Hemagglutinating 7S subunits of 19S cold agglutinins.

Two highly purified IgM cold agglutinins have been mildly reduced yielding 7S subunits, with interchain covalent bonds intact. These subunits retained most of the cold-agglutinin activity as well as the specificity of the parent antibodies. However, as might be anticipated from theories of the importance of antibody size and number of subunits for complement binding, the IgM subunits were only very weakly lytic compared with the intact cold agglutinins. The findings are consistent with the presence of ten antibody-combining sites on the IgM molecule.

Loss of strain specificity of the TA3-St subline: evidence for the role of epiglycanin in mouse allogeneic tumor growth.

We described the in vivo conversion of the strain-specific ascites murine mammary adenocarcinoma subline TA3-St to a new ascites subline designated TA3-MM. This conversion occurred during passage in a syngeneic A/HeHa mouse infected with pneumonia-producing microorganisms. The mode number of chromosomes of the TA3-MM cell (82) was greater than that of the parental TA3-St cell (69) or the other non-strain-specific subline TA3-Ha (42). The TA3-MM subline could grow in and kill mice of various allogeneic strains. In addition, the TA3-MM cell possessed numerous receptors for the lectin of Vicia graminea seeds, which were hardly detectable at the surface of the parent TA3-St subline but were present in abundance at the cell surface of the non-strain-specific subline TA3-Ha. These lectin receptors of the TA3-Ha cell were previously demonstrated to be present in a unique high-molecular-weight endogenous cell surface glycoprotein termed epiglycanin. The V. gramines lectin receptors on the new TA3-MM subline also were present on an epiglycanin-like molecule. This finding provides further evidence for the hypothesis that allogeneic growth in the TA3 system is a direct result of these membrane glycoproteins.

Reversible loss in suspension culture of a major cell-surface glycoprotein of the TA3-Ha mouse tumor.

A major cell-surface glycoprotein of the TA3-Ha ascites mammary adenocarcinoma diminished during transfer from ascites growth to cell growth in suspension culture. A sensitive, hemagglutination-inhibition assay that used a lectin from Vicia graminea seeds indicated approximately a 50% loss after 7-10 days of culture and a 90% loss after 2 months. These findings were corroborated by carbohydrate and amino acid analysis with gas-liquid chromatography of trypsin glycopeptides released from the cell surface. Repassage of the cultured cells in vivo caused the reappearance of the surface glycoprotein.

Further studies on the relationship between large glycoprotein molecules and allotransplantability in the TA3 tumor of the mouse: studies on segregating TA3-HA hybrids.

In a series of six TA3-HA/A.CA hybrid cell lines formed by the fusion of the TA3-HA mammary carcinoma of a strain A mouse with a normal embryonic fibroblast of an A.CA mouse and then converted to the ascites form in parental strain A, the capacity to grow in foreign strains was inversely related to the ability to absorb anti-H-2a antibody. The absorptive capacities of the hybrid cell lines were intermediate between the low absorptive capacity of the non-strain-specific parent TA3-HA ascites cell line and the much higher absorptive capacity of the strain-specific ascites line TA3-St of the same tumor. Each hybrid cell line possessed an abundance of large endogenous cell-surface glycoprotein molecules similar to epiglycanin, a glycoprotein detected at the surface of the parent TA3-HA cell. The results suggested that the amount of epiglycanin-like material at the hybrid cell surfaces, determined by chemical and immunochemical methods, may have been directly related to the capacities of the cells to grow in foreign mouse strains and inversely related to their capacities to absorb anti-H-2a antibody.

Isolation and partial characterization of an epiglycanin-like glycoprotein from a new non-strain-specific subline of TA3 murine mammary adenocarcinoma.

A new non-strain-specific ascites subline of the TA3 mammary adenocarcinoma TA3-MM, which arose in vivo from the strain-specific TA3-St subline during an acute respiratory illness of the syngeneic mouse strain A/HeHa hosts, possessed at its surface a glycoprotein not found on the parent TA3-St cell. This glycoprotein, termed TA3-MM epiglycanin, was characterized by a high molecular weight (500,000), by potent inhibition of hemagglutination by the Vicia gramines lectin, and by carbohydrate and amino acid compositions nearly identical to those of the glycoprotein epiglycanin present at the surface of the allotransplantable TA3-Ha ascites cell. By electron microscopic examination, TA3-MM epiglycanin appeared as long extended rods with widths (2.5 nm) and lengths (450--500 nm) similar to those of TA3-Ha epiglycanin. Incubation of each of two sublines of the TA3-MM ascites cell, TA3-MM/1 and TA3-MM/2, with a modified trypsin followed by column chromatography produced approximately 1.0- and 0.2-fold as much epiglycanin-like material, respectively, as was obtained from the TA3--a ascites cell. Continuous growth of the TA3-MM cell in suspension culture resulted in an almost complete disappearance of epiglycanin in a manner demonstrated earlier for the TA3-Ha cell grown under similar conditions. Allotransplantability in the TA3-MM cell may be due, at least in part, to masking a histocompatibility antigens by epiglycanin-like molecules.

