Assessing humoral and cell-mediated immune response in Hawaiian green turtles, Chelonia mydas.

Seven immature green turtles, Chelonia mydas, captured from Kaneohe Bay on the island of Oahu were used to evaluate methods for assessing their immune response. Two turtles each were immunized intramuscularly with egg white lysozyme (EWL) in Freund's complete adjuvant, Gerbu, or ISA-70; a seventh turtle was immunized with saline only and served as a control. Humoral immune response was measured with an indirect enzyme linked immunosorbent assay (ELISA). Cell-mediated immune response was measured using in vitro cell proliferation assays (CPA) using whole blood or peripheral blood mononuclear cells (PBM) cultured with concanavalin A (ConA), phytohaemagglutinin (PHA), or soluble egg EWL antigen. All turtles, except for one immunized with Gerbu and the control, produced a detectable humoral immune response by 6 weeks which persisted for at least 14 weeks after a single immunization. All turtles produced an anamnestic humoral immune response after secondary immunization. Antigen specific cell-mediated immune response in PBM was seen in all turtles either after primary or secondary immunization, but it was not as consistent as humoral immune response; antigen specific cell-mediated immune response in whole blood was rarely seen. Mononuclear cells had significantly higher stimulation indices than whole blood regardless of adjuvant, however, results with whole blood had lower variability. Both Gerbu and ISA-70 appeared to potentiate the cell-mediated immune response when PBM or whole blood were cultured with PHA. This is the first time cell proliferation assays have been compared between whole blood and PBM for reptiles. This is also the first demonstration of antigen specific cell-mediated response in reptiles. Cell proliferation assays allowed us to evaluate the cell-mediated immune response of green turtles. However, CPA may be less reliable than ELISA for detecting antigen specific immune response. Either of the three adjuvants appears suitable to safely elicit a detectable immune response in green turtles.

Anti-microbial activity and anti-complement activity of extracts obtained from selected Hawaiian medicinal plants.

Selected plants having a history of use in Polynesian traditional medicine for the treatment of infectious disease were investigated for anti-viral, anti-fungal and anti-bacterial activity in vitro. Extracts from Scaevola sericea, Psychotria hawaiiensis, Pipturus albidus and Eugenia malaccensis showed selective anti-viral activity against Herpes Simplex Virus-1 and 2 and Vesicular Stomatitis Virus. Aleurites moluccana extracts showed anti-bacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa, while Pipturus albidus and Eugenia malaccensis extracts showed growth inhibition of Staphylococcus aureus and Streptococcus pyogenes. Psychotria hawaiiensis and Solanum niger inhibited growth of the fungi Microsporum canis, Trichophyton rubrum and Epidermophyton floccosum, while Ipomoea sp., Pipturus albidus, Scaevola sericea, Eugenia malaccensis, Piper methysticum, Barringtonia asiatica and Adansonia digitata extracts showed anti-fungal activity to a lesser extent. Eugenia malaccensis was also found to inhibit the classical pathway of complement suggesting that an immunological basis for its in vivo activity was identified. This study has confirmed some of the ethnobotanical reports of Hawaiian medicinal plants having curative properties against infections using biological assays in vitro.

pp60src-mediated phosphorylation of connexin 43, a gap junction protein.

Several laboratories have demonstrated a decrease in gap junctional communication in cells transformed by the src oncogene of the Rous sarcoma virus. The decrease in gap junctional communication was associated with tyrosine phosphorylation of the gap junction protein, connexin 43 (Cx43). This study was initiated to determine if the phosphorylation of Cx43 is the result of a direct kinase-substrate interaction between the highly active tyrosine kinase, pp60v-src, and Cx43. Previous biochemical studies have been limited by the low levels of Cx43 protein in fibroblast cell lines. To obtain larger quantities of Cx43, we constructed a recombinant baculovirus expressing Cx43 in Spodoptera frugiperda (Sf-9) cells and subsequently purified the expressed Cx43 by immunoaffinity chromatography. We observed that this partially purified Cx43 was phosphorylated on tyrosine in vitro in the presence of kinase-active pp60src. Phosphotryptic peptide mapping indicated that the in vitro phosphorylated Cx43 contained phosphopeptides which comigrated with a subset of tryptic peptides prepared from Cx43 phosphorylated in vivo. Furthermore, coinfection of Sf-9 cells with recombinant baculoviruses encoding pp60v-src and Cx43 resulted in the accumulation of phosphotyrosine in Cx43. Taken together, the evidence presented in this paper demonstrates that kinase active pp60c-src is capable of phosphorylating Cx43 in a direct manner. Since the presence of phosphotyrosine on Cx43 is correlated with the down-regulation of gap-junctional communication, these results suggest that pp60v-src regulates gap junctional gating activity via tyrosine phosphorylation of Cx43.

Retinoid-enhanced gap junctional communication is achieved by increased levels of connexin 43 mRNA and protein.

Natural and synthetic retinoids are potent inhibitors of experimental carcinogenesis in animals and cause reversion of premalignant lesions in humans. In the model C3H 10T1/2 cell system, retinoids enhance postconfluent growth control, reversibly inhibit carcinogen-induced transformation, and enhance gap junctional intercellular communication. These effects are highly correlated. 10T1/2 cells were found to express low levels of connexin 43, a gap junctional protein first found in the heart. After treatment of confluent 10T1/2 cells with the synthetic retinoid tetrahydrotetramethylnapthalenylpropenylbenzoic acid (TTNPB), levels of connexin 43 mRNA and protein increased within 6 h of treatment, while elevation of junctional communication was detected within 12-18 h. The maximally effective concentration of TTNPB (10(-8) M) caused an approximate 10-fold elevation of connexin 43 gene transcripts after 72 h. Indirect immunofluorescence microscopy using a polyclonal antibody to the synthetic C-terminal region of connexin 43 demonstrated that TTNPB induced many fluorescent plaques in regions of cell-cell contact. These results provide a molecular basis for the retinoid-enhanced junctional communication in 10T1/2 cells. It is proposed that one action of retinoids is to modulate the intercellular transfer of signal molecules. These could mediate many of the physiological actions of retinoids on growth control and carcinogenesis.

Exposure of an arginine-rich protein at surface of cells in S, G2, and M phases of the cell cycle.

Phenylglyoxal (PG) is shown to be a cell surface probe specific for arginine moieties in protein: (1) It does not enter the cell as evidenced by lack of PG in the cytoplasm. (2) It does not cause excessive cell leakage as measured by release of 51Cr. (3) It reacts with positively-charged groups in proteins at the cell surface but not with those of phospholipids at the surface; since pronase removes PG from the surface, but phospholipase C does not. (4) Under the conditions used in these experiments, it reacts virtually exclusively with arginine moieties in protein (Freedman et al., '68; Takahashi, '68; Werber and Sokolovsky, '72). Synchronized cells were exposed to radioactive PG to assess quantity of arginine moieties in protein at the surface. There is a sharp decrease in arginine at the cell surface at entry into G1 phase from M and a 24-fold increase upon entry into S phase. There is a slight drop in exposed arginine in late S phase followed by an increase to 26 times the G1 level immediately prior to mitosis. Lactoperoxidase-catalyzed iodination of tyrosine moieties in protein at the surface of synchronized cells shows a very gradual increase in protein as the cells move through the cycle and increase in size. Since the increase in arginine moieties in protein at the surface does not reflect a similar increase in total protein at the surface, an arginine-rich protein appears to be exposed at the cell surface during the division-related phases of the cell cycle.