A reliable method for detecting complexed DNA in vitro.

Quantification of eluted nucleic acids is a critical parameter in characterizing biomaterial based gene-delivery systems. The most commonly used method is to assay samples with an intercalating fluorescent dye such as PicoGreen®. However, this technique was developed for unbound DNA and the current trend in gene delivery is to condense DNA with transfection reagents, which interfere with intercalation. Here, for the first time, the DNA was permanently labeled with the fluorescent dye Cy5 prior to complexation, an alternative technique hypothesized to allow quantification of both bound and unbound DNA. A comparison of the two methods was performed by quantifying the elution of six different varieties of DNA complexes from a model biomaterial (collagen) scaffold. After seven days of elution, the PicoGreen® assay only allowed detection of three types of complexes (those formed using Lipofectin™ and two synthesised copolymers). However, the Cy5 fluorescent labeling technique enabled detection of all six varieties including those formed via common transfection agents poly(ethylene imine), poly-L-lysine and SuperFect™. This allowed reliable quantification of the elution of all these complexes from the collagen scaffold. Thus, while intercalating dyes may be effective and reliable for detecting double-stranded, unbound DNA, the technique described in this work allowed reliable quantification of DNA independent of complexation state.

Isolated growth hormone deficiency type II caused by a point mutation that alters both splice site strength and splicing enhancer function.

A heterozygous single base mutation in the human growth hormone (GH) gene (GH-1) was identified in a family presenting with isolated GH deficiency type II (IGHD II). Affected individuals have a guanine to adenine transition at the first nucleotide of exon 3 (E3+1 G-->A) that results in exon skipping and production of a dominant-negative 17.5-kDa isoform. We show that the mechanistic basis for exon skipping is due to the unique position of this mutation because it weakens the 3' splice site and simultaneously disrupts a splicing enhancer located within the first seven bases of exon 3. A G-->T mutation at this same position not only affects splicing but also results in a premature stop codon for those transcripts that include exon 3. Thus, mutations that alter the first nucleotide of exon 3 illustrate the various mechanisms by which changes in sequence can cause disease: splice site selection, splicing enhancer function, messenger RNA decay, missense mutations, and nonsense mutations. For IGHD II, only exon skipping leads to production of the dominant-negative isoform, with increasing skipping correlating with increasing disease severity.

Simultaneous irradiation for prostate cancer: intermediate results with modern techniques.

PURPOSE: In this study of men with early stage prostate cancer we evaluated treatment outcome after modern simultaneous irradiation, comprising transperineal implantation followed by external beam radiation. Disease-free survival rates were calculated according to an undetectable prostate specific antigen (PSA) nadir. MATERIALS AND METHODS: From 1992 to 1996, 689 men with clinical stage T1-T2, N0, Nx prostate cancer were treated with ultrasound guided transperineal 125iodine seed implantation followed 3 weeks later by external beam radiation. Disease-free status was defined as the achievement and maintenance of a PSA nadir of 0.2 ng./ml. or less. Median followup was 4 years (range 3 to 7). None of these men received neoadjuvant or adjuvant hormonal therapy. RESULTS: Overall 5-year disease-free survival was 88%. The 5-year rate according to PSA 4.0 ng./ml. or less, 4.1 to 10.0, 10.1 to 20.0 and greater than 20.0 was 94%, 93%, 75% and 69%, respectively. Multivariate analysis revealed that pretreatment PSA was the strongest indicator of subsequent disease-free status in regard to Gleason score or clinical stage. CONCLUSIONS: Intermediate treatment outcome analysis of modern simultaneous radiation supports the principles of radiation dose intensification for intracapsular disease plus the treatment of potential microscopic capsular penetration.

Prostate specific antigen bounce after radioactive seed implantation followed by external beam radiation for prostate cancer.

