Evaluation of antibody inhibition assays of melanoma antigens in sera to monitor tumor growth in melanoma patients

Summary It was reported previously that melanoma leukocyte-dependent antibody (LDA) in the sera of melanoma patients was inhibited by small-molecular-weight (small-mol.-wt.) glycoproteins which were similar to cell surface antigens identified in cell membrane extracts of melanoma cells. The present study was to determine whether measurement of the levels of these factors in sera may be a useful monitor of tumor growth in melanoma patients. Small-mol.-wt. fractions were obtained by gel filtration or membrane chromatography of acidified sera and tested for their ability to inhibit LDA in ⁵¹Cr release cytotoxic assays. A panel of LDA was used, consisting of three antisera from melanoma patients, which appeared relatively specific for melanoma, and three non-melanoma antisera against carcinoembryonic antigen, ₂ microglobulin, and fetal antigens. The results showed that in patients with melanoma, approximately 70% had melanoma LDA-inhibitory activity detected in the small-mol.-wt. fractions of their sera when these were tested against the panel of melanoma LDA. The specificity of the inhibitory activity for melanoma LDA was shown by failure of the serum fractions to inhibit non-melanoma LDA and by absence of inhibitory activity in equivalent serum fractions from non-melanoma carcinoma patients for melanoma LDA. The levels of melanoma LDA-inhibitory activity in the serum fractions appeared to correlate with tumor growth, as shown by clearance of the inhibitory activity after surgical removal of melanoma and reappearance in the serum of patients who subsequently developed recurrent melanoma. The 30% false-negative rate indicated that the assays could not be used to reliably exclude melanoma, but the close correlation with tumor growth and the low number of false-positive results suggested that in 70% of patients detection of these small-mol.-wt. antigens would be of value to detect recurrence from melanoma and to monitor the effectiveness of therapy.     

Valine-sensitive acetohydroxy acid synthases in Escherichia coli K-12: unique regulation modulated by multiple genetic sites.

Summary Spontaneous mutants (146) of Escherichia coli K-12 were selected that were resistant to inhibition of growth by 1.2 mM L-valine (Val^r). The Val^r isolates, containing acetohydroxy acid synthase resistant to feedback inhibition by L-valine (AHAS^r), were classed according to cotransduction of the mutation with leu. Several mutations resulting in an AHAS^r phenotype were found to be cotransducible with glyA. However, no mutations causing a Val^r phenotype were linked to ilv. AHAS activity was more closely examined in representatives of three classes of mutants with Val^r linked to leu, labeled ilv-660, ilv-661, and ilv-662. The ilvE503 allele in E. coli K-12, known to cause a two- to three-fold derepression of AHAS, was found to affect regulation of synthesis of both valine-sensitive AHAS (AHAS^s) and AHAS^r in the mutants containing ilv-660 and ilv-661, whereas it affected repression of AHAS^s, only, in the mutant containing ilv-662. Further, both AHAS^s and AHAS^r in the ilv-661 mutant were repressed by valine, whereas valine did not repress AHAS^r synthesis in the strain carrying ilv-660 and only partially repressed AHAS^r in the strain carrying ilv-662. Unexpectedly, AHAS^r synthesis in strains carrying ilv-660 or ilv-662 was repressible by leucine. The ilv-660 locus appears to be similar in position to ilvH and encodes a product that confers valine-sensitivity upon AHAS activity in the wild-type E. coli K-12. The ilv-660 and ilv-662 loci may normally encode products that influence both the feedback sensitivity of AHAS and control of AHAS biosynthesis.     

Polyamines in Paul's Scarlet rose suspension cultures

Polyamine concentrations have been determined at intervals in suspension cultures of Paul's Scarlet rose cells during a culture period of 2 weeks. The mean concentrations of the putrescine, spermidine and spermine in the cells of the inocula were respectively 73, 70 and 13 nmol/g fresh weight. Putrescine at fitst increased with a peak (160 nmol/g) after 6 h, declined to a minimum (14 nmol/g) after 2–3 days, increased to a second peak (180 nmol/g) after 5–6 days, and then declined slowly to the concentration of the inoculum (taken on day 14). Spermidine rose slowly (×2.6) to a broad peak over 3–6 days (180 nmol/g), then declined slowly to the concentration in the inoculum. Spermine showed a rapid increase to a peak (130 nmol/g) after 2–3 days, and then declined rapidly, reaching the inoculum concentration by day 6. In one experiment the three amines showed a minor peak at day 11. Changes in spermine and RNA contents appeared to be correlated. DNA content reached a peak after that of the RNA (day 3) and did not appear to be correlated with the content of putrescine or the polyamines.     

