast deficient for both PNP and HPRT has only a marginal elevation of PPRibP content, 1.5 times normal values. The elevated PPRibP content of HPRT-deficient cells reflects the efficient, unilateral reutilization of the ribose moiety of purine ribonucleotides and is not a cause of purine overproduction. Purine overproduction characterizing PNP-deficient lymphoblasts appears similar to overproduction from deficiency of HPRT, i.e. a break in the inosinate cycle rather than overactive de novo purine synthesis.     

Characterization of renin-like activity in human plasma protein IV-4 fraction.

Cohn's fraction IV-4 of human plasma protein generates by itself a substance with slight pressor activity when incubted without any additives. This renin-like activity was readily inactivated by alkaline treatment. Furthermore, the generated pressor substance, when purified, was found very similar to [Aspl]-[Ile5]-angiotensin-I in the following characteristics; 1) heat-stability, 2) dializability, 3) inactivation by trypsin, 4) Rf value and 5) electrophoretic mobility at various pHs, 6) marked enhancement in pressor and oxytocic activities after incubation with normal human plasma or rabbit's lung extract, and 7) cross reactivity with [AspI]-[Ile5]-angiotensin-I in radioimmunoassay. It is concluded that renin-like activity in the preparation of fraction IV-4 of human plasma protein must be renin or an extremely similar enzyme.     

Analysis of mutational effects at the HPRT locus in human G(0) phase lymphocytes irradiated in vitro with gamma rays

The mutational effects of ionising radiation at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus were studied in human peripheral blood G(0) phase lymphocytes irradiated in vitro with gamma rays. The presence of radiation induced mutants was assessed by selecting the HPRT mutants every week on the basis of 6-thioguanine resistance up to 1 month after irradiation. A dose-related increase of 14.25x10(-6) mutants/Gy was measured after an expression time of 7 days. After 2 weeks from culture starting the fraction of clonable cells in irradiated and control cell populations decreased, limiting the measurements of mutant frequency. The mutational spectrum of the HPRT gene was determined by PCR analyses in a total of 99 mutant clones derived from irradiated lymphocytes. The independent origin of mutant clones carrying the same mutation was assessed by analysing the TCR gamma gene rearrangements. The results showed a dose-related increase of deletion mutants up to 3Gy, whereas point mutation frequency increased only up to 2Gy. Two preferentially deleted regions were identified; one involving the HPRT exon 3, and another one the 3'-terminal and the 3'-flanking region of the gene. One complex mutation involving a non-contiguous deletion of exons 2-5 and 7/8 was observed among the mutants isolated after 3Gy irradiation.     

Expression and characterization of glycolipid-anchored B7-1 (CD80) from baculovirus-infected insect cells: protein transfer onto tumor cells.

Tumor cells can be modified to express immunostimulatory molecules such as B7-1 by protein transfer using purified glycosylphosphatidylinositol-anchored B7-1 (GPI-B7-1). In this study recombinant baculovirus encoding GPI-B7-1 (vBacB7-1(GPI)) was established to obtain large quantities of purified GPI-B7-1 to modify tumor cells by protein transfer. vBacB7-1(GPI)-infected insect cells showed high-level cell surface expression of GPI-B7-1 that was susceptible to PIPLC treatment. GPI-B7-1 expressed in insect cells (Bac-GPI-B7-1) mediated T cell proliferation, indicating that the GPI-B7-1 retains costimulatory activity. Moreover, Bac-GPI-B7-1 was completely solubilized in Triton X-100 at 4 degrees C compared to 22% solubilization of GPI-B7-1 expressed in CHOK1 cells, suggesting that GPI-anchored proteins expressed in insect cells may not be clustered into the detergent-insoluble fraction. SDS-PAGE analysis of Bac-GPI-B7-1 showed faster mobility (45 kDa) compared to GPI-B7-1 from CHOK1 (68 kDa) and this difference may be due to a difference in glycosylation. Cell binding assays showed that immunoaffinity-purified Bac-GPI-B7-1 retained its functional ability to bind CD28(+) cells. Moreover, when human tumor cells were incubated with this functionally active purified GPI-B7-1, an efficient transfer of B7-1 onto tumor cells was observed. These results demonstrate that GPI-B7-1 can be expressed in insect cells in a functionally active form and can be used to modify tumor cells for immunotherapeutic applications.     