PURPOSE: Prostate specific antigen (PSA) may temporarily increase following radiotherapy for prostate cancer without signaling cancer recurrence. We describe this phenomenon which is called PSA bounce. MATERIALS AND METHODS: From 1984 to 1995, 779 stage T1T2N0 cancer cases were treated with simultaneous radiotherapy with a 125iodine prostate implant followed by external beam radiation. Median pretreatment PSA was 7.7 ng./ml. (range 0.3 to 188). PSA bounce was defined as an increase of 0.1 ng./ml. or greater above the preceding PSA level after simultaneous radiation followed by a subsequent decrease below that level. Disease-free status was defined as the ability to achieve and maintain posttreatment PSA 0.2 ng./ml. or less. RESULTS: PSA bounce was observed in 35% of men (273 of 779). Median time to PSA bounce was 18 months from the time of implant and 92% of bounces were observed within 36 months. Median pre-bounce PSA was 0.7 ng/ml. (range 0.1 to 8.9) and median bounce height (increase above the pre-bounce level) was 0.4 ng./ml. (range 0.1 to 15.8). No distinguishing characteristics were observed between men with PSA bounce and those with cancer recurrence, and bounce had no prognostic significance relative to recurrence. CONCLUSIONS: PSA bounce is common following seed implantation for prostate cancer. It produces anxiety in men previously treated for prostate cancer and confounds the diagnosis of recurrence.

Post-treatment PSA < or = 0.2 ng/mL defines disease freedom after radiotherapy for prostate cancer using modern techniques.

OBJECTIVES: The prostate-specific antigen (PSA) definition of disease freedom after radiotherapy for prostate cancer is still in dispute. This report focuses on the PSA nadir achieved in men treated by modern radiotherapy techniques. METHODS: From 1984 to 1994, 489 consecutive men with clinical Stage T1 -T2 prostate cancer were treated by simultaneous radiation: prostate iodine-125 implant followed by external beam radiation. A transperineal implant was performed on 143 men with Stage T1-T2NX, the focus of this study; 346 men with Stage T1-T2N0 had a retropubic implant. The median pretreatment PSA was 8.3 ng/mL (range 0.3 to 188). A rising PSA was defined as one that rose on three consecutive occasions above whatever nadir was achieved. A minimum 5-year follow-up (range 5 to 15) was reached by 453 men. RESULTS: After a minimum 5-year follow-up, 336 men had a nonrising PSA, and of this group, 107 had undergone simultaneous radiation by the transperineal implant technique. A PSA nadir of 0.2 ng/mL or less was achieved by 97% of the transperineally implanted men, and 3% had a nadir of 0.3 to 1.0 ng/mL. Of the 489 men, those who had a nadir of 0.2 ng/mL or less had a 92% nonrising PSA rate (P = 0.001) 10 years after treatment compared with a 41% rate for men who had a nadir of 0.3 to 1.0 ng/mL. All men whose nadir was greater than 1.0 ng/mL had recurrence. The median time to achieve the PSA nadir of 0.2 ng/mL was 27 months (range 3 to 102). CONCLUSIONS: Primarily on the basis of the results from men treated with simultaneous radiation using the transperineal technique, the definition of disease freedom for radiotherapy should be men who achieve and maintain a PSA nadir of 0.2 ng/mL or less.

Prostate specific antigen nadir achieved by men apparently cured of prostate cancer by radiotherapy.

PURPOSE: The role of prostate specific antigen (PSA) nadir in the definition of disease freedom after radiotherapy of prostate cancer is controversial. We evaluate post-irradiation PSA nadir in men apparently cured of this disease. MATERIALS AND METHODS: From 1984 to 1993, 354 men with clinical stage T1T2N0 prostate cancer were treated with radioactive 125iodine prostate implant followed by external beam radiation. Median pretreatment PSA was 8.4 ng/ml (range 0.3 to 188). Of these men 250 are disease-free and median pretreatment PSA was 6.5 ng/ml (range 0.3 to 123). Treatment failure is defined as 3 consecutive PSA increases above nadir. Median followup is 7 years (range 5 to 14 years) for the 250 disease-free men and 6 years (range 0.5 to 14) for all 354 men. RESULTS: PSA nadir 0.5 ng/ml or less was achieved by 98% of all disease-free men (244 of 250) with minimum 5-year followup, including 87% (217) who achieved nadir 0.2 ng/ml or less. All 27 disease-free men with minimum 10-year followup had a PSA nadir of 0.5 ng/ml or less. PSA nadir significantly correlated with disease-free survival by receiver operator characteristics curve analysis (0.93 area under the curve) in all 354 men. CONCLUSIONS: PSA nadir is the fundamental measurement that determines possible cure after radiotherapy. Except for perhaps rare occasions, men must at least achieve a nadir of 0.5 ng/ml. or less to be cured of prostate cancer by irradiation. However, the prognostic value of this nadir level depends on most men achieving a nadir of 0.2 ng/ml or less. Disease freedom for radiotherapy, defined as achievement and maintenance of PSA nadir 0.5 ng/ml or less, is reasonable.