Application of CNDO2 theoretical calculations to interpretation of the chemical reactivity and biological activity of the syn and anti diolepoxides of benzo[a]pyrene

$\text{CNDO}\text{2}$ molecular orbital theoretical calculations performed on the anti and syn diolepoxides (1 and 2) of the potent carcinogen benzo[a]pyrene provide insight into the molecular structure and reactivity of these mutagenic and carcinogenic hydrocarbon metabolites. Hydrogen-bonded interaction between the 7-HO proton and the epoxide oxygen atom of 2 is shown to be absent in the normal semichair conformation of the tetrahydro ring, (H…O bond distance = 2.7 Å), but is energetically favored in a somewhat distorted puckered structure (H…O bond distance = 1.7 Å). Unexpectedly, internal H-bonding alters the relative electron density at C₉ and C₁₀, leading to prediction of the former as the more electrophilic center. Since all reactions of 2 take place exclusively at C₁₀, transannular H-bonding is concluded not to contribute significantly to the structure of 2. Diolepoxide reactions with both weak and strong nucleophiles and with DNA are discussed and the mechanisms interpreted in terms of molecular structure as determined by the theoretical calculations.     

Conversion of type II procollagen to collagen in Vitro: Removal of the carboxy-terminal extension is inhibited by several naturally occurring amino acids,polyamines, and structurally related compounds

Chick embryo sterna, which actively synthesize type II procollagen, were pulse-labeled with radioactive proline; protein synthesis was then inhibited by unlabeled proline and cycloheximide. After the inhibition of protein synthesis, several amino acids, polyamines, or structurally related compounds were added to the incubation medium. The conversion of procollagen, first to two intermediates, pC-collagen and pN-collagen, and then to collagen, was monitored by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The addition of 50 mm β-alanine, arginine, asparagine, glutamine, hydroxylysine, lysine, or ornithine, as well as agmatine, ?-aminocaproic acid, S-2-aminoethylcysteine, cadaverine, canavanine, putrescine, or spermine clearly inhibited the removal of the carboxy-terminal extension and pC-collagen accumulated; the removal of the amino-terminal extension was not affected. The inhibition of the conversion was reversible and unaffected by fetal calf serum. The results suggest that the conversion of type II procollagen to collagen requires at least two separate proteinases for the removal of amino-terminal and carboxy-terminal extensions. The results further suggest that naturally occurring molecules may be used to modulate the rate of conversion of procollagen to collagen, and development of analogs of these compounds may provide the means to interfere with excessive deposition of collagen in diseases with tissue fibrosis.     

Initiation of DNA synthesis in the parotid salivary gland of adult mice by a factor isolated from acellular fluid of the Ehrlich ascites carcinoma

The rate of DNA synthesis in the parotid salivary gland of adult mice is very low. We have purified about 5 000-fold a mitogen from the aceIlular ascitic fluid of the Ehrlich ascites carcinoma which stimulates DNA synthesis in the parotid salivary gland in vivo. This stimulation of DNA synthesis was produced with a protein concentration of this mitogen of 3 μg per 25 g of body weight. The purification procedure included ammonium sulfate fractionation and DEAE Sephacel column chromatography. This potent, heat-labile mitogen is presumed to be a protein with a mol.wt, of 3.5×10³ to 1.3×10⁴. The data indicate that this new factor is quite different from epidermal growth factor and tumor growth factor. Hypophysectomy did not prevent the stimulatory effect of this mitogen on the parotid salivary gland, indicating that the pituitary gland is not involved directly in mediating the mitogenic effect.     

Loss of transferrin receptors following induced differentiation of HL-60 promyelocytic leukemia cells

Human promyelocytic leukemia (HL-60) and lymphoblastoid (Daudi) cells were studied: for transferrin receptors before and after induced differentiation with dimethyl sulfoxide (DMSO), sodium butyrate or retinoic acid. None of these reagents affected the morphology or presentation of receptors in Daudi cells, but many HL-60 morphologically matured to banded neutrophils and demonstrated a concomitant loss of transferrin binding, suggesting an important role for transferrin receptors in cellular differentiation.