Intraspecies transfer via total cellular DNA of the gene for hypoxanthine phosphoribosyltransferase into cultured mouse cells

Summary Under selective growth conditions a revertant of mouse cells, defective in hypoxanthine phosphoribosyltransferase activity (HPRT, EC-No. 2.4.2.8), was isolated, which contained an electrophoretically abnormal form of HPRT activity. The specific HPRT activity in crude extracts of the revertant cells is about 30% of the level determined in normal wild type cells. The variant HPRT reacts with antiserum against normal mouse HPRT but the rate of heat inactivation of the variant activity is different from the wild type form. By isozyme and karyotype analyses of somatic cell hybrids between the revertant mouse cells and Chinese hamster cells we found that the abnormal HPRT activity is coded for by the mouse X-chromosome as expected for a mutation in the structural HPRT gene.DNA has been purified from the abnormal HPRT revertant cells and incubated with mouse A9 cells (HPRT^-). After growth in selective medium one clone was isolated which expressed the electrophoretically abnormal form of HPRT. Six clones showed the normal form of HPRT due to reversion of the defective HRRT locus in A9 cells. This result indicates DNA-mediated transfer of the mouse HPRT gene at a frequency of about 0.5×10^-7. A similar frequency has been found for transfer of the variant HPRT locus via isolated metaphase chromosomes to A9 recipient cells. When placed in non-selective media the DNA-mediated transferent cells gradually lost their ability to express the HPRT transgenome at a rate of about 6% per average cell generation.     

Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells.

The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.     

A 55,000-60,000 Mr receptor protein for urokinase-type plasminogen activator. Identification in human tumor cell lines and partial purification

The iodinated Mr approximately equal to 15,000 amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (u-PA) molecule bound specifically to the cell surface of all of seven cultured human tumor cell lines studied. Cross-linking of iodinated ATF to the cell surface using a bifunctional amino-reactive reagent followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed with the four cell lines studied the occurrence of a single band migrating with an Mr of 70,000-75,000, indicating complex formation with an Mr of 55,000-60,000 u-PA receptor protein (u-PA-R). In the human monocyte cell line U937 cultivated in the presence of phorbol ester, the amount of complex was strongly increased, and a fraction of the complex had a slower electrophoretic mobility. Comparison between autoradiograms of reduced and unreduced samples suggests that u-PA-R consists of one polypeptide chain. Two forms of u-PA-R, which differed with respect to affinity to concanavalin A, were identified. u-PA-R retained its ability to bind to ATF after cell lysis, and it was purified approximately 2,200-fold from biosynthetically labeled U937 cells by affinity chromatography with proenzyme u-PA coupled to Sepharose. The purified Mr 55,000-60,000 protein was specifically bound and cross-linked to u-PA, proenzyme u-PA, and ATF, but not to tissue-type plasminogen activator or other unrelated proteins.     

Comparison of the cytolytic effects in vitro on Trypanosoma brucei brucei of plasma, high density lipoproteins, and apolipoprotein A-I from hosts both susceptible (cattle and sheep) and resistant (human and baboon) to infection.