Overexpression of the G-protein inwardly rectifying potassium channel 1 (GIRK1) in primary breast carcinomas correlates with axillary lymph node metastasis.

The acquisition of genetic alterations in tumor cells is a hallmark of cancer progression. Genetic alterations, including chromosomal sequence alterations and abnormal gene expression, increase the malignant potential of tumors by affecting pathways that regulate cell growth, cell death, tumor angiogenesis, and invasion/metastasis. We used an expression profiling technique, representational difference analysis, to identify genes the expressions of which are aberrantly increased in invasive breast carcinomas as compared with adjacent normal breast tissue from the same individual. Among the genes we identified was GIRK1, which encodes a 501 amino acid, G-protein inwardly rectifying potassium channel protein. We then measured GIRK1 mRNA expression in benign breast tissues, primary invasive breast carcinomas, and metastatic breast carcinomas from axillary lymph nodes using quantitative TaqMan reverse transcription-PCR and correlated the results with clinical parameters. We found that GIRK1 overexpression correlated with lymph node metastasis (P < 0.0029), and overexpression was greatest in tumors with more than one positive lymph node. These results indicate that GIRK1 may be useful as a biomarker for lymph node metastasis and possibly a pharmaceutical target.

Anitroxide-sterol derivative potently modifies cholesterol biosynthesis by normal and neoplastic guinea pig lymphocytes.

Leukemic guinea pig lymphocytes (L2C) synthesise cholesterol in vitro at a forty-fold greater rate than normal cells. Equilibration (18 h) with lecithin or lecithin-cholesterol liposomes, respectively, enhances or suppresses sterol manufacture by normal lymphocytes but does not influence sterol production by L2C cells. In contrast, greater than 5-10(9) molecules/cell of a nitroxide-derivative of androstane, (17 beta-hydroxy-4',4'-dimethylspiro [5 alpha-androstan-3,2'-oxazolidin]-3'-yloxyl), commonly used as a membrane spin-probe, drastically inhibit sterol roduction by both normal and leukemic cells (maximum within 2 H). At less than 5-10(9) molecules/cell, this sterol stimulates cholesterol synthesis. 25-Hydroxycholesterol at low concentrations also stimulates sterol manufacture, whereas high concentrations are also inhibitory in both cell types.

Pulmonary hemorrhage in lupus erythematosus without evidence of an immunologic cause.

Intra-alveolar hemorrhage is a known complication of lupus erythematosus (LE), but its cause is controversial. Some authors have shown immune complexes (ICs) deposited at various sites in the alveolar septae and postulated that these deposits result in pulmonary hemorrhage (PH). A patient with LE and PH had no detectable IC deposits at a time when IC disease was present in the kidney and vasculitis was active in the skin. Reviewing the literature, we show that IC deposits in the lung are nonspecific and are not correlated with PH. We propose that classification schemes that differentiate between IC-mediated PH and idiopathic PH are arbitrary, and that patients thought to have idiopathic PH should be followed up prospectively to monitor the development of possible immunologic disease.

Mediastinal lymph node evaluation by computed tomography in lung cancer. An analysis of 345 patients grouped by TNM staging, tumor size, and tumor location.

To more clearly characterize the role of computed tomography in staging the mediastinal lymph nodes of patients with lung cancer, we analyzed computed tomographic and surgical findings in the chest in 345 consecutive patients with lung cancer who underwent operative staging. Patients were grouped according to the TNM staging system of the American Joint Commission, central or peripheral location of the primary tumor, lobar location of the tumor, and maximum tumor diameter as determined by computed tomography or gross pathology. One third of patients with abnormal findings on the computed tomographic scan did not have mediastinal lymph node metastases. Mediastinal metastases occurred frequently in patients with central cancers (38%). The predictive value of a negative scan in all patients was high (greater than or equal to 90%) except for patients with central T3 lesions (72%), left upper lobe lesions (83%), and central adenocarcinomas (75%). However, only the differences between central T3 and central T2 or T1 lesions, and between central adenocarcinomas and central squamous cell carcinomas, were unlikely to be due to chance alone (p less than 0.05). None of the lobar differences were statistically significant. The frequency of mediastinal metastases in patients with peripheral lesions was 15% (28 of 192 patients); computed tomography correctly identified enlarged mediastinal lymph nodes in all but seven patients. However, there were no true-positive computed tomographic scans in 59 patients with peripheral lesions 2 cm in diameter or smaller; accordingly, we suggest that computed tomography is not indicated for the sole purpose of mediastinal staging in this group. Ninety-four percent of patients in this series undergoing thoracotomy with a curative intent had a curative resection. Only 4% had unresectable lesions; palliative resections were done in 2%.