Simultaneous radiotherapy for prostate cancer: 125I prostate implant followed by external-beam radiation.

PURPOSE: Using a rigorous prostate-specific antigen definition of disease-freedom, the 10-year disease-free survival rates after simultaneous radiation of prostate cancer are presented. PATIENTS AND MATERIALS: From January 1984 through December 1996, 1020 men with clinical stage T1T2N0 prostate cancer were treated by simultaneous radiation: radioactive 125I prostate implantation followed by external-beam radiation. The median pretreatment prostate-specific antigen was 7.5 ng/mL (range, 0.2-188 ng/mL). Implantation was performed by both the retropubic and the transperineal technique, always followed by external-beam radiation. None received hormone treatment. Disease freedom is defined as achieving and maintaining a posttreatment prostate-specific antigen of < or = 0.5 ng/mL. The median follow-up is 3 years (range, 1-14 years). RESULTS: The overall 5- and 10-year disease-free survival rates are 79% and 72%, respectively, after which a plateau is reached. At 5 years posttreatment, significantly better disease-free survival results are documented with simultaneous radiation by the ultrasound technique (92%) compared with the retropubic implant technique (73%). On multivariate analysis, pretreatment prostate-specific antigen is the most significant factor associated with disease-free survival, followed by implant technique. DISCUSSION: The 10-year disease-free survival rate after simultaneous radiation is comparable to the 10-year results after radical prostatectomy. Disease freedom is defined by the same prostate-specific antigen criteria used for surgery. A plateau in the disease-free curve suggests cure. Of equal importance, the information described in this report should form only a baseline relative to future results as men treated with simultaneous radiation using the transperineal implant technique reach longer follow-up.

Prostate-specific antigen nadir of 0.5 ng/mL or less defines disease freedom for surgically staged men irradiated for prostate cancer.

OBJECTIVES: This report describes treatment results of men with prostate cancer staged with a pelvic lymph node dissection. Disease freedom was defined by a prostate-specific antigen (PSA) level nadir of 0.5 ng/mL or less. METHODS: Since 1984, 363 men with clinical Stage T1 or T2, surgical stage node-negative prostate cancer were simultaneously irradiated with a retropubic iodine 125 prostate implant followed by external-beam radiation. The average pretreatment PSA level was 13.6 ng/mL (median 8.5, range 0.3 to 188). Disease freedom was defined as the achievement and maintenance of a nadir of 0.5 ng/mL or less. Treatment failure was defined as a nadir of more than 0.5 ng/mL or a PSA rise above this level. The median follow-up is 5 years (average 5.5, range 1 to 12.5). RESULTS: For all men, the 5- and 10-year disease-free survival results are 78% and 65%. Of 201 men with a minimum 5-year follow-up, 140 (70%) are disease free. The 5-year disease-free survival rate by pretreatment PSA is 4.0 ng/mL or less, 93%; 4.1 to 10.0 ng/mL, 87%; 10.1 to 20.0 ng/mL, 72%; and greater than 20.0 ng/mL, 45%. CONCLUSIONS: The 10-year disease-free survival results of retropubic implantation, a technique considered a failure by many investigators, followed by external-beam radiation appear to be better than either technique given separately and are comparable to the results following radical prostatectomy. These results are valuable because they form a baseline that may be improved upon in the future by simultaneous irradiation using the transperineal implant technique.