The African trypanosome, Trypanosoma brucei brucei causes a fatal wasting disease in livestock but does not ordinarily infect humans, apparently because this unicellular parasite is lysed by high density lipoproteins (HDL) in human serum. To assess whether there is a specific active constituent in trypanolytic HDL, we have systematically compared the cytotoxic action on T.b.brucei in vitro of native and delipidated HDL, and of individual apolipoproteins, from nonpermissive hosts (human and baboon) with their counterparts from susceptible hosts (cattle and sheep). When suspensions of trypanosomes were incubated for 2 h at 37 degrees C with human or baboon plasma most cells were lysed, but not with bovine or sheep plasma. Similarly, HDL isolated from human and baboon plasma were trypanolytic (typically about 95% and 60% lysis, respectively, at 1 mg protein/ml), whereas bovine and sheep HDL were benign (less than 8% lysis). Subfractionation of human HDL by serial isopycnic ultracentrifugation and by heparin-Sepharose affinity chromatography established that the denser and smaller particles had greater trypanolytic activity both in vitro and in vivo. When human HDL was delipidated, the trypanocidal activity was associated with the water-soluble protein (apolipoprotein) fraction and not with the lipid constituents. Bovine apolipoproteins were also weakly trypanolytic in free solution (20-40% lysis), but not when complexed with cholesterol-phospholipid liposomes (less than 10% lysis). The major apolipoprotein of human HDL, apolipoprotein (apo) A-I had full trypanolytic activity (89-95% lysis at 1 mg protein/ml) when purified, whether in solution or incorporated into liposomes, but other apolipoproteins isolated from human HDL, including apoA-II, apoC, and apoE, were nontrypanolytic. Purified baboon apoA-I was also trypanolytic, though less potent than human apoA-I, but apoA-I from permissive hosts (cattle and sheep) was inactive when presented in liposomes. Incubation of bovine or sheep HDL with purified human apoA-I, and subsequent separation of the HDL by ultracentrifugation, produced chimeric HDL containing significant amounts of the human apolipoprotein; these particles showed appreciable trypanolytic activity. By contrast, human HDL particles in which about 70% of the apoA-I had been displaced with apoA-II had markedly reduced lytic properties compared to the native HDL (30% versus 80% lysis at 0.6 mg total protein/ml). We tentatively conclude that the trypanolytic activity of native human or baboon plasma resides in the apoA-I content of the HDL particles and that, conversely, bovine and sheep plasma are inactive because the apoA-I polypeptide present in their HDL lacks trypanocidal activity.     

The kringle domain of tissue-type plasminogen activator inhibits in vivo tumor growth

The two-kringle domain of tissue-type plasminogen activator (t-PA) has previously been shown to contain anti-angiogenesis activity. In this study, we explored the potential in vivo anti-tumor effects of the recombinant kringle domain (TK1-2) of human t-PA. Anti-tumor effects of purified Pichia-driven TK1-2 were examined in nude mice models by subcutaneous implantation of human lung (A-549) and colon (DLD-1, HCT-116) cancer cell lines. Mice bearing the tumors were injected with PBS or purified TK1-2 (30 mg/kg) i.p. every day for 22 days. TK1-2 treatment suppressed the A-549, DLD-1, and HCT-116 tumor growth by 85.3%, 52.4%, and 62.5%, respectively. Immunohistological examination of the tumor tissues showed that TK1-2 treatment decreased the vessel density and also the expression of angiogenesis-related factors including angiogenin, VEGF, alpha-SMA, vWF, and TNF-alpha, and increased the apoptotic fraction of cells. TK1-2 neither inhibited in vitro growth of these cancer cells nor affected t-PA-mediated fibrin clot lysis. These results suggest that TK1-2 inhibits the tumor growth by suppression of angiogenesis without interfering with fibrinolysis.     

Elevated levels of erythrocyte hypoxanthine phosphoribosyltransferase associated with allelic variation of murine Hprt

Murine stocks with wild-derived hypoxanthine phosphoribosyltransferase (HPRT) A alleles (Hprt a) have erythrocyte HPRT activity levels that are approximately 25-fold (Mus musculus castaneus) and 70-fold (Mus spretus) higher than those of laboratory strains of mice with the common Hprt b allele (Mus musculus: C3H/HeHa or C57B1/6). Since the purified HPRT A and B enzymes have substantially similar maximal specific activities (64 and 46 units/mg of protein, respectively), we infer that these HPRT activity levels closely approximate the relative levels of HPRT protein in these cells. Red blood cells of HPRT A and B mice have similar levels of adenine phosphoribosyltransferase activity (APRT; EC 2.4.2.7) and reticulocyte percentages, which suggests that the elevated levels of HPRT in erythrocytes of HPRT A mice are not secondary consequences of abnormal erythroid cell development. The HPRT activity levels in reticulocytes of HPRT B mice are approximately 35-fold higher than the levels in their erythrocytes and approach the HPRT activity levels in reticulocytes of HPRT A mice. Thus, the marked differences in the levels of HPRT protein in erythrocytes of HPRT A and B mice result from differences in the extent to which the HPRT A and B proteins are retained as reticulocytes mature to erythrocytes. The substantial and preferential loss of HPRT B activity from reticulocytes is paralleled by an equivalent loss of HPRT immunoreactive protein (i.e., CRM) from that cell, and we infer that the HPRT B protein is degraded or extruded as reticulocytes mature to erythrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Natural killer cells in rhesus monkeys: properties of effector cells which lyse Raji targets