Extended ex vivo preservation of the heart and lungs. Effects of acellular oxygen-carrying perfusates and indomethacin on the autoperfused working heart-lung preparation.

The autoperfused working heart-lung preparation has been proposed as a method for long-term heart-lung preservation. We investigated the effects of acellular oxygen-carrying perfusates (study 1) and the effect of donor pretreatment with indomethacin (study 2) on the working ex vivo heart-lung block. In study 1 perfusion with stroma-fee hemoglobin resulted in significantly reduced survival (118 +/- 46 minutes) compared with autologous blood (561 +/- 125 minutes, p less than 0.05) or perfluorocarbon (438 +/- 114 minutes, p less than 0.05). Decrease in survival with stroma-free hemoglobin perfusate is associated with a marked decrease in left ventricular performance and a significant increase in pulmonary vascular resistance. Perfusion with autologous blood is associated with a significant increase in pulmonary vascular resistance after 240 minutes of explantation, which is significantly delayed by perfusion with perfluorocarbon. Perfusion for 6 hours with blood pretreated with indomethacin (study 2) resulted in a decrease in the concentration of prostacyclin and thromboxane A2 metabolites but an increase in the prostaglandin/thromboxane A2 metabolite ratio. This is associated with abrogation of the increase in pulmonary vascular resistance (12,787 +/- 1682 dynes/sec/cm-5, T = 0; 13,134 +/- 2654 dynes/sec/cm-5, T = 360 minutes) observed in preparations perfused with autologous blood (13,194 +/- 1942 dynes/sec/cm-5, T = 0; 24,768 +/- 3325 dynes/sec/cm-5, T = 360 minutes, p less than 0.05). We conclude that alteration of the cellular and humoral components of autologous blood may prove advantageous for increasing the utility of the autoperfused working heart-lung preparation as a preservation technique.

Further characterization of the glycoproteins and glycosaminoglycans released from TA3 murine adenocarcinoma cells in culture.

TA3 murine ascites adenocarcinoma cells were compared for their ability to release radioactive glucosamine and 35SO4-labeled glycoproteins and glycosaminoglycans into the culture medium. Both TA3-Ha and TA3-St cells contained cell-surface heparan sulfate that was released into culture, but not chondroitin sulfate. Both cells released a membranous aggregate of labeled components from the cell surface and hyaluronic acid from inside the cells that fractionated in the void volume of Sepharose CL-4B. This void-volume fraction from the TA3-Ha cells contained glucosamine-labeled epiglycanin at a higher concentration relative to other glucosamine-labeled components than that found on plasma membranes. Glycoproteins associated with epiglycanin found on the cell surface, as well as released into culture medium, contained sulfate that could not be removed by chondroitinase ABC, heparinase, or keratinase. Kinetic analysis of the glucosamine-labeled material released from TA3-Ha cells indicated that hyaluronic acid was released rapidly with a 45-min half-life, whereas the other membranous components were released much more slowly.

Assessing the expression of two genes simultaneously in surgical specimens using polymerase chain reaction.

We developed a novel polymerase chain reaction (PCR)-based method to analyze simultaneously the relative expression of two genes in a single PCR reaction. The method, relational PCR (R-PCR), utilizes special PCR primers that enable a PCR reaction to be converted from a standard uniplex reaction to a multiplex reaction in which all products are dependent on the same reaction components for amplification. We show that the quantitative ability of R-PCR is unaffected by sample nucleic acid input concentration over a range of 25-fold (30 to 750 ng of total RNA) and demonstrate excellent interexperimental reproducibility. We used R-PCR to analyze estrogen receptor gene expression in a series of invasive breast carcinomas, and our results show an excellent correlation between estrogen receptor mRNA expression and protein product accumulation determined by standard immunocytochemistry on paraffin sections.