The PSA nadir that indicates potential cure after radiotherapy for prostate cancer.

OBJECTIVES: The prostate-specific antigen nadir that indicates potential cure by radiotherapy has never been established. We determined this nadir level and used it to define precisely disease freedom after radiotherapy. METHODS: Combination radioactive 125I prostate implant followed by external-beam radiation was administered to 660 men with clinical Stage T1T2N0 prostate cancer. The average pretreatment prostate-specific antigen level (Tandem R Assay) was 11.7 ng/mL (median 8.0 ng/mL, range 0.3 to 188 ng/mL). To analyze these data, recurrence was defined as a prostate-specific antigen level rising above whatever nadir was achieved. The median follow-up is 42 months (range 12 to 150 months). RESULTS: Eighty-one percent of all men are calculated to achieve a prostate-specific antigen nadir of 0.5 ng/mL or less and to have a 5- and 10-year disease-free survival rate of 93% and 83%, respectively, as compared with a 5-year disease-free survival rate of 26% for those achieving a nadir of 0.6 to 1.0 ng/mL--a significant difference (P = 0.0001). All men with a nadir greater than 1.0 ng/mL ultimately failed treatment. Of 201 men with a minimum 5-year follow-up, 143 are disease-free and 140 (98%) achieved and maintained a nadir of 0.5 ng/mL or less. CONCLUSIONS: For possible cure of prostate cancer with radiotherapy, a prostate-specific antigen nadir of 0.5 ng/mL or less should be achieved. With this nadir level, disease freedom after irradiation is defined as achievement and maintenance of a nadir of 0.5 ng/mL or less. A nadir greater than 0.5 ng/mL or subsequent increase above 0.5 ng/mL is defined as irradiation treatment failure. This definition may help resolve the controversy about the potential for cure of prostate cancer by irradiation.

Arachidonic acid stimulates interleukin-6 release from rat peritoneal macrophages in vitro: evidence for a prostacyclin-dependent mechanism.

Interleukin-6 (IL-6) is a cytokine involved in the differentiation of B-cells to antibody secreting plasma cells, the activation of T-cells, and the stimulation of hepatocyte production of acute phase proteins. Because of the pro-inflammatory effects of this cytokine, we investigated the ability of the fatty acid arachidonic acid (AA) to regulate the release of IL-6 from rat resident peritoneal macrophages (M phi) in vitro. AA (0.5-16 microM) stimulated IL-6 release during a 4 h incubation period in a biphasic manner, with 4 microM AA generating a peak of IL-6 release (3-5-fold). AA (0.5-16 microM) also induced an increasing release of the AA metabolite thromboxane B2 (TXB2). The AA-induced release of IL-6 occurred within 1-2 h of incubation, whereas TXB2 concentrations were elevated within 5 min of AA treatment. The TX synthetase inhibitor CGS 12970 (4.0 microM and 40.0 microM) effectively blocked the generation of TXB2, but increased prostacyclin (PGI2) generation and potentiated the release of IL-6. In addition, PGI2, as well as the PGI2 agonists iloprost and cicaprost, stimulated IL-6 release from M phi by greater than 5-fold over vehicle-treated basal levels. These data suggest that PGI2 (but not TXA2) is involved in AA-induced IL-6 release from peritoneal M phi.

Lipopolysaccharide induces interleukin-6 release from rat peritoneal macrophages in vitro: evidence for a novel mechanism.