Spontaneously occurring natural killer cell activity of rhesus monkey peripheral blood mononuclear cells was assayed against five human cell lines, three of which were Epstein-Barr virus (EBV) positive, including the human B cell line Raji. The lytic activity to Raji cells was high, significantly higher than to any other cell line tested. Raji cells are normally insensitive to spontaneous lysis by human NK cells, and the contrasting vigor of the rhesus monkey cytolytic activity to Raji prompted us to investigate the properties of this effector cell. We found the effector cell-mediating lysis of Raji to be nonadherent and phagocytic with lytic activity slightly enhanced in the E-rosette-forming cell (ERFC+) fraction and decreased in the ERFC- fraction. Further isolation of FcIgG receptor-positive and FcIgG receptor-negative subsets by rosetting resulted in significant enrichment of NK activity to Raji in the positive fraction and a loss of activity in the negative fraction. Depletion studies with various monoclonal antibodies (mAb's) confirmed that nearly all lytic activity was contained in the CD16+ (Leu 11b+) population, while subsets of effector cells expressed CD2 (9.6) and CD8 (OKT8). Depletion of CD4 (OKT4)-, HLADR (OKIa)-, or LFA1 (MAC-1)-positive populations failed to reduce NK activity. We compared the phenotypic properties of alloimmune effector cells exhibiting specificity for allogeneic donor targets with those exhibiting lysis of Raji targets. Results indicated that allospecific cytotoxic T lymphocytes expressed a CD16-, CD2+ phenotype, a pattern distinct from that of the effector cell population recognizing Raji targets. The presence of CD2 mAb's in the culture had no effect on NK lytic activity. In contrast, mAbs CD8 and Leu 11b were inhibitory. This would suggest a functional role for CD8 and FcIgG molecules in the lysis of Raji cells by rhesus effectors. In summary, these studies describe a distinct population of effector cells in the blood of rhesus monkeys which exhibit spontaneous lytic activity to Raji cells and exhibit the properties of NK cells.     

Inhibition by monochloramine of the transport of glutamine and glucose in HeLa cells and lymphocytes.

1. Chloramine was previously shown to inhibit glutamine uptake by human lymphoblast tumour cells. In the present study, the effect of monochloramine on the glutamine and glucose transport systems in HeLa cells and rat mesenteric lymphocytes was investigated. 2. Initial exposure to monochloramine slightly inhibited both the glutamine and glucose transport systems in HeLa cells. However, pre-exposing the cells to monochloramine increased its inhibitory action. 3. Similar results were obtained using rat mesenteric lymphocytes, which suggests that monochloramine's effects are not cell specific. 4. Only the Na(+)-independent (system L) component of glutamine transport activity in HeLa cells was inhibited by monochloramine. 5. Dithiothreitol protected both the glucose and glutamine transport carriers in HeLa cells against monochloramine inhibition. 6. Monochloramine did not inhibit HeLa cell metabolism, nor enhance cell lysis, which, in conjunction with other experimental data, suggests that monochloramine inhibits cellular transport activity by binding to thiol groups present on the membrane.     

Regulation of purine de novo synthesis in cultured human fibroblasts: the role of P-ribose-PP.

