Immunohistochemical localization of adenosine deaminase complexing protein in intestinal mucosa and in colorectal adenocarcinoma as a marker for tumour cell heterogeneity

Summary Adenosine deaminase complexing protein (ADCP), a dimeric glycoprotein, has been reported to be decreased or deficient in transformed or cancer-derived cell lines, indicating its potential significance as an indicator of malignant transformation. A similar deficiency was reported in total homogenates of tumours of colon, kidney, lung and liver. In previous biochemical studies we failed to confirm the consistent reduction in ADCP concentration in cancer tissues. A possible explanation for our findings was thought to be intercellular heterogeneity in ADCP expression in individual tumour cells. To study ADCP expression in individual cells we developed an immunohistochemical method which was applied to tissue sections. Paraformaldehyde-lysine-periodate (PLP) solution was found to be a suitable fixative. Fixed tissue samples were paraffin-embedded, sectioned and stained for ADCP, using an indirect peroxidase-labelled antibody procedure. The protein was localized in normal colonic mucosa, mainly in the brush border region of the luminal epithelium and in cytoplasmic granules. Intense ADCP immunoreactivity was found also in the basal part of some cells. In cancer cells, three staining patterns were observed: membranous, diffuse cytoplasmic and granular cytoplasmic. The adenocarcinomas exhibited significant intratumour and intertumour heterogeneity in their staining types.Further studies on ADCP expression in colorectal cancer in relation to clinical and histopathological characteristics are warranted in order to fully evaluate the potential significance of ADCP as a cancer associated antigen.     

Roots as an enhancing factor for the production of artemisinin in shoot cultures of Artemisia annua

Artemisinin was produced in differentiated shoot cultures of Artemisia annua L. but was undetected in callus or cell cultures. The growth regulators benzyladenine, kinetin, chlormequat, and daminozide, at concentrations which severely reduced rooting, reduced artemisinin production. A highly significant correlation (1% level) was observed between shoot artemisinin content and number of roots (r=0.775^**), but shoot number and artemisinin content were unrelated (r=-0.198). Benzyladenine increased shoot proliferation at 0.5 and 5.0 M, but decreased root production at 0.5, 5.0, and 50 M. The highest levels of artemisinin production (0.287% DW) were obtained in hormone-free medium when root production was maximized. Removal of roots from shoots cultured in hormone-free liquid medium reduced shoot artemisinin by 53% and shoot arteannuin B by 60%. Neither artemisinin, arteannuin B, or artemisinic acid were detected from roots developed in semi-solid or liquid medium.Abbreviations BA benzyladenine - CCC chlormequat - DW dry weight - FW fresh weight - GA₃ gibberellic acid - GC/MS gas chromatography/mass spectrometry - HPLC-EC high-performance liquid chromatography with electrochemical detection - MS Murashige & Skoog basal medium - 2,4-d 2,4-dichlorophenoxyacetic acid Journal paper no. 14558 of Purdue Agricultural Research Progress     

Effects of glucose-supplemented diets on food intake, nymphal development, and fecundity of glucose-averse, non-glucose-averse, and heterozygous strains of the German cockroach,Blattella germanica

Life history parameters were determined for glucose-averse (glu/glu), wild-type (glu+/glu+) and heterozygous (glu/glu+) genotypes of Blattella germanica (L.) (Blattodea: Blattellidae) fed diets supplemented with glucose. Glu/glu nymphs consumed less glucose-supplemented diet, gained less weight, developed slower and had a lower rate of survival than glu/glu nymphs fed the same diet without added glucose, or glu+/glu+ and glu/glu+ fed either diet. Prior to formation of the first oötheca, female glu/glu consumed less glucose-supplemented diet per day than glu+/glu+ and glu/glu+, which presumably delayed egg case production. Oötheca-bearing glu/glu and glu/glu+ females consumed less glucose-supplemented diet than glu+/glu+ females. Despite a difference in female total diet intake, there was no effect of diet or genotype on fecundity. However, the intrinsic rate of increase (r) for glulglu on unsupplemented diet was less than that of glu+/glu+ and glu/glu+, suggesting that individuals with both glu alleles may be at a selective disadvantage in environments lacking diets containing glucose plus a toxicant.     

Binding parameters of monoclonal antibodies reacting with ovarian carcinoma ascites cells

Summary Binding parameters were determined for four mouse monoclonal antibodies reacting with three antigens on the surface of fresh human ovarian carcinoma ascites cells, under nearly physiological conditions. The object of these experiments was to aid in the selection of the optimal monoclonal antibodies for intraperitoneal immunotherapy. The number of antigenic sites per cell, the effective equilibrium association constant (affinity) and the half-life for dissociation were: for Ab MH99, 1.2×10⁶ sites/cell, (1.9–4.1)×10⁸M^–1, and 4 h; for Ab MX35, (3.2–4.1)×10⁵ sites/cell, (3.4–4.8)×10⁸M^–1, and >10 h; and for Ab MW207, 1.3×10⁵ sites/cell, (3.6–4.1)×10⁹M^–1, and 3.1 h, respectively. One of the antigens, MH99, is recognized by five different monoclonal antibodies, and competitive inhibition experiments demonstrated that two distinct determinants are present; this antigen is also recognized by the previously described Ab 17-1A. These binding data will aid the rational design of immunotherapy strategies.     

Serial immune testing in surgically resected lung cancer patients

Summary Immunoregulation of phytohemagglutinin (PHA) responsiveness by glass-adherent cells and prostaglandin-synthesizing cells was serially monitored in the peripheral blood mononuclear cells (PBMC) of surgically resected stage I and stage II lung cancer patients entered on a trial of adjuvant immunotherapy. The relationship between immunoregulatory cell function, immunocompetence, and disease relapse was determined. Immunoregulatory activity was measured in PHA-stimulated cultures in the presence and absence of 2 g/ml indomethacin and in the presence and absence of glass-adherent cells. In each instance of disease relapse seen, an increase in immunoregulatory cell function to a level significantly different from normal was observed 3 months prior to or coincident with clinical confirmation of disease recurrence. This was usually associated with a decline in PHA responsiveness. In the patients who did not relapse, the levels of PHA responsiveness and immunoregulatory function persisted within normal limits throughout the course of study. Percentages and numbers of leukocytes and leukocyte subsets and delayed cutaneous hypersensitivity were also monitored in this study, but could not be consistently correlated with early changes in clinical disease status. These data suggest that the development of indomethacin-sensitive and glass-adherent suppressor cells may precede and predict for tumor recurrence in surgically resected lung cancer patients.     

Effect of chemotherapy on NK function in the peripheral blood of cancer patients

Summary The effects of cytotoxic chemotherapy on NK cell function and on glass adherent cell regulation of NK cell function was evaluated in the peripheral blood mononuclear cells of 20 previously untreated solid tumor patients. Most of the patients studied had lung cancer and received one of four combination chemotherapy treatment regimens. In addition, one patient with colon carcinoma and one patient with melanoma were studied, each of whom received treatment with a single agent. The results demonstrated that chemotherapy exerted a differential influence on NK activity which correlated with the pretreatment NK level of function in the individual patient. In patients with depressed NK levels prior to treatment, chemotherapy augmented NK function; in patients with normal levels prior to treatment, chemotherapy depressed NK function. The effects observed appeared to be associated with the capacity of chemotherapy to influence glass adherent cell regulation of NK activity. There was no apparent correlation between the effects of chemotherapy on numbers of NK effector cells, Leu11+ cells, or latex-ingesting cells. Also, there was no correlation between the effects seen and the type of drug treatment that was administered; rather, this was dependent on the pretreatment NK level of function which in turn was associated with glass adherent cell regulation of NK function.Supported in part by PHS Grant No. 27598     

Precocious Meiotic Centromere Separation of a Novel Yeast Chromosome

In Saccharomyces cerevisiae, a reciprocal translocation between chromosome II and a linear plasmid carrying a centromere (CEN6) has split chromosome II into two fragments: one, approximately 530 kilobase pairs (kbp) in size, has the left arm and part of the right arm of chromosome II; the other, a telocentric fragment approximately 350 kbp in size, has CEN6 and the rest of the right arm of chromosome II. A cross of this yeast strain with a strain containing a complete chromosome II exhibits a high frequency of precocious centromere separation (separation of sister chromatids during meiosis I) of the telocentric fragment. Precocious centromere separation is not due to the position of the centromere per se, since diploids that are homozygous for both fragments of chromosome II segregate the telocentric fragment with normal meiotic behavior. The precocious centromere separation described here differs from previously described examples in that pairing and synapsis of this telocentric chromosome seem to be normal. One model of how centromeres function in meiosis is that replication of the centromere is delayed until the second meiotic division. Data presented in this paper indicate that replication of the centromere is complete before the first meiotic division. The precocious separation of the centromere described here may be due to improper synapsis of sequences flanking the centromere.     

RNA-Dependent DNA Polymerase Activity in Preparations of a Mutant of Newcastle Disease Virus Arising from Persistently Infected L Cells

RNA-dependent DNA polymerase activity was found in peparations of a mutant of Newcastle disease virus. The enzyme activity was not found in wild-type virus preparations.