Interleukin-6 (IL-6) is a cytokine involved in the terminal differentiation of B-cells, T-cell activation, and secretion of hepatic acute phase proteins. The production of IL-6 is regulated by many factors, including IL-1 and lipopolysaccharide (LPS). Because IL-6 may be an important contributor to the effects of LPS in inflammation and septic shock, we investigated the ability of LPS to induce IL-6 release from peritoneal macrophages (m phi) in vitro. M phi were isolated from male Long-Evans rats, and cultured in 96-well tissue culture plates at 1 x 10(5) cells/well in serum-free RPMI-1640 medium. Following a 2-hr attachment period, the cells were rinsed twice to remove the nonadherent cells. LPS (0.006-100 ng/ml) stimulated IL-6 release by six- to 12-fold during a 4 hr incubation. In contrast, IL-1 beta (0.006-100 ng/ml) had no effect. Because cyclooxygenase metabolites of arachidonic acid are increased by LPS, we determined the effects of indomethacin (a cyclooxygenase inhibitor) and CGS8515 (a 5-lipoxygenase inhibitor) on LPS-induced IL-6 release. Neither indomethacin (10 microM) nor CGS8515 (2.5 microM) had any effect on basal or LPS-induced IL-6 release. Very low concentrations of LPS (0.01-1,000 pg/ml) stimulated IL-6 by two- to threefold. Pertussis toxin (10 ng/ml), which inactivates Gi protein, had no effect on LPS-induced IL-6 release from mø. Thromboxane B2 (TXB2) concentrations were also elevated with as little as 0.1 pg/ml LPS; however, pertussis toxin inhibited LPS-stimulated TXB2 release.(ABSTRACT TRUNCATED AT 250 WORDS)

Granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein (pIXY 321) enhances high-dose Ara-C-induced programmed cell death or apoptosis in human myeloid leukemia cells.

High dose Ara-C (HIDAC) induces programmed cell death (PCD) or apoptosis in vitro in human myeloid leukemia cells, which correlates with the inhibition of their clonogenic survival. Hematopoietic growth factors (HGFs) granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) have been demonstrated to enhance the metabolism and cytotoxic effects of HIDAC against leukemic progenitor cells. We examined the effect of pIXY 321 (a GM-CSF/IL-3 fusion protein) on HIDAC-induced PCD and related gene expressions as well as HIDAC-mediated colony growth inhibition of human myeloid leukemia cells. Unlike the previously described effects of HGFs on normal bone marrow progenitor cells, exposure to pIXY 321 alone for up to 24 hours did not suppress PCD in HL-60 or KG-1 cells. However, exposure to pIXY 321 for 20 hours followed by a combined treatment with Ara-C plus pIXY 321 for 4 or 24 hours versus treatment with Ara-C alone significantly enhanced the oligonucleosomal DNA fragmentation characteristic of PCD. This was temporally associated with a marked induction of c-jun expression and a significant decrease in BCL-2. In addition, the treatment with pIXY 321 plus HIDAC versus HIDAC alone produced a significantly greater inhibition of HL-60 colony growth. These findings highlight an additional mechanism of HIDAC-induced leukemic cell death that is augmented by cotreatment with pIXY 321 and may contribute toward an improved antileukemic activity of HIDAC.

Effect of combined treatment with interleukin-3 and interleukin-6 on 4-hydroperoxycyclophosphamide-mediated reduction of glutathione levels and cytotoxicity in normal and leukemic bone marrow progenitor cells.

4-Hydroperoxycyclophosphamide (4-HC) is widely used as an ex vivo bone marrow purging agent for acute myeloid leukemia (AML) blasts. We have determined the effect of a combined treatment with interleukin 3 (IL-3) plus IL-6 on 4-HC cytotoxicity against normal (CFU-GEMM) versus AML (L-CFU) bone marrow progenitor cells. Following an 18 h exposure to IL-3 plus IL-6, treatment with 4-HC in conjunction with IL-3 and IL-6 for one hour resulted in a significantly greater inhibition of L-CFU versus CFU-GEMM colony growth. In addition, treatment with IL-3 plus IL-6 reduced the inhibitory effects of higher concentrations of 4-HC on CFU-GEMM but not L-CFU growth. IL-3 and IL-6 did not protect the self-renewing, clonogenic, AML blast progenitor cells from the cytotoxic effects of 4-HC. While the total intracellular glutathione (GSH) levels were not significantly different between untreated normal bone marrow mononuclear cells (NBMMC) and AML blasts, greater intracellular GSH-S transferase activity was observed in the NBMMC. 4-HC produced a marked reduction in GSH levels in NBMMC as well as AML blasts. But treatment with IL-3 plus IL-6 in conjunction with 4-HC resulted in significantly higher GSH levels in NBMMC. These differences in intracellular GSH levels and GST activity may offer an explanation for the differential protective effects of IL-3 plus IL-6 treatment against the cytotoxic effects of 4-HC on CFU-GEMM colony growth.