Procedures for assaying the rate of purine de novo synthesis in cultured fibroblast cells have been compared. These were (i) the incorporation of [(14)C]-glycine or [(14)C]formate in alpha-N-formylglycinamide ribonucleotide (an intermediate in the purine synthetic pathway) and (ii) the incorporation of [(14)C]-formate into newly synthesised cellular purines and purines excreted by the cell into the medium. Fibroblast cells, derived from patients with a deficiency of hypoxanthine phosphoribosyltransferase (HPRT-) (EC 2.4.2.8) and increased rates of purine de novo synthesis, were compared with fibroblasts from healthy subjects (HPRT+). Fetal calf serum, which was used to supplement the assay and cell growth medium, was found to contain sufficient quantities of the purine base hypoxanthine to inhibit purine de novo synthesis in HPRT+ cells. This inhibition was the basis of differentiation between HPRT- and HPRT+ cells. In the absence of added purine base, both cell types had similar capacities for purine de novo synthesis. This result contrasts with the increased rates of purine de novo synthesis reported for a number of human HPRT- cells in culture but conforms recent studies made on human HPRT- lymphoblast cells. The intracellular concentration and utilisation of 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP), a substrate and potential controlling factor for purine de novo synthesis, were determined in HPRT- and HPRT+ cells. The rate of utilisation of P-Rib-PP in the salvage of free purine bases was far greater than that in purine de novo synthesis. Although HPRT- cells had a 3-fold increase in P-Rib-PP content, the rate of P-Rib-PP generation was similar to HPRT+ cells. Thus, in fibroblasts, the concentration of P-Rib-PP appears to be critical in the control of de novo purine synthesis and its preferential utilisation in the HPRT reaction limits its availability for purine de novo synthesis. In vivo, HPRT+ cells, in contrast to HPRT- cells, may be operating purine de novo synthesis at a reduced rate because of their ability to reutilise hypoxanthine.     

Purification and characterization of human lymphoblast N-acetylglucosamine-1-phosphotransferase.

N-Acetylglucosamine-1-phosphotransferase (GlcNAcPTase) was solubilized with 2% Tergitol NP-10 from cultured human lymphoblast cells and purified 3840-fold with 14% recovery using lentil lectin-Sepharose 4B, DEAE-Sephacel and Sephacryl S-400 chromatographies. The partially purified enzyme requires the non-ionic detergent Tergitol NP-10 and a divalent cation, Mn2+ or Mg2+, for its activity and exhibits an optimal pH at 7.2-7.5 in Tris-maleate buffer. Kinetic studies demonstrated an apparent Km of 24 microM for the donor UDP-N-acetylglucosamine and of 117 mM for the artificial acceptor alpha-methylmannoside. The GlcNAcPTase is inhibited by UDP and UDP-glucose, and by negatively charged phospholipids including phosphatidylserine, phosphatidylglycerol and phosphatidic acid. The apparent mol. wt of the human lymphoblast GlcNAcPTase is approximately 1000 kDa, which is analogous to that reported for the partially purified enzyme from rat liver (Waheed et al., 1982).     

Identification of RNA as a complement inhibitory component in an extract of Ehrlich ascites tumor cells.

A factor capable of inhibiting complement was obtained from intact Ehrlich ascites tumor cells by mild extraction with phosphate-buffered saline (PBS). The inhibitor caused a decrease in extent of lysis of EAC14 with a concomitant extension of Tmax. EA, EAC1, EAC4 and EAC142 were all less susceptible to complement-mediated lysis after treatment with the tumor cell extract. Partial purification of a complement inhibitor was accomplished. The inhibitor was rich in RNA and its activity was totally destroyed by RNAase but not DNAase. RNA from mouse tissues, yeast, and Escherichia coli also inhibited complement hemolytic activity. The partially purified material only inhibited lysis of EAC1 and EAC14. Slow inhibition of fluid phase C1 was also demonstrated. In addition, RNA-rich partially purified tumor cell extract was capable of precipitating with purified human C1q.     

Interleukin-2- and mitogen-activated NK-like killer cells from highly purified human peripheral blood T cell (CD3+ N901-) cultures