Deoxycytidine protects normal bone marrow progenitors against Ara-C and gemcitabine cytotoxicity without compromising their activity against cisplatin-resistant human ovarian cancer cells.

The intracellular metabolism and cytotoxic effects of Ara-C and 2'-difluorodeoxycytidine (dFdC or Gemcitabine) administered with or without deoxycytidine (dCyd) were examined in cisplatin-resistant (2008/C13) and -sensitive (2008) human ovarian cystadenocarcinoma cells. Compared to 2008 cells, 2008/C13 cells possess 2.1-fold higher glutathione (GSH) levels, enhanced expressions of GSH S-transferase (GST)-pi mRNA and protein, and significantly greater activity of GST, GSH peroxidase, and GST reductase. Although 2008/C13 cells were slightly cross-resistant to 4-hydroperoxycyclophosphamide, the drug displayed a steep dose-response (colony growth inhibition) effect toward these cells. 2008/C13 cells expressed greater sensitivity toward Ara-C and Gemcitabine. This was associated with intracellular Ara-CTP and dFdCtriphosphate levels in 2008/C13 significantly higher than those in 2008 cells. Against bone marrow progenitor cells, the cytotoxic effects of submicromolar levels of Ara-C or dFdC, produced in plasma following intraperitoneal administration of the drugs, were significantly reversed by cotreatment with high levels of dCyd achieved in plasma following intravenous administration. In contrast, the metabolism and cytotoxic effects of Ara-C and dFdC in 2008 and 2008/C13 cells were not significantly altered by dCyd concentrations that are reached in the peritoneum following intravenous administration. These in vitro data suggest that systematically administered dCyd might protect bone marrow progenitor cells against Ara-C cytotoxicity without impairing antitumor activity of intraperitoneal Ara-C.

Treatment with interleukin-3 plus granulocyte-macrophage colony-stimulating factors improves the selectivity of Ara-C in vitro against acute myeloid leukemia blasts.

Hematopoietic growth factors (HGFs) interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) individually have been shown to increase the percentage of acute myeloid leukemia (AML) blasts in S phase and enhance the cytotoxic effects of Ara-C against these blasts in culture. We compared in vitro the effects of a combined treatment with GM-CSF (10 ng/mL) plus IL-3 (10 ng/mL) on the metabolism and cytotoxicity of Ara-C in normal bone marrow mononuclear cells (NBMMC) and AML blasts. NBMMC from six healthy volunteers and AML blasts from 10 patients were incubated for 20 hours with or without IL-3 plus GM-CSF, followed by a concurrent treatment with Ara-C for 4 additional hours. Exposure to the HGFs and Ara-C produced significantly higher intracellular Ara-CTP levels as well as higher Ara-CTP/dCTP pool ratios in AML blasts as compared with NBMMC. Treatment with HGFs resulted in [3H] Ara-C DNA incorporation that was significantly higher in AML blasts versus NBMMC. This selective improvement of Ara-C metabolism in AML blasts was associated with an enhanced Ara-C-mediated leukemia colony-forming unit (CFU) growth inhibition. In contrast, exposure to HGFs resulted in an improved colony growth of normal CFU granulocyte-monocyte and CFU-granulocyte, erythroid, monocyte, megakaryocyte. These in vitro studies indicate that a combined treatment with IL-3 plus GM-CSF may improve the selectivity of Ara-C against AML blasts.

Hormonal regulation of protein kinase C in the mouse mammary gland.