In this study, highly purified (HP) CD3-positive N901-negative T lymphocytes could be induced to become natural killer (NK)-like in culture in the presence of recombinant interleukin-2 (rIL-2) and phytohemagglutinin (PHA). Thus, purified CD3+ N901- T cells from fresh human peripheral blood were obtained by negative selection using an indirect panning technique. To ensure that T lymphocyte fractions were completely devoid of any detectable NK cells, two additional purification procedures were employed: incubation of post-pan T cells with the NK-cytotoxic lysomotropic agent L-leucinemethylester, and complement-mediated lysis using the NK cell specific NKH1a monoclonal antibody. Purity of CD3+ N901- cells could be confirmed by surface marker analysis, whereby two NK-associated antigens, N901 and H-25, were undetectable, while 94 +/- 1% of cells expressed the CD3 (Leu-4) antigen. On functional analysis, fresh HP CD3+ N901- cells exhibited no cytotoxic activity against the standard NK target K562. When HP NK-depleted T lymphocytes were cultured for 7 days in the presence of rIL-2 (100 U/ml), neither surface antigen expression nor cytotoxic activity against K562 changed significantly. However, significant cytotoxicity against K562 [18 +/- 5% specific lysis at 25:1 effector:target (E/T) ratio] could be induced when HP CD3+ N901- cells were grown for 7 days in the presence of rIL-2 and PHA (0.5% v/v). Concomitantly, antigens N901 and H-25 were found to be coexpressed on a minor proportion (22 +/- 16 and 22 +/- 6%, respectively) of CD3+ (88 +/- 2% on day 7) cells. Four-week long-term culture of HP NK-depleted T cells in the presence of rIL-2 and PHA yielded a continuous increase in cytotoxicity against K562 cells (0 up to 46% specific lysis at 25:1 E/T ratio). Of particular interest was the emergence of cytotoxicity against the NK-resistant Daudi cell target (15 +/- 8% specific lysis at 25:1 E/T ratio on day 21). Expression of antigens N901 and H-25 as well as CD3 remained essentially unchanged in long-term culture. In sorting experiments, the H-25+ cell fraction was significantly enriched for cytotoxicity against K562, when compared to both H-25- and unseparated cell fractions. In summary, our results suggest that a proportion of HP CD3+ N901- T lymphocytes may give rise to cells that exhibit NK-like functional and phenotypic properties.     

Human alveolar macrophage FcR-mediated cytotoxicity. Heteroantibody- versus conventional antibody-mediated target cell lysis

Human alveolar macrophage have three distinct receptors for IgG: FcRI, FcRII, and FcRIII. In order to compare the ability of these receptors to mediate target cell lysis, three different assay systems were examined. First, we studied lysis of chicken E (CE) opsonized with heteroantibodies, which are synthetic antibodies composed of Fab fragments with anti-FcR activity covalently linked to Fab fragments with anti-CE activity. We found alveolar macrophage readily lysed heteroantibody-opsonized CE via each of the three FcR classes (FcRI, 20 +/- 5%; FcRII, 27 +/- 7%; and FcRIII, 13 +/- 13%, p less than 0.05). Non-FcR-dependent lysis of anti-beta 2-microglobulin x anti-CE heteroantibody-opsonized CE was not detected. Second, lysis of hybridoma cell lines bearing anti-FcR antibodies on their cell surface was examined to assess killing of "tumor-like" target cells. Whereas peripheral blood monocytes and lymphocytes were able to lyse hybridoma cell lines bearing surface anti-FcR mAb, alveolar macrophages were not. Third, activity of alveolar macrophage FcR was examined in a conventional antibody-dependent cellular cytotoxicity assay by using O+ (R1,R2) human RBC opsonized with human anti-D and anti-CD serum as target cells. We found lysis of anti-D and anti-CD opsonized human RBC was mediated exclusively via FcRI. No activity of FcRII or FcRIII was detected in these latter assays even if performed under conditions that impair FcRI activity. Thus, all three FcR present on alveolar macrophage mediate lysis of heteroantibody-opsonized CE; in contrast, with the use of a conventional antibody-dependent cellular cytotoxicity assay, only FcRI activity was detected. We were unable to demonstrate lysis of anti-FcR-bearing hybridoma cell lines by alveolar macrophages.     

用人DNA介导的基因转移修复中国仓鼠细胞的HPRT缺陷

 中国仓鼠卵巢细胞(CHO-K1)经N-甲基-N'-硝基-N-亚硝基胍(MNNG)诱变和6-巯基鸟嘌呤(6-TG)选择,得到稳定的次黄嘌呤磷酸核糖转移酶(HPRT)缺陷细胞株,酶活性仅为野生型的6.5％。用磷酸钙共沉淀法和电脉冲法向HPRT-细胞转移人宫颈癌细胞(HeLaS_3)基因组DNA,纠正了CHO细胞的HPRT缺陷。酶活性提高了6.9倍,达到野生型的45％。用Alu序列探针进行分子杂交,证实经过基因转移并连续传代15次以上的受体细胞中含人DNA序列。表明人的有关基因已稳定地整合到CHO细胞的染色体中。