The hormonal regulation of protein kinase C (PKC) induction over 3 to 14 days was investigated in the mouse mammary gland in vitro and in vivo. In intact mice, estradiol (1 microgram/mouse injected daily for 2 weeks) stimulated PKC activity 70%, while progesterone (1 mg/mouse injected daily) inhibited it by 30%. Prolactin, whose levels were elevated for 2 weeks by two pituitary isografts, had no effect. When mammary gland explants were cultured in insulin and cortisol, the further addition of estradiol (1 ng/ml), progesterone (1 microgram/ml), or prolactin (1 microgram/ml) did not alter PKC activity after 3 days. These data suggest the following conclusions: although previous studies have implicated prolactin in the transient, calcium-phospholipid activation of PKC, it does not appear to elevate total levels of this kinase over prolonged periods. In contrast, the sex steroids do appear to affect long-term levels of this kinase; furthermore, this latter effect may be indirect.

Immunofluorescence comparisons of anti-actin specificity.

The abilities of antibody populations against brain actin and two immunogenic forms of cardiac actin to react with sarcomeric muscle actin and cytoplasmic non-muscle actin were tested by indirect immunofluorescence, by using isolated skeletal muscle myofibrils and cultured non-neuronal dorsal root ganglion cells as the test systems. All three antibody preparations stained the I-bands of myofibrils, a result that demonstrated the presence of antigenic determinants shared among skeletal, cardiac, and brain actins. However, although antibodies against cytoplasmic brain actin stained the stress fibers of cultured cells, those against glutaraldehyde cross-linked cardiac actin did not, a result that implies that cardiac actin possesses determinants common to sarcomeric actins but not present on cytoplasmic actin. Finally, antibodies against SDS-treated cardiac actin readily stained the stress fibers of cultured cells, in contrast to those against glutaraldehyde cross-linked cardiac actin, a result that suggests that the state of the original immunogen can affect the actin type specificity of the resulting antibody population.

Distribution of tubulin and actin in neurites and growth cones of differentiating nerve cells.

Embryonic chick nerve cells, from dissociated dorsal root ganglia, were cultured on polylysine substrata and examined for tubulin and actin distribution by indirect immunofluorescence. Antibodies generated against chick brain tubulin produced specific fluorescence in growth cones, neurites, and cell bodies without revealing distribution differences or substructure in the nerve cells. However, at reduced antitubulin concentrations, differences were resolved. Tubulin fluorescence remained uniform and intense in neurites and cell bodies, but exhibited reduced intensity and patterning in growth cones. Nonneuronal cells in the cultures served as controls for typical cytoplasmic tubulin fluorescence distribution. Straining controls demonstrated that fluorescence resulted from tubulin-antitubulin binding. Analogous studies, using antibodies generated against chick brain actin, demonstrated distribution differences at reduced antiactin concentrations, including "hot spots" of intense fluorescence in growth cones and a paucity of fluorescence in neurites.

Immunological differences between actins from cardiac muscle, skeletal muscle, and brain.

The antigenic similarities and differences between various actins were explored by use of antisera against purified bovine cardiac actin and chicken embryo brain actin. In double-antibody coprecipitation tests, purified iodinated actins from bovine cardiac muscle, rabbit skeletal muscle, chicken embryo brain, and bovine brain all bound to antiserum against chicken embryo brain actin. This result demonstrates the presence of shared antigenic determinants among these actins. Cardiac actin antiserum, on the other hand, bound cardiac and skeletal actin, but failed to bind significantly either brain actin. In radioimmunoassay, all four unlabeled actins were capable of some degree of inhibition of binding of (125)I-labeled chicken embryo brain actin to homologous antiserum. The results confirm the existence of shared or similar antigenic determinants, but also show that the molecules are not antigenically identical. In the cardiac actin radioimmunoassay, unlabeled cardiac and skeletal muscle actins inhibited the binding of (125)I-labeled cardiac actin to homologous antiserum, but neither brain actin inhibited the binding. Thus, the muscle actins possess at least one antigenic determinant not expressed by the brain actins, in addition to the shared determinants. Furthermore, cardiac actin and skeletal actin generated different inhibition curves in the cardiac actin radioimmunoassay, demonstrating that, although antigenically related, they are not identical. Correlations with existing sequence data imply that substitutions in only a few residues alter the antigenic properties